CN116535521B - BCMA-targeted chimeric antigen receptor and its application - Google Patents

Abstract

The invention provides a BCMA-targeted chimeric antigen receptor, which comprises a human-derived BCMA-targeted ScFv structure, wherein a heavy chain variable region ScFv-VH and a light chain variable region ScFv-VL of the ScFv are connected through one or more G4S sequences. Novel second generation BCMA-targeted CAR-T cells were successfully constructed in the present invention by retroviral vector transduction. In vitro and in vivo experiments show that BCMA positive tumor cells can effectively activate BCMA CAR31-T cells after being stimulated, secrete cytokines such as IFN-gamma, TNF-alpha and the like, and promote apoptosis of tumor cells. BCMA CAR31-T cells are able to effectively and specifically kill BCMA positive tumor cells. Therefore, the novel BCMA-targeted second-generation CAR-T cells have high-efficiency and specific anti-tumor activity, and can become a novel therapy for clinically treating MM.

Description

Chimeric antigen receptor of targeted BCMA and application thereof

Technical Field

The invention relates to the field of cellular immunotherapy of tumors, in particular to a targeted BCMA chimeric antigen receptor.

Background

Multiple Myeloma (MM) is a malignant tumor of abnormal proliferation of terminally differentiated plasma cells, the second most common hematological malignancy. In recent years, with the development of new therapeutic drugs such as proteasome inhibitors and immunomodulators, the prognosis of patients has been significantly improved. MM is still an incurable condition and almost all patients eventually face the risk of relapse and drug resistance. Thus, new treatment regimens are critical for these patients. Immunotherapy represented by chimeric antigen receptor T (CHIMERIC ANTIGEN receptor modified T cell, CAR-T) cells shows good therapeutic effects in hematological tumors, and is expected to be a novel and effective therapeutic approach for MM.

Unlike traditional T cell activation pathways, CAR-T cell activation is independent of MHC presentation. The CAR-T cells recognize and bind to tumor target cell surface antigens via extracellular single chain variable regions (ScFv-chain variable fragment). In the original design, the extracellular antigen binding domain is linked to the intracellular signaling domain via a transmembrane domain (first generation CAR) which, upon recognition and binding to antigen, directly induces T cell activation. The binding of the antigen binding domain to the ligand on the cell surface provides a first signal and once bound to the ligand, the intracellular co-stimulatory molecule is then activated to provide a second signal, which is transmitted to the intracellular activation domain to activate CAR-T, exerting antitumor activity. The first generation CAR structure was found to have little tumor clearing effect and no proliferative activity, so researchers constructed a second generation CAR comprising co-stimulatory domains such as CD28 or 4-1BB, which has greater anti-tumor activity. CARs that incorporate more than one intracellular co-stimulatory domain (e.g., CD28, OX40, 4-1BB, CD27, etc.) become third generation CARs. Fourth generation CARs are CARs capable of secreting cytokines such as IL-12 and expressing cell surface markers such as co-stimulatory ligands. Currently, the second generation CAR structure shows better safety and clinical effectiveness, so that the clinical application is wider.

Typical CAR-T cell production requires peripheral blood extraction from a patient, peripheral Blood Mononuclear Cells (PBMCs) separation, T cell stimulation and activation, and then cell surface expression of CARs capable of specifically recognizing tumor target antigens by genetic engineering, and successful CAR-T cell preparation is returned to the patient after massive expansion, thereby exerting antitumor function. Production of CAR-T cells typically takes 10-14 days. Since 2017, 5 CAR-T products have been marketed with FDA approval for the treatment of hematological malignancies, with Abecma targeting BCMA marketed in the present year as the first CAR-T cell product for the treatment of MM.

Recognition of tumor-specific antigens is critical for the success of CAR-T cell therapy. First, the antigen must be expressed on the surface of tumor cells. Second, the antigen must be expressed uniformly on the tumor cells and should ideally be necessary for tumor survival. Most importantly, the target antigen cannot be expressed in the relevant healthy tissue to avoid potential on-target extra-tumor toxicity.

BCMA, also known as CD269 or TNFRSF17, is a 27kDa type III transmembrane glycoprotein expressed by mature B lymphocytes, plasma cells and most patients with multiple myeloma. BCMA can bind to a variety of ligands, including BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand), and mediates cell survival via downstream NF- κb and MAPK/JNK signaling pathways, BCMA is a member of tumor necrosis factor, playing a vital role in supporting plasma cell differentiation and survival. It is selectively expressed on normal plasma cells and MM cells, and BCMA expression is not detected in cells of other tissues. BCMA, together with transmembrane activator, calcium modulator and cyclophilin ligand interacting factor (TACI), is also a receptor for proliferation-inducing ligands (APRIL). In multiple myeloma, the APRIL/BCMA pathway plays a key role in supporting a growing, drug-resistant and immunocompromised environment. BCMA has become an ideal target for the treatment of multiple myeloma due to its unique expression on plasma cells and its important role in multiple myeloma.

Although BCMA CAR-T cells targeting MM have been reported, clinical trials on novel CAR-T cells are still needed to advance the treatment of MM.

Disclosure of Invention

In order to solve the above problems, the present invention provides a chimeric antigen receptor targeting BCMA, which comprises a human-derived BCMA-targeting ScFv structure, wherein the heavy chain variable region ScFv-VH and the light chain variable region ScFv-VL of the ScFv are connected by one or more G4S sequences, wherein the amino acid sequence of the heavy chain variable region ScFv-VH has at least 90% homology, preferably at least 95% homology, more preferably at least 98% homology with the following SEQ ID NO.8 ：SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGS IYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYF EPAYWGQGTLVTVSS;

The amino acid sequence of the light chain variable region ScFv-VL has at least 90% homology, preferably at least 95% homology, more preferably at least 98% homology with SEQ ID NO.9 as follows ：QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYE VSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGT KLTVLG.

In one embodiment, the ScFv structure of the receptor comprising human-derived BCMA is ScFv-VH- (G4S) n-ScFv-VL, wherein n is an integer of 1 or more, preferably 3 or 4.

In one embodiment, the receptor comprises human-derived BCMA with an ScFv-VH- (G4S) 3-ScFv-VL having the amino acid sequence SEQ ID NO.8 and a heavy chain variable region ScFv-VL having the amino acid sequence SEQ ID NO.9.

In one embodiment, the ScFv structure of the receptor comprising human-derived BCMA is ScFv-VH- (G4S) n-ScFv-VL- (G4S) n-ScFv-VH, wherein n is an integer of 1 or more, preferably 3 or 4.

In one embodiment, the receptor comprises a human-derived BCMA-targeted ScFv having the structure ScFv-VH- (G4S) 3-ScFv-VL- (G4S) 4-ScFv-VL- (G4S) 3-ScFv-VH, the heavy chain variable region ScFv-VH having the amino acid sequence of SEQ ID NO.8, and the light chain variable region ScFv-VL having the amino acid sequence of SEQ ID NO.9.

In one embodiment, the receptor comprises an upstream signal peptide and myc tag for detection in tandem, a ScFv structure comprising a human targeted BCMA of heavy and light chain variable regions, a CD8 hinge-transmembrane domain, a CD28 or 4-1BB co-activation domain and a CD3 zeta intracellular signaling domain.

In one embodiment, the invention provides a chimeric antigen receptor T cell targeted to BCMA, which expresses the chimeric antigen receptor described above.

In one embodiment, the invention provides a medicament for treating tumors, which comprises the chimeric antigen receptor T cells described above.

In one embodiment, the present invention provides the chimeric antigen receptor described above for use in the preparation of chimeric antigen receptor T cells and their use in tumor therapy.

In one embodiment, the tumor is a surface BCMA positive tumor.

In one embodiment, the tumor is multiple myeloma.

In one embodiment, the invention provides the use of the chimeric antigen receptor described above, wherein a gene fragment encoding the chimeric antigen receptor is inserted into a viral expression vector, packaged into viral vector particles, and infected with human T cells to prepare chimeric antigen receptor T cells for surface BCMA positive tumor treatment.

In the present invention, the plasmids of pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 which are successfully constructed are transiently transfected into Phoenix-ECO cells, the transfection efficiency is higher than 50%, the transduction efficiency is higher than 95% by using transiently transfected retroviral vector particles to transduce PG13 cell lines, pPCR detection shows that the retroviral vector particles have higher viral titer, and the highest titer is higher than 1X 10 ⁷ copies/mL, which indicates that the PG13 cell lines which stably express and produce BCMA CAR retroviral vectors are successfully prepared. Transduction of human primary T cells with successfully prepared amphotropic retroviral vectors BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19 and BCMA CAR20 and detection of CAR expression indicated that we have successfully prepared BCMA CAR-T cells.

In an in vitro tumor killing experiment, the invention utilizes flow cytometry to detect apoptosis of target cells, and a luciferase bioluminescence method to detect survival of target cells, wherein BCMA CAR16-T shows relatively better anti-tumor capability. However, since the killing effect was inferior to the positive control group, we showed that the BCMA CAR16 screened could be further optimized to increase the binding capacity to the target antigen.

Because BCMA is in a trimer structure and the molecular weight of protein is smaller than 32.3kDa, BCMA CAR16 is subjected to series connection of ScFv structures, and BCMA CAR31, BCMA CAR32 and BCMA CAR33 are designed to increase the flexibility of an extracellular antigen binding domain ScFv region, better capture antigen and improve the killing capacity on tumors.

The amphotropic retroviral vectors were prepared from BCMA CAR31, BCMA CAR32 and BCMA CAR33 and transduced human primary T cells with transduction efficiencies exceeding 50% and qPCR assays showed successful integration into the T cell genome.

In an in vitro tumor killing experiment, the BCMA CAR31-T cells with the best killing effect are screened by the in-vitro tumor killing experiment through real-time dynamic living cell imaging, and are further compared with BCMA CAR16-T cells. CD69 is a mark of T cell activation, and the BCMA CAR31-T and BCMA CAR16-T cells have no obvious self-activation phenomenon when no tumor cell stimulation is found, so that the BCMA CAR-T cells can be effectively activated when the tumor cells are stimulated, and the anti-tumor effect can be exerted.

To determine the antitumor capacity of BCMA CAR31-T cells in vitro, we selected RPMI-gfp-luc expressing human BCMA positive tumor cells. The survival result of the target cells detected by the luciferase bioluminescence method shows that compared with the BCMA CAR16-T group, under different effect target ratios, the BCMA CAR31-T cells have stronger killing power on two different BCMA positive tumor cells, which indicates that the killing power of the BCMA CAR31-T after structure optimization on the tumor cells is improved.

To verify the antigen specificity of the antitumor effect of BCMA CAR31-T cells, BCMA CAR31-T cells were co-incubated with different kinds of tumor cell lines K562-hbma-gfp, PMI-gfp-luc, K562-cBCMA and K562, and as a result, it was shown that BCMA CAR31-T cells had stronger killing power against both different BCMA positive tumor cells at different potency ratios compared to BCMA CAR16-T group. However, BCMA CAR31-T exhibited an indistinguishable killing capacity from Pan-T for non-human BCMA expressing K562-cBCMA and BCMA negative K562 cells, indicating BCMA antigen specificity for killing tumor cells by BCMA CAR31-T cells.

When the CAR-T cells bind to tumor antigens, the CAR-T cells recruit other immune cells, releasing a large amount of cytokines, producing an anti-tumor immune response. In cytokine secretion experiments, BCMA CAR31-T cells secreted more pro-inflammatory cytokines TNF- α, IFN- γ, IL-6, IL-17A and aFasL under stimulation by tumor cells than BCMA CAR16-T, thereby promoting apoptosis of tumor cells.

Generating a strong and durable anti-tumor immune response requires not only the triggering of cytotoxicity and cytokine production, but also the stimulation of CAR-T cell proliferation. We found that BCMA CAR31-T cells proliferated faster than BCMA CAR16-T by CFSE assay.

In addition, the invention establishes a tumor xenograft model of NPG mice to confirm the anti-tumor ability of BCMA CAR31-T cells in vivo. After two days of the second tail vein injection of BCMA CAR31-T, secretion of IFN-gamma cytokines is obviously enhanced compared with BCMA CAR16-T, which indicates that BCMA CAR31-T cells are activated better, the general condition of mice is good, and symptoms of cytokine release syndromes such as fever, nausea, vomiting and the like are not generated. During the period of continuously observing the tumor change of the mice, the BCMA CAR31-T group tumor signals are obviously weakened compared with BCMA CAR16-T, more effective anti-tumor capability is shown, the flow cytometry continuously detects the T cell content in the peripheral blood of the mice, and the result shows that the BCMA CAR31-T cell content is higher than the BCMA CAR16-T cell content until 37 days after tumor inoculation, so that the BCMA CAR31-T cell has longer lasting survival time in the mice and longer lasting anti-tumor capability.

Thus, novel second generation BCMA-targeted CAR-T cells were successfully constructed in the present invention by retroviral vector transduction. In vitro experiments show that BCMA positive tumor cells can effectively activate BCMA CAR31-T cells after being stimulated, secrete cytokines such as IFN-gamma, TNF-alpha and the like, and promote apoptosis of tumor cells. BCMA CAR31-T cells are able to effectively and specifically kill BCMA positive tumor cells. BCMA CAR31-T cells have good proliferative capacity in vitro. Further in vivo anti-tumor experiments show that BCMA CAR31-T cells can be rapidly activated to secrete IFN-gamma, and have a certain tumor effect. Thus, the novel BCMA-targeted second generation CAR-T cells have high-efficiency and specific antitumor activity and are likely to become a new therapy for clinically treating MM.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram of an experimental scheme of the present invention;

fig. 2 is a schematic structural diagram of a BCMA CAR of the present invention;

FIG. 3 is a schematic representation of a BCMA CAR retroviral vector plasmid preparation scheme;

FIG. 4 is a technical roadmap for BCMA CAR retroviral vector packaging;

FIG. 5 is a technical roadmap for the preparation of BCMA CAR-T cells;

FIG. 6 is a technical roadmap of BCMA CAR-T in vitro anti-tumor function;

FIG. 7 is a graph showing the results of flow cytometry detection of BCMA CAR-T cell killing K562-hBCMA-gfp cell efficiency;

Figure 8BCMA CAR-T cell killing RPMI-gfp-luc cell efficiency assay (n=3) (^* P <0.05 compared to bb2121CAR-T cells);

Fig. 9 is a schematic structural diagram of an optimized BCMA CAR;

FIG. 10 shows a gel electrophoresis pattern of the digestion assay, wherein A is the digestion assay fragment of pMFG-BCMA CAR31 and pMFG-BCMA CAR32, B is the digestion assay fragment of pMFG-BCMA CAR 33;

Fig. 11 is a graph of the results of optimized BCMA CAR amphotropic retroviral vector particle transduction efficiency for human primary T cells (n=3) (ns: no statistical difference);

FIG. 12 is a graph of an in vitro killing assay protocol for optimized BCMA CAR-T cells;

Fig. 13 is a graph of real-time fluorescence monitoring results of in vitro killing of K562-hbma-gfp by the BCMA CAR-T cells optimized of fig. (n=3);

FIG. 14 is a graph of results of CAR-T cell CD69 expression (n=3), wherein A: BCMA CAR-T cell surface CD69 expression flow chart, B: BCMA CAR-T cell surface CD69 expression histogram without tumor cell stimulation, C: BCMA CAR-T cell surface CD69 expression histogram with BCMA+ tumor cell stimulation (P <0.05, ns: no statistical difference compared to BCMA CAR16-T cells);

fig. 15 is a graph of the results of luciferase bioluminescence assay for BCMA CAR-T cell killing ability in vitro (n=3) (P <0.05, P <0.01, P <0.001 compared to BCMA CAR16-T cells);

fig. 16 is a graph of the results of different tumor cell killing efficiency assays (n=3) for BCMA CAR-T cells (P <0.05, P <0.01, P <0.001, P <0.0001, ns: no statistical difference compared to BCMA CAR16-T cells);

Fig. 17 is a graph of BCMA CAR-T cytokine secretion levels (n=3) results (P <0.05, P <0.01, P <0.001, P < 0.0001) compared to BCMA CAR16-T cells;

FIG. 18 is a graph of BCMA CAR-T cell proliferation potency assay results;

FIG. 19 is a graph of an in vivo anti-tumor assay protocol for BCMA CAR-T;

FIG. 20 is a graph of results of in vivo imaging (n=6) of a xenograft model of a mouse, A: a graph of in vivo imaging of a mouse, B: a statistical graph of mean signal intensity of a tumor of a mouse (ns: no statistical difference);

fig. 21 is a graph of results of in vivo imaging (n=6) of a mouse xenograft model, a: a mouse in vivo imaging picture, B: a mouse tumor mean signal intensity statistic (P <0.05, P <0.01, P <0.001 compared to BCMA CAR16-T cells);

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application.

It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

6 Novel human-source BCMA-targeted ScFv structures are screened out by a phage display method. Specific experimental protocol as shown in fig. 1, to verify whether ScFv of these 6 BCMA-targeted cells were able to efficiently and specifically recognize BCMA-positive tumor cells, we designed and constructed a BCMA CAR structure of second generation human origin using CD8 as transmembrane domain, CD28 and CD3 zeta as intracellular stimulatory domain, see fig. 2, and preparing BCMA CAR-T cells by retroviral vector. And (3) screening BCMA CAR16-T cells with the strongest killing capacity to tumor cells through in-vitro tumor cell killing experiments. We further concatenated ScFv of BCMA CAR16 to further increase antigen binding capacity to BCMA. In vitro experiments, tumor cells expressing human BCMA antigens are used as target cells, activation conditions of BCMA CAR-T after optimization are detected, killing specificity and effectiveness contrast of the BCMA CAR-T cells to the target cells are verified, proliferation capacity and cytokine secretion level contrast of the BCMA CAR-T are detected, verification is further carried out in an in vivo xenograft tumor model, secretion conditions and anti-tumor capacity of the cytokines are detected, and maintenance conditions of the CAR-T cells in vivo after treatment are detected.

In the present invention the amino acid sequence is as follows:

SP amino acid sequence MEWSWVFLFFLSVTTGVHSDI (SEQ ID NO. 1);

EQKLISEEDL (SEQ ID NO. 2) Myc amino acid sequence;

CD8 amino acid sequence:

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA(SEQ ID NO.3);

CD28 amino acid sequence:

PRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO.4)

CD3 zeta amino acid sequence:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.5)

BCMA CAR15 ScFv-VH amino acid sequence:

SQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMDLSSLTSEDTAVYYCARDRGNSADFDSWGQGTLVTVSS(SEQ ID NO.6)

BCMA CAR15 ScFv-VL amino acid sequence:

DIVMTQSPSSLSASVGDRVTITCRASRDINRWLAWYRRKPGKAPELLIYAASDLKHGVPSRFSGSGSGTDFTLTISSLEPEDFATYYCQQGDSWPFTFGRGTKLEIKR(SEQ ID NO.7)

BCMA CAR16 ScFv-VH amino acid sequence:

SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSS(SEQ ID NO.8)

BCMA CAR16 ScFv-VL amino acid sequence:

QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG(SEQ ID NO.9)

BCMA CAR17 ScFv-VH amino acid sequence:

SQVQLLESHWVGTTSYAQNGKPGASVRSEDTAVYYQRDTSTSTVYMELCACKEHATSYYIFSPGQIVAGTFGLEWASGYSFVKMGIIRVSSSLRAEDGRVRQNPSVTMTYWGQGTLVTVSS(SEQ ID NO.10).

BCMA CAR17 ScFv-VL amino acid sequence:

QSALDRDSSSTLVFGGFSGSSSEVVSGTLRPGQSITWYTITQPASVSLSKRGPSGVPTQPASVSLYNYVSGSKSGNYYCCTGTSSMIYPKLSYQATYSKRGPSGVPSGLQGEDEAQHPGKAGQLTVLG(SEQ ID NO.11);

BCMA CAR18 ScFv-VH amino acid sequence:

SQVQLVESGGGVVSRDNSKNTVSCAASGFISYDGEDTAVYYYLQMNSLRQPGRSLRSNKYYADSVKGRAFSSYGMHWVRQAPGFISYDGEDGLEWVALGKFTIVLCARDLFGGGDVLRDSWGQGTLVTVSS(SEQ ID NO.12)

BCMA CAR18 ScFv-VL amino acid sequence:

DIQMTQSPSPSRFSGSASQPEDFATYYCQQSYSTLFTAPKLLIYAASQSISSYFTLTISSSLSASVGDLNWYPGKGTDQGSFGPTITCRKSGVLRVSLQQGTKVEIKR(SEQ ID NO.13)

BCMA CAR19 ScFv-VH amino acid sequence:

SQVQLVESGGGFVLNWVRLAPGKGFISRDNSKNTLYLQMNSLYYDSVKGRFYLVQPGGANTRVEDTAVYSGISGSGGLSLTFEWVTRLSCAASDSTCANLWTAAGIDYWGQGTLVTVSS(SEQ ID NO.14)

BCMA CAR19 ScFv-VL amino acid sequence:

QSALTQPASVASHRFSTTNNDESGSKISCTASTLGTSSDIGKSGSMYDVSSRPSGAPKFIYDRVSWYQQHPVFGLQADLTISGNTGSPGQSITADYYCNSYGGGTKLTVLG(SEQ ID NO.15)

BCMA CAR20 ScFv-VH amino acid sequence:

SQVQLVESGNSTAVYDVWLVCAACAKEVWGGLVRKNTLYISGSGGSTYYADQMNSLRAEYVQPEWSVGSSGFSGFLWSRLSTSYAMSLEKGRFTISRDDQAPGKGGKGTIVTVSS(SEQ ID NO.16)

BCMA CAR20 ScFv-VL amino acid sequence:

NIVMTQSPSTYLTISNLQPVGDRVITCRAKPGQSLSASVGDRVTWYQVPSRFSGSGSHSAPKLLIYFNTSLQEDSFPWSGGTTQSIRDFTLATYYCQQYHGALGHGTKLEIKR(SEQ ID NO.17)

BCMA CAR31 ScFv amino acid sequence:

SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSS(SEQ ID NO.18)

BCMA CAR32 ScFv amino acid sequence:

SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG(SEQ ID NO.19), Wherein the underline in the sequence is (G4S) n;

BCMA CAR33 ScFv amino acid sequence:

SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG(SEQ ID NO.20), Wherein the underlined sequence is the (G4S) n sequence.

EXAMPLE BCMA CAR retroviral vector plasmid construction

6 BCMA-targeting ScFv sequences were screened and synthesized by general biosystems to pUC57 vector. Plasmid containing CD28-CD8-CD3 zeta sequence and retrovirus vector pMFG plasmid can be purchased commercially or prepared by oneself, scFv fragment and CD28-CD8-CD3 zeta fragment are amplified by PCR method, scFv-CD28-CD8-CD3 zeta fragment is amplified by homologous recombination method, recombined target fragment and vector are cut by XhoI and NotI double enzyme, and then connection is made, so that the complete pMFG-BCMA CAR plasmid is constructed. Specific experimental protocol figure 3 shows.

Experimental procedure

1. Vector plasmid and plasmid extraction of interest

Taking out pUC57-BCMA CAR15、pUC57-BCMA CAR16、pUC57-BCMA CAR17、pUC57-BCMA CAR18、pUC57-BCMA CAR19、pUC57-BCMA CAR20、pMFG-CD8-CD28-CD3ζ and vector plasmid pMFG strain stored at-80 ℃, adding 50 mu L of each strain into 25mL of LB liquid medium (the final concentration of Amp is 50 mu g/mL), and shaking the strain in a shaking table at 37 ℃ and 200rpm for 12-16h to 3-4 multiplied by 10 ⁹/mL of cell density. Bacterial pellet was harvested, passed through centrifugation, suspension, filtration column, 4mL of isopropanol was added to the eluate and mixed well. Immediately, the supernatant was discarded by centrifugation at 15000g for 30min at 4 ℃. The DNA pellet was washed with 2mL of 75% ethanol, centrifuged at 15000g for 10min at room temperature, and the supernatant carefully discarded. Drying the precipitate for 5-10min, adding appropriate volume of nuclease-free water, flicking, and mixing. The DNA concentration was measured and the plasmid was stored at-20℃for further use.

2. PCR amplification of fragments of interest

The pUC57-BCMA CAR15、pUC57-BCMA CAR16、pUC57-BCMA CAR17、pUC57-BCMA CAR18、pUC57-BCMA CAR19、pUC57-BCMA CAR20 plasmid obtained above was subjected to amplification of the target bands by PCR, each target fragment being about 800bp. The pMFG-CD8-CD28-CD3 zeta plasmid obtained above was subjected to amplification of the target band by PCR method, and the fragment was about 800bp.

TABLE 1 primer sequence listing

The obtained target fragments were named CAR15-ScFv, CAR16-ScFv, CAR17-ScFv, CAR18-ScFv, CAR19-ScFv, CAR20-ScFv and CD8-CD28-CD3 zeta, respectively. The obtained CAR15-ScFv, CAR16-ScFv, CAR17-ScFv, CAR18-ScFv, CAR19-ScFv and CAR20-ScFv were subjected to homologous recombination PCR with CD8-CD28-CD3 zeta, respectively, to obtain complete ScFv-CD8-CD28-CD3 zeta fragments, each fragment being about 1600bp. And (3) enzyme cutting the vector plasmid and homologous recombination target fragment.

The purified CAR15-ScFv-CD8-CD28-CD3ζ、CAR16-ScFv-CD8-CD28-CD3ζ、CAR17-ScFv-CD8-CD28-CD3ζ、CAR18-ScFv-CD8-CD28-CD3ζ、CAR19-ScFv-CD8-CD28-CD3ζ、CAR20-ScFv-CD8-CD28-CD3ζ was double digested with XhoI and NotI restriction enzymes, and the pMFG vector fragment was electrophoresed and recovered.

And connecting the target fragment and the carrier fragment, and connecting the XhoI and NotI double-enzyme-cut target fragment CAR15-ScFv-CD8-CD28-CD3ζ、CAR16-ScFv-CD8-CD28-CD3ζ、CAR17-ScFv-CD8-CD28-CD3ζ、CAR18-ScFv-CD8-CD28-CD3ζ、CAR19-ScFv-CD8-CD28-CD3ζ、CAR20-ScFv-CD8-CD28-CD3ζ recovered by the gel with the pMFG carrier enzyme-cut fragment, wherein the molar ratio of the target fragment to the carrier fragment is 3:1-5:1.

The ligation product was transformed, transformed with DH 5. Alpha. Competent cells, followed by plasmid DNA extraction and restriction enzyme identification. The extracted plasmid DNA thus constructed was digested with XhoI and NotI restriction enzymes to determine whether the band was correct, and the correct plasmid DNA was identified by digestion. The restriction enzyme was identified correctly and the purified plasmid DNA was sequenced by the engine, sequencing primers as shown in the following table:

TABLE 2 sequencing primer Table

Experimental results

BCMA CAR structure schematic diagram

The ScFv region of BCMA CAR is humanized as a monoclonal antibody sequence targeting BCMA, the SP gene and myc gene for detecting expression efficiency are added before ScFv gene sequence. The transmembrane region employs a CD8 hinge-transmembrane domain. The intracellular co-stimulatory molecule employs a CD28 molecule as the co-stimulatory domain and the intracellular signaling molecule employs CD3 zeta. Six BCMA CAR expression plasmid maps screened are shown in figure 2.

The result of amplifying gel electrophoresis of target fragments shows that plasmids pUC-BCMA CAR15, pUC-BCMA CAR16, pUC-BCMA CAR17, pUC-BCMA CAR18, pUC-BCMA CAR19 and pUC-BCMA CAR20 containing ScFv fragments are amplified by a PCR method, the sizes of the plasmids are about 800bp, the plasmids containing CD8-CD28-CD3 zeta fragments are amplified by PCR, the fragment sizes are about 830bp, and the gel electrophoresis result shows that the target bands are correct.

2. Results of gel electrophoresis of homologous recombination of target fragment

The CAR15-ScFv, the CAR16-ScFv, the CAR17-ScFv, the CAR18-ScFv, the CAR19-ScFv and the CAR20-ScFv are recombined by using a homologous recombination PCR method, the sizes of fragments after recombination are about 1.6kb, and gel electrophoresis junctions show that the sizes of the bands are correct.

3. Gel electrophoresis result of enzyme cutting carrier fragment

The plasmid of pMFG vector is digested with XhoI and NotI, the fragment size is about 7kb, and gel electrophoresis result shows that the fragment size is correct, and the plasmid can be used for connection experiment with target band.

4. Enzyme digestion identification result

The constructed plasmids are subjected to XhoI and NotI double enzyme digestion identification, the fragment sizes are 7kb and 1.6kb, and gel electrophoresis results show that the constructed plasmids pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 are correct in size and can be further verified by sequencing.

5. Successful construction of BCMA CAR plasmid

The plasmid with correct enzyme cutting identification is purified and then is sent to sequencing. Sequencing results indicated that the pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 sequences were completely correct.

EXAMPLE two BCMA CAR retroviral vector packaging

On the basis of constructing and completing pMFG-BCMA CAR plasmid, packaging the-BCMA CAR retrovirus vector, and constructing a cell line for stably producing the retrovirus vector. The specific experimental procedure is shown in FIG. 4, where the pMFG-BCMA CAR plasmid was first transiently transfected into Phoenix ECO cells and BCMA CAR avirulent retroviral vector supernatant particles were collected. The collected BCMA CAR amphotropic retroviral vector particles were transduced into PG13 cell lines for production of amphotropic retroviral vector supernatant particles, and PG13 cell lines were constructed that stably produced BCMA CAR amphotropic retroviral vector supernatant particles, and viral vector titers were detected using qPCR to verify whether BCMA CAR amphotropic retroviral vector supernatant particles were successfully produced.

Material (B)

Human eosinophil packaging cell line Pheonix-ECO, gibbon leukemia virus packaging cell line PG13 was purchased from American type culture Collection (AMERICAN TYPE culture collection, ATCC).

DMEM complete medium is prepared by operating in a biosafety cabinet, adding 56mL FBS (10%) and 5.6mL of green streptomycin double antibody (1%) into every 500mL of DMEM (89%), vacuum filtering the prepared DMEM complete medium by using a 500mL filter with the thickness of 0.22 μm, marking the bottle body as a reagent, and storing in a refrigerator with the temperature of 4 ℃ for standby.

Cell frozen stock solution is prepared by preparing FBS and DMSO in a ratio of FBS to DMSO=9:1 in a biosafety cabinet, and filtering the prepared cell frozen stock solution with a 0.22 mu m filter membrane for later use.

Staining buffer PBS (1×) and FBS were prepared in the ratio PBS to fbs=9:1 and placed at 4 ℃ for use.

Second, experimental method

Phoenix ECO and PG13 cell culture

1.1. Cell resuscitation

The cells to be resuscitated are taken out from the liquid nitrogen tank for resuscitating, then the cell pellet is resuspended in DMEM complete medium, and the cell suspension is transferred to a proper culture flask and placed in a 37 ℃ and 5% CO ₂ incubator for culture.

1.2 Passage of cells and liquid exchange

The cell adhesion and growth state were observed under a microscope, and if the cell density was <80%, but the color of the medium became yellow, a cell liquid change treatment was required. If the cell density is >80%, then cell passaging is required. Cells that were not successfully adherent were washed by shaking the flask several times with the appropriate volume of 1 XPBS to wash the cells. The washed PBS is gently removed, a proper volume of trypsin is added for cell digestion, and the culture flask is gently shaken to enable the trypsin to be fully contacted with the cells for accelerating the cell digestion, and the cells can be placed into a 37 ℃ incubator for accelerating the cell digestion. Cells were observed under a microscope for shedding and after digestion until cells shed about 90%, digestion was stopped by adding three volumes of trypsin in DMEM complete medium. The cell suspension was transferred to a centrifuge tube and centrifuged at 300g for 5min. The supernatant was carefully pipetted. The cell pellet was resuspended by adding an appropriate volume of fresh DMEM complete medium. The cell suspension was transferred to an appropriate flask and placed in a 37 ℃ 5% CO ₂ incubator for culture. The cell growth state was observed under an inverted microscope every 24 hours.

1.3 Cryopreservation of cells

Cells were digested by adding an appropriate volume of trypsin, digested at 37℃for about 5min, and observed under a mirror every 1 min. When the cells had fallen off by about 90%, digestion was stopped by adding 3 times the volume of trypsin in DMEM complete medium. The cell suspension was gently blown into a single cell suspension with a pipette, and 20. Mu.L of the single cell suspension was removed and mixed with an equal volume of trypan blue solution. Drop onto a cell counter plate and count with a cell counter. The cell suspension was transferred to a centrifuge tube and centrifuged at 300g for 5min. The supernatant was aspirated with a pipette and discarded, taking care not to contact the cell pellet, and the cells were resuspended in cell cryopreservation solution (typically 5X 10 ⁶-1×10⁷/mL). The cell cryopreservation suspension was added to a 2mL cryopreservation tube, and the procedure was followed to transfer the incubator immediately into a-80 ℃ refrigerator overnight.

2. Preparation of the supernatant of the philic retroviral vector

2.1 Plating of Phoenix-ECO cells old medium was discarded from well-grown Phoenix-ECO cells. Cells were digested by trypsin addition and incubated at 37 ℃ for about 5min to accelerate cell digestion. Digestion was stopped by adding 3 volumes of trypsin in DMEM complete medium and centrifuged at 300g for 5min. The cells were resuspended in DMEM complete medium, and 20. Mu.L of single cell suspension was added to an equal volume of trypan blue solution for cell counting.

The single cell suspension required for seeding a six well cell culture plate was calculated and formulated at 1X 10 ⁶ per well, 2.5mL per well. Single cell suspensions diluted to 1 x 10 ⁶ per well were added to six well plates. The six-hole culture plate is gently shaken back and forth or left and right to uniformly distribute cells. Six-well plates were placed in a 37 ℃ CO2 incubator containing 5% of the total gas for overnight incubation.

2.2Phoenix-ECO cell transfection

After 24h of cell inoculation, the six-hole culture plate is taken out, observed under an inverted microscope, and transfected when the cell growth state is good and the density reaches about 80%. The required plasmid pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 volumes, and Fugene HD volumes were calculated.

Six well plates were changed and 2.3mL DMEM complete medium was added to each well. 200. Mu.L of the mixed solution was gently added dropwise to a six-well plate, and the total volume was made 2.5mL per well, and the plate was gently shaken well. Six well plates were placed in a 37 ℃ CO2 incubator containing 5% CO2 for cultivation.

2.3 Harvesting of the supernatant of the aviphilic retroviral vector

After 24h of transfection, the six-well plates were removed from the incubator, the culture supernatant carefully removed, fresh DMEM complete medium was slowly added, 2.5 mL/well, and the six-well plates were moved to a 32℃incubator containing 5% CO ₂ for cultivation. After 48h of transfection, carefully collecting the supernatant into a centrifuge tube, slowly adding new DMEM complete medium at 2.5 mL/well, transferring the six-well culture plate to a 32 ℃ and 5% CO2 incubator for culture, filtering the collected culture supernatant by a 0.45 μm low-adsorption virus filter membrane, and sub-packaging and storing at-80 ℃ for later use. After 72h of transfection, the six-hole culture plate is taken out and placed into a biosafety cabinet, the supernatant is carefully collected into a centrifuge tube, the collected culture supernatant is filtered by a low-adsorption virus filter membrane with the thickness of 0.45 mu m, and the culture supernatant is subpackaged and stored at-80 ℃ for standby.

2.4Phoenix-ECO cell transfection efficiency assay

The Phoenix-ECO cells 72h after transfection were trypsinized and the cell suspension was collected after termination. Centrifugation at 300g for 5min, discarding, collecting cell pellet, and washing cells with PBS. Centrifuge at 300g for 5min at room temperature and discard the supernatant. Cells were resuspended in 50L staining buffer, stained with anti-hc-Myc PE antibody, and untransfected Phoenix-ECO cells served as negative control. After staining at 4℃for 1h in the dark, cells were washed by adding 900L PBS. Centrifugation at 300g for 5min, the supernatant was discarded and the cell pellet was resuspended in staining buffer. Transfection efficiency was measured using a flow cytometer.

3. Preparation of amphotropic retroviral vectors

PG13 cell transduction, pretreatment of non-treated 12 well plates with RetroNectin 1 day prior to transduction, dilution of RetroNectin with PBS to a final concentration of 10 μg/mL, 1mL per well. The PG13 cell control group, BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19, and BCMA CAR20 experimental group. The avidity retroviral vector supernatant collected 72h after transfection with Phoenix-ECO was removed from-80℃and re-thawed at room temperature and 1mL was added per well to a 12-well plate. The culture plates were centrifuged at 30℃for 1h at 12 wells, to which the supernatant of the philic retroviral vector was added. PG13 cells were removed from the 37℃incubator, the cell state was observed under a mirror, trypsinization was terminated, and a pipette was blown into a single cell suspension. Cell count, 1X 10 ⁶ cells per well, transfer to centrifuge tube, centrifuge at 300g for 5min.

After 48 hours of transduction, a part of digested cells are taken for transduction efficiency detection, and the rest PG13 cells are subjected to passage expansion culture to a T75 culture flask. When the cell density reached 80%, the amphotropic retroviral vector supernatant was designated as H0 and fresh DMEM complete medium was added and the plates were transferred to a 32℃and 5% CO ₂ incubator for cultivation, and the viral vector supernatant was extirpated at-80 ℃. Viral vector supernatants were then harvested 4 consecutive days and designated H1-H4. The harvested viral vector supernatant was stored at-80℃for later use. And detecting the transduction efficiency of the PG13 cells, taking a small amount of PG13 cells after 48 hours of transduction, and detecting the transduction efficiency. qPCR detects BCMA CAR retroviral vector titres and detects the viral titres of BCMA CAR amphotropic retroviral vector supernatants produced by the above-described collected PG13 cell lines to verify whether BCMA CAR amphotropic retroviral vector particles were successfully prepared.

Third, experimental results

1. Successful preparation of BCMA CAR avirulent retroviral vectors

The Phoenix-ECO cells were transfected with pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 plasmids, the transfection efficiencies were examined by 72h post-flow cytometry, the transfection efficiencies were 56.70% for pMFG-BCMA CAR15, 64.91% for pMFG-BCMA CAR16, 59.11% for pMFG-BCMA CAR17, 57.88% for pMFG-BCMA CAR18, 59.85% for pMFG-BCMA CAR19, 57.95% for pMFG-BCMA 20, and six plasmids were all successfully transfected in Phoenix-ECO cells. The supernatant of the amphotropic retroviral vector collected 72h after transfection was used to transduce PG13 cells.

2. Successful construction of cell lines stably producing BCMA CAR amphotropic retroviral vectors

Successfully prepared BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19 and BCMA CAR20 avirulent retroviral vector supernatants were transduced into PG13 cells and the transduction efficiencies were examined by flow cytometry after 48 h. BCMA CAR15 transduction efficiency was 96.91%, BCMA CAR16 transduction efficiency was 97.53%, BCMA CAR17 transduction efficiency was 98.50%, BCMA CAR18 transduction efficiency was 98.69%, BCMA CAR19 transduction efficiency was 97.82%, and BCMA CAR20 transduction efficiency was 98.62%, which indicated successful production of amphotropic retroviral vector particles and successful construction of PG13 cell lines that stably produced amphotropic retroviral vector particles.

3. Successful preparation of BCMA CAR amphotropic retroviral vectors

Expanding the PG13 cell line transduced by BCMA CAR amphotropic retroviral vector into a T75 culture flask, and continuously harvesting the amphotropic retroviral vector particles for 5 days for qPCR detection, wherein the titer of the BCMA CAR amphotropic retroviral vector is 1.40X10 ⁷±1.13×10⁶ copies/mL, the titer of the BCMA CAR 16H 1 is 1.33X10 ⁷±7.07×10⁴ copies/mL, the titer of the BCMA CAR 17H 2 is 3.09 X10 ⁷±1.70×10⁶ copies/mL, the titer of the BCMA CAR 18H 2 is 2.44X10 ⁷±2.55×10⁶ copies/mL, the titer of the BCMA CAR 19H 2 is 1.95X10 ⁷±2.36×10⁴ copies/mL, and the titer of the BCMA CAR 20H 2 is 2.68X10 ⁷±4.24×10⁶ copies/mL, which are higher than that of a positive control. This result suggests that we successfully prepared BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19, BCMA CAR20 amphotropic retroviral vectors, each BCMA CAR selecting the highest titer amphotropic retroviral vector particles for human primary T cell transduction experiments.

Example preparation of triple BCMA CAR-T cells

The BCMA CAR amphotropic retroviral vector particles are transduced into human primary T cells to construct BCMA CAR-T cells based on the preparation of BCMA CAR amphotropic retroviral vector particles. The technical route is shown in figure 5, wherein human peripheral blood PBMC is firstly separated by a density gradient centrifugation method, primary T cells are activated under the stimulation of a CD3 monoclonal antibody and a cytokine IL-2, BCMA CAR is transduced by the activated T cells in a centrifugal way, BCMA CAR-T cells are constructed, and the expression of a cell surface Myc label is detected by using a flow cytometry, so that whether the BCMA CAR is expressed on the surface of the T cells is detected.

Experimental method

1. Construction of BCMA CAR-T cells

Non-treated 12 well plates were pre-treated with retroNectin 1 day prior to transduction, diluted with PBS to a final concentration of 10 μg/mL, 1mL per well.

The T cell non-transduction control group (Pan-T group), BCMA CAR15-T group, BCMA CAR16-T group, BCMA CAR17-T group, BCMA CAR18-T group, BCMA CAR19-T group, BCMA CAR20-T group and bb2121 CAR-T group (BCMA CAR currently marketed, except for ScFv, the other structures are the same as those of other BCMA CARs we construct). The experiment was repeated for transduction by T cells isolated from different volunteers.

The RetroNectin solution was aspirated, 1mL of PBS was added to the 12-well plate, and the solution was discarded. Amphotropic retroviral vector supernatants collected from PG13 cell lines were removed from-80℃and re-thawed at room temperature, and 1mL was added to each well of a 12-well culture plate. The culture plates were centrifuged at 30℃for 1h at 12 wells to which the amphotropic retroviral vector supernatant was added. T cells were removed from the 37 ℃ incubator, observed under the mirror for cell status, and pipetted into single cell suspensions. Cell counts, 1×10 ⁶ cell suspensions per well, transferred to centrifuge tubes and centrifuged at 300g for 5min. The cells were resuspended in 1mL of amphotropic retroviral vector supernatant per well, and the control was resuspended in AIM-V complete medium. Cells were blown off as a single cell suspension and slowly added dropwise to a 12 well culture plate. The amphotropic retroviral vector supernatant was centrifuged at 30℃for 1h in a 12-well culture plate. Placing the 12-hole culture plate into a 37 ℃ and 5% CO ₂ incubator for culturing for not less than 1h. The 12-well plates were removed, the cells were collected by digestion, centrifuged at 300g for 5min, and the supernatant carefully removed. New amphotropic retroviral vector supernatant was slowly added dropwise, 1mL per well, and new AIM-V complete medium was added to the control. Cells were collected and centrifuged at 300g for 5min and the supernatant carefully removed. Cells were resuspended in fresh AIM-V complete medium. The 12-well plates were placed in a 37℃and 5% CO ₂ incubator for cultivation.

T cell transduction efficiency assay

The primary human T cells after 48h transduction were mixed with a pipette and gently blown off to form a single cell suspension. 300. Mu.L of the cell suspension was taken into a 1.5mL centrifuge tube. Centrifugation at 300g for 5min, cell pellet was collected and cells were washed with PBS. Centrifuge 300g for 5min at room temperature and discard supernatant. Cells were resuspended in 50. Mu.L of staining buffer, stained with APC anti-human CD3 antibody and anti-hc-Myc PE antibody, and untransduced T cells served as negative controls. After staining at 4℃for 1h in the dark, the cells were washed by adding 900. Mu.L of PBS. Centrifugation was performed at 300g for 5min, the supernatant was discarded and the cell pellet was resuspended using staining buffer. And detecting transduction efficiency by using a flow cytometer, wherein the percentage of the CD3 positive cells and the Myc positive cells is the transduction efficiency of the BCMA CAR transduced T cells.

Experimental results

BCMA CAR retroviral vectors successfully transduced human primary T cells. The BCMA CAR amphotropic retrovirus vector is transduced into human primary T cells, the transduction efficiency is detected after 48 hours, the transduction efficiency of bb2121 CAR-T is 55.30%, the transduction efficiency of BCMA CAR15-T is 43.18%, the transduction efficiency of BCMA CAR16-T is 55.35%, the transduction efficiency of BCMA CAR17-T is 65.87%, the transduction efficiency of BCMA CAR18-T is 62.66%, the transduction efficiency of BCMA CAR19-T is 55.42%, and the transduction efficiency of BCMA CAR20-T is 54.59%, and the research result shows that the BCMA CAR-T cells are successfully constructed and can be used for subsequent experiments to verify the killing capacity of the BCMA CAR-T cells.

Example four BCMA CAR-T in vitro anti-tumor function validation

On the basis of constructing successful BCMA CAR-T cells, the killing capacity of the BCMA CAR-T cells on tumor cells is verified. BCMA CAR-T cells were co-incubated with target cells and apoptosis of the target cells was detected using luciferase assay flow cytometry to verify the killing capacity of BCMA CAR-T cells against tumor cells, the technical route shown in fig. 6.

Detection of in vitro anti-tumor ability of BCMA CAR-T by luciferase bioluminescence method

Firefly luciferase is a monomeric protein with a size of about 61kDa, and the substrate ATP-Mg ²⁺ catalyzes the oxidation of luciferin, and chemical energy in the oxidation process is converted into electronic transitions to generate light energy, forming the product molecule oxidized luciferin. The RPMI-gfp-luc cells stably expressing the luciferase reporter gene stored in the laboratory can generate chemical signals under the catalysis of the substrate to detect the survival of tumor cells.

1. The packets are Pan-T group, BCMA CAR15-T group, BCMA CAR16-T group, BCMA CAR17-T group, BCMA CAR18-T group, BCMA CAR19-T group and BCMA CAR20-T group.

2. Plating, namely blowing RPMI-gfp-luc into single cell suspension, counting, diluting the cells into 4X 10 ⁴/50 mu L when the cell growth state is good, taking 50 mu L/hole, and inoculating the cells into 96 Kong Quanbai culture plates.

3. Pan-T, BCMA CAR15-T, BCMA CAR16-T, BCMA CAR17-T, BCMA CAR18-T, BCMA CAR19-T and BCMA CAR20-T cells were mixed with target cells at different effective target ratios (1:4, 1:2,1:1,2:1, 4:1) at 50. Mu.L/well, respectively (tumor cell group was set as blank). Culturing for 12h in a CO ₂ incubator at 37 ℃.

4. ONE-Glo ^TM luciferase detection reagent with the same volume as the culture medium is added into each hole, and the mixture is fully mixed.

5. The following formula was used for analysis, cell lysis ratio = 1- (experimental lysis-blank lysis)/(maximum release Kong Liejie-blank lysis) ×100%. Each experiment was repeated three times.

6. Data processing statistical analysis was performed using GRAPHPAD PRISM software, the metering data were expressed in (x+ -s), the comparison between the two groups was performed using t-test, and the difference was statistically significant when P < 0.05.

Flow cytometry detection of BCMA CAR-T in vitro anti-tumor capability

Annexin V is a phospholipid binding protein with high affinity to Phosphatidylserine (PS), and can specifically bind to the envelope of early apoptotic cells through PS exposed outside the cell, so Annexin V is a sensitive indicator for detecting early apoptosis.

1. The grouping is Pan-T group, BCMA CAR15-T group, BCMA CAR16-T group, BCMA CAR17-T group, BCMA CAR18-T group, BCMA CAR19-T group and BCMA CAR20-T group.

2. Plating, namely blowing K562-hBCMA-gfp into single cell suspension, counting, diluting the cells into 4X 10 ⁴/100 mu L when the cell growth state is good, taking 100 mu L/hole, and inoculating the cells into a 96-well plate.

3. Effector cells were mixed with target cells at different effect-target ratios (1:4, 1:2,1:1,2:1, 4:1) respectively (tumor cell groups were set as blank), and cultured in a 37 ℃ 5% CO ₂ incubator for 12h.

4. Cells were washed by adding 130. Mu.L of staining buffer to each well, centrifuging at 300g for 5min, and discarding the supernatant.

5. BV421 anti-human CD3 antibody is added to each well, and the wells are dyed for 40min at 4 ℃ in a dark place.

6. The staining was stopped by adding 150. Mu.L of staining buffer per well, centrifuging at 300g for 5min, and discarding the supernatant.

7. Annexin V-Alexa Fluor 647 staining was applied to each well to detect apoptosis of tumor cells and stained from light at 4℃for 40min.

8. The staining was stopped by adding 150. Mu.L of staining buffer per well, centrifuging at 300g for 5min, and discarding the supernatant.

9. Cells were resuspended with 200 μl staining per well.

10. Data were collected by flow cytometry and analyzed by FlowJo. The apoptosis rate of tumor cells was calculated as the percentage of CD3 negative and Annexin V positive cells in total cells. Experiments were repeated three times from different volunteers. Screening BCMA CAR-T with the best killing effect.

Flow cytometry further verifies BCMA CAR-T in vitro anti-tumor capability

1. Grouping was Pan-T group, positive control group (bb 2121 CAR-T group), BCMA CAR16-T group.

2. Plating, namely blowing RPMI-gfp-luc into single cell suspension, counting, diluting the cells into 4X 10 ⁴/100 mu L when the cell growth state is good, taking 100 mu L/hole, and inoculating the cells into a 96-well plate.

3. Pan-T, bb2121 CAR-T and BCMA CAR16-T cells were mixed with target cells at different effect target ratios (1:4, 1:2,1:1, 2:1) respectively (tumor cell group was set as blank), and cultured in a 37℃and 5% CO ₂ incubator for 12h.

4. The flow detection method and the data processing are the same as the steps.

Fourth, experimental results

1. Tumor cell surface BCMA expression detection results

The tumor cells K562-hBCMA-gfp, RPMI-gfp-luc, K562-cBCMA and K562 were stained with BV421anti-human BCMA antibody, and the results of the flow assay showed that the cell surface BCMA of K562-hBCMA-gfp and RPMI-gfp-luc were highly expressed to different extents, wherein the cell surface BCMA of K562-hBCMA-gfp was 85.5% and the cell surface BCMA of RPMI-gfp-luc was 58.5%, which served as target cells for the study, and the cell surfaces of K562-cBCMA and K562 were not substantially expressing BCMA, which served as negative control cells.

2. BCMA CAR-T cell in vitro tumor killing activity detected based on luciferase bioluminescence method

To verify the killing ability of BCMA CAR-T to tumor cells in vitro, we incubated BCMA CAR15-T, BCMA CAR16-T, BCMA CAR17-T, BCMA CAR18-T, BCMA CAR19-T and BCMA CAR20-T cells with target cells at different potency target ratios, respectively. The apoptosis rate of target cells is detected by a luciferase bioluminescence method after 12 hours, and the result shows that compared with a Pan-T group, the killing capacity of a BCMA CAR16-T group and a BCMA CAR17-T group on tumor cells is enhanced, the BCMA CAR16-T group is positively correlated with the effective target ratio, the BCMA CAR16-T shows the strongest killing effect on the tumor cells, and three independent repeated experiments show the same result.

3. BCMA CAR-T cell in vitro tumor killing activity based on flow cytometry detection

To re-verify the killing ability of BCMA CAR-T in vitro on tumor cells, we incubated BCMA CAR15-T, BCMA CAR16-T, BCMA CAR17-T, BCMA CAR18-T, BCMA CAR19-T and BCMA CAR20-T cells with target cells K562-hbma-gfp at different potency target ratios, respectively. The result of the flow detection of the apoptosis rate of the target cells after 12 hours is shown in the figure, compared with the Pan-T group, the killing capacity of the BCMA CAR16-T group and the BCMA CAR17-T group on the tumor cells is enhanced, the killing capacity is positively correlated with the effective target ratio, the BCMA CAR16-T shows the strongest killing effect on the tumor cells, three independent repeated experiments show the same result, and the detection result is consistent with that of a firefly bioluminescence method, as shown in figure 7. BCMA CAR16-T with the best killing effect was selected and compared with positive BCMA CAR (bb 2121 CAR-T).

The result is further verified by BCMA CAR-T in vitro anti-tumor capability

Screening BCMA CAR16-T with most obvious killing effect on tumor cells and positive bb2121 CAR-T cells further compares the killing effect, and the result is shown in the following figure 8, wherein the killing effect of BCMA CAR16-T on the tumor cells is obvious compared with that of Pan-T group, but the effect is not as obvious as that of positive control group bb2121 CAR0-T, and the difference has statistical significance. On the basis of the screened BCMA CAR16, further optimization is needed, and the killing efficiency on tumor cells is improved.

Example five optimization of construction of BCMA CAR retroviral vector plasmids and BCMA CAR retroviral vector packaging

Construction of optimized BCMA CAR retroviral vector plasmid

The BCMA CAR16-T cells with the strongest killing ability to tumor cells are successfully screened. But the killing capacity against tumor cells was to be enhanced compared to the bb2121 CAR-T cells already on the market. Because BCMA CAR16-T and bb2121 CAR-T are identical in structure except ScFv, we designed that ScFv of two or three BCMA CAR16-T are connected in series to increase antigen affinity, so as to enhance antitumor ability.

Schematic of BCMA CAR31, BCMA CAR32, BCMA CAR33 Structure

The ScFv region of the BCMA CAR adopts a humanized monoclonal antibody sequence BCMA CAR16 of the target BCMA screened by the prior laboratory phage display, the three optimized ScFv regions of the BCMA CAR structure are respectively connected by two SvFv, the three ScFv regions are connected, and the rest structure is unchanged. The map of the newly constructed BCMA CAR expression plasmid is shown in FIG. 9

2. Gel electrophoresis results of fragments of interest and vector fragments

After double digestion of XhoI and NgoMIV, the vector pMFG fragment is about 7kb, the target fragment BCMA CAR31 fragment is about 1.7kb, the BCMA CAR32 fragment is about 1.7kb, and the BCMA CAR33 fragment is about 2.5kb, and the electrophoresis result shows that the vector fragment and the target fragment have correct band sizes.

Enzyme digestion identification gel electrophoresis result of pMFG-BCMA CAR

The constructed pMFG-BCMA CAR31, pMFG-BCMA CAR32 and pMFG-BCMA CAR33 are subjected to plasmid extraction and then identified by double digestion (XhoI/NgoMIV), the target fragment BCMA CAR31 fragment is about 1.7kb, the BCMA CAR32 fragment is about 1.7kb, the BCMA CAR33 fragment is about 2.5kb, the vector pMFG fragment is about 7kb, the electrophoresis result is shown in figure 10, the plasmid digestion identification ①②③④⑤ of the pMFG-BCMA CAR31 is correct, the plasmid digestion identification ①④⑤ of the pMFG-BCMA CAR32 is correct, and the plasmid digestion identification ②③⑤ of the pMFG-BCMA CAR33 is correct. The plasmids identified as correct were sent to sequencing for further identification. The plasmid with correct enzyme cutting identification is purified and then is sent to sequencing. Sequencing results showed that pMFG-BCMA CAR31, pMFG-BCMA CAR32, pMFG-BCMA CAR33 sequences were perfectly correct.

Optimizing BCMA CAR retroviral vector packaging

And (3) packaging the BCMA CAR retrovirus vector on the basis of constructing the optimized pMFG-BCMA CAR plasmid, and constructing a cell line for stably producing the retrovirus vector. The specific experimental procedure is shown in FIG. 4, wherein the optimized pMFG-BCMA CAR plasmid is firstly transiently transfected into Phoenix ECO cells, and BCMA CAR avirulent retroviral vector supernatant particles are collected. The collected BCMA CAR amphotropic retroviral vector supernatant particles were transduced into PG13 cell lines for production of amphotropic retroviral vector supernatant particles, and PG13 cell lines were constructed that stably produced BCMA CAR amphotropic retroviral vector supernatant particles, and viral vector titers were detected using qPCR to verify whether BCMA CAR amphotropic retroviral vector supernatant particles were successfully produced. The following results were obtained using a similar method as before.

1. Successful preparation of BCMA CAR avirulent retroviral vectors

The Phoenix-ECO cells were transfected with plasmids pMFG-BCMA CAR31, pMFG-BCMA CAR32, pMFG-BCMA CAR33, and the transfection efficiencies were examined by 72h post-flow cytometry, namely, pMFG-BCMA CAR31 was 59%, pMFG-BCMA CAR32 was 58.07%, pMFG-BCMA CAR33 was 60.62%, and all three plasmids were successfully transfected in Phoenix-ECO cells.

2. Successful construction of cell lines stably producing BCMA CAR amphotropic retroviral vectors

Successfully prepared BCMA CAR31, BCMA CAR32, BCMA CAR33, the amphotropic retroviral vector supernatant was transduced into PG13 cells and the transduction efficiency was examined by flow cytometry after 48 h. The results were 93.66% BCMA CAR31 transduction efficiency, 95.35% BCMA CAR32 transduction efficiency, 98.24% BCMA CAR33 transduction efficiency, indicating successful preparation of amphotropic retroviral vector particles and successful construction of PG13 cell lines that stably produced amphotropic retroviral vector particles.

3. Successful preparation of BCMA CAR amphotropic retroviral vectors

The PG13 cell lines transduced by BCMA CAR31, BCMA CAR32 and BCMA CAR33 are expanded and cultured in a T75 culture flask, and the amphotropic retroviral vector particles are continuously harvested for 5 days for qPCR detection, so that the titer of the BCMA CAR31 retroviral vector is 2.29 multiplied by 10 ⁷±1.06×10⁶ copies/mL, the titer of the BCMA CAR 32H 3 is 1.64 multiplied by 10 ⁷±1.12×10⁶ copies/mL, and the titer of the BCMA CAR 33H 4 is 2.40 multiplied by 10 ⁷±4.53×10⁶ copies/mL, which are all higher than that of a positive control. This result shows that we successfully prepared BCMA CAR31, BCMA CAR32, BCMA CAR33 amphotropic retroviral vectors, and selected the highest titer amphotropic retroviral vector particles for human primary T cell transduction.

EXAMPLE six preparation of optimized BCMA CAR-T cells

Upon successful preparation of BCMA CAR amphotropic retroviral vector particles, the BCMA CAR amphotropic retroviral vector particles are transduced into human primary T cells to construct BCMA CAR-T cells. The technical route is shown in figure 5, wherein human peripheral blood PBMC is firstly separated by a density gradient centrifugation method, primary T cells are activated under the stimulation of a CD3 monoclonal antibody and a cytokine IL-2, BCMA CAR is transduced by the activated T cells in a centrifugal way, BCMA CAR-T cells are constructed, the expression of a cell surface Myc label is detected by using a flow cytometry method, so that whether the BCMA CAR is expressed on the surface of the T cells is detected, and further, the copy number of a BCMA CAR retroviral vector in each human primary T cell genome is detected by a qPCR method, and whether the BCMA CAR retroviral vector is successfully integrated into the human primary T cell genome is detected. The following results were obtained using a similar method as before.

Successful transduction of human primary T cells by BCMA CAR retroviral vectors

The BCMA CAR amphotropic retroviral vector is transduced into human primary T cells, the transduction efficiency is detected after 48 hours, the result is shown in figure 11, the BCMA CAR16-T transduction efficiency is 61.10 +/-3.65%, the BCMA CAR31-T transduction efficiency is 61.97 +/-2.98%, the BCMA CAR32-T transduction efficiency is 62.20 +/-1.59%, the BCMA CAR33-T transduction efficiency is 57.80+/-3.02%, the result shows that the transduced human primary T cells successfully express the BCMA CAR, and the transduction efficiency among the three groups is not statistically different from that of the BCMA CAR16-T of the control group, so that the BCMA CAR-T cells are successfully prepared.

BCMA CARs successfully integrate into the human primary T cell genome. The BCMA CAR amphotropic retroviral vector transduces human primary T cells, and the copy number of the BCMA CAR16-T integrated into the T cell genome is detected after 48 hours, and the result shows that the copy number of the BCMA CAR16-T integrated into the genome is 0.44+/-0.05 copies/T cell, the copy number of the BCMA CAR31-T integrated into the genome is 0.48+/-0.02 copies/cell, the copy number of the BCMA CAR32-T integrated into the genome is 0.43+/-0.04 copies/T cell, and the result shows that the BCMA CAR33-T integrated into the genome is 0.32+/-0.04 copies/T cell, and the BCMA CAR is successfully integrated into the human primary T cell genome.

Example seven in vitro anti-tumor function validation of optimized BCMA CAR-T

On the basis of successfully constructing 3 optimized BCMA CAR-T cells, the killing function of the BCMA CAR-T cells on tumor cells in vitro is verified. The method comprises the steps of using different tumor cells expressing human BCMA antigens as target cells, using target cells not expressing the human BCMA antigens as a control, monitoring the survival of the tumor cells through a luciferase bioluminescence experiment and an incucyte real-time dynamic living cell imaging, detecting apoptosis of the tumor cells by utilizing a flow cytometry to verify the killing specificity and effectiveness of BCMA CAR-T cells on the target cells, detecting the secretion cytokine level of the BCMA CAR-T cells through a CBA experiment, and detecting the proliferation capability of the BCMA CAR-T through a CFSE experiment. The specific experimental protocol is shown in fig. 12. The following experimental results were obtained using a similar method as before.

BCMA CAR31-T has significant killing ability in vitro

The BCMA CAR16-T, the BCMA CAR31-T, the BCMA CAR32-T and the BCMA CAR33-T are respectively incubated with K562-hBCMA-gfp cells according to the effective target ratio of 1:1, the fluorescence change of the tumor cells is continuously monitored by real-time fluorescence, the tumor cells are recorded every 2 hours, the continuous monitoring is carried out for 48 hours, and the result is shown in figure 13 that the killing effect of the BCMA CAR31-T on the tumor cells is stronger than that of the BCMA CAR16-T of a control group. Thus, in the next experiments we chose BCMA CAR31-T to continue tumor killing, cytokine secretion and proliferation capacity comparison with BCMA CAR16-T.

BCMA CAR31-T can be activated effectively

The flow cytometry detects BCMA CAR-T cell surface CD69 expression, and the result is shown in figure 14, the BCMA CAR31-T has no statistical difference compared with BCMA CAR16-T in no tumor cell stimulation, the BCMA CAR31-T has higher expression than BCMA CAR16-T cell surface CD69 under the stimulation of K562-hBCMA-gfp cell expressing BCMA, the difference has statistical significance, and the research result shows that the optimized BCMA CAR31-T can be activated by tumor cells better.

BCMA CAR31-T has high-efficiency in vitro anti-tumor capability

To determine the lytic ability of BCMA CAR31-T cells to BCMA positive tumor cells, BCMA CAR31-T cells, BCMA CAR16-T cells, or Pan-T were incubated with RPMI-gfp-luc or cells at different potency target ratios for 12h for chemiluminescent signal intensity detection, experimental results are shown in fig. 15, where BCMA CAR31-T showed a greater ability to kill tumor cells in vitro at each potency target ratio than BCMA CAR16-T, and the differences were statistically significant.

BCMA CAR31-T has high and specific in vitro antitumor ability. To verify the antigen specificity of the antitumor effect of BCMA CAR31-T cells, BCMA CAR31-T cells were co-incubated with K562-hbma-gfp, PMI-gfp-luc cells expressing human BCMA antigen at different potency target ratios. K562-cBCMA expressing cynomolgus BCMA and K562 cells not expressing BCMA served as negative target cell controls. BCMA CAR16-T control. The sample is detected by flow cytometry. The results are shown in figure 16, where BCMA CAR31-T cells had greater killing power against both different BCMA positive tumor cells at different potency target ratios than BCMA CAR16-T group. However, for non-human BCMA expressing K562-cBCMA and BCMA negative K562 cells, BCMA CAR31-T exhibited an indistinguishable killing capacity from Pan-T, indicating BCMA antigen specificity of BCMA CAR31-T cells for tumor cells.

BCMA CAR31-T has a potent cytokine secretion capacity

Cytokine production is a marker for efficient activation of CAR-T cells. BCMA CAR31-T cells were co-incubated with K562-hbma-gfp cells expressing human BCMA antigen at a 1:1 potency target ratio for 12h. Cell culture broth is harvested and cytokines such as TNF- α, IFN- γ, IL-6, IL-17A, aFasL are measured using CBA kit. The results are shown in FIG. 17, where the cytokines TNF- α, IFN- γ, IL-6, IL-17A, aFasL secreted by BCMA CAR31-T cells were significantly increased compared to BCMA CAR16-T cells upon stimulation by BCMA positive tumor cells. Release of these cytokines suggests that BCMA CAR31-T cells can be effectively activated, further confirming that BCMA CAR31-T cells have more potent antitumor activity.

BCMA CAR31-T has a strong proliferation capacity in vitro. To assess the proliferative capacity of BCMA CAR31-T cells in vitro, we used CFSE-based detection methods to measure proliferation, pan-T combined BCMA CAR16-T group as control. The results are shown in FIG. 18, where the CFSE green fluorescent signal of BCMA CAR16-T cells was significantly reduced compared to BCMA CAR31-T group cells after 72h of cell culture, indicating a faster proliferation rate of the BCMA CAR31-T cells.

Example eight optimization of anti-tumor function validation in BCMA CAR-T in vivo

The BCMA CAR31-T cells are found to have high efficiency and specificity on killing tumor cells, and can have strong proliferation capacity and cytokine secretion capacity in vitro. The in vivo anti-tumor capacity of BCMA CAR31-T cells was verified by further utilizing xenograft tumor models. The experimental protocol is shown in figure 19.

Mouse xenogeneic tumor transplantation model construction success

NPG mice were injected 2X 10 ⁶ RPMI-gfp-luc cells intravenously and subjected to in vivo imaging 10 days later, and the results are shown in FIG. 20, in which tumor signals were seen in all mice, and the tumor formation was uniform and indicated that the mouse xenograft model was successfully constructed. The mice were randomly divided into four groups with no statistical difference in tumor signal between BCMA CAR31-T group and BCMA CAR16-T group.

BCMA CAR-T cell in vivo effective anti-tumor

From the sixth day after the first injection of BCMA CAR-T, live imaging of mice is carried out once a week, and the change of the tumor signal intensity of the mice is detected, and the result is shown in figure 21, wherein after the first injection of BCMA CAR31-T, the tumor signal of the mice is not statistically different from that of BCMA CAR16-T, the imaging of the mice after the second injection shows that the tumor signal of the BCMA CAR31-T group is obviously weakened compared with that of BCMA CAR16-T, the difference has statistical significance, and the result shows that the BCMA CAR31-T has effective in vivo anti-tumor capability.

Increased secretion of IFN-gamma by BCMA CAR-T

The peripheral blood of the mice is collected 48h after two BCMA CAR-T cell injections, serum is separated, and the human IFN-gamma secretion level in the serum is detected by ELISA, wherein the result shows that the IFN-gamma secretion level of BCMA CAR31-T after the first BCMA CAR-T injection is 180.29 +/-31.63 pg/mL, the BCMA CAR16-T (157.74 +/-45.51 pg/mL) is not different, and the result is consistent with the in-vivo imaging result of the mice, and no obvious anti-tumor effect exists. After the second BCMA CAR-T injection, compared with BCMA CAR16-T (1254.03 +/-1523.75 pg/mL), the BCMA CAR31-T secretion IFN-gamma level is obviously increased (3778.37 +/-934.68 pg/mL), and the result is consistent with the living imaging of the mice, and has obvious anti-tumor effect. The results of this study showed that BCMA CAR31-T was effectively activated after the second CAR-T injection.

BCMA CAR31-T has durable anti-tumor effect in vivo

The method comprises the steps of taking blood on 24 days, 31 days and 37 days after tumor cell inoculation, detecting CD3 ⁺ T cells injected into peripheral blood of the mice, wherein the result is shown as follows, the content of CD3 ⁺ T cells in peripheral blood of each 100 mu L of mice is in a model group (-1474.00 +/-3670.66) or a Pan-T group (-288.33 +/-8110.37) on 24 days, 31 days and 37 days after tumor cell inoculation, the content of BCMA CAR31-T groups (29850.00 +/-82348) is remarkably higher than that of BCMA CAR16-T groups in peripheral blood of the mice, the difference has statistical significance, the content of CD3 ⁺ T cells in peripheral blood of each 100 mu L of mice on 31 days after tumor cell inoculation is in a model group (-1474.00 +/-3670.66) or a Pan-T group (-288.33 +/-8110.37), the content of BCMA CAR16-T groups (8000.00) is remarkably higher than that of BCMA CAR31-T groups (39343+/-82348), and the content of BCMA CAR31-T groups (8000.00) is remarkably higher than that of BCMA CAR31-T groups (8000.00) in peripheral blood of the mice on 8000.00 days after tumor cell inoculation, and the content of BCMA CAR31-T groups (8000.00) is remarkably higher than that of BCMA CAR31-T groups (8000.00) is in peripheral blood of the group 8000.00, and the difference has statistical significance. The study results show that the BCMA CAR31-T group survived longer in vivo than the BCMA CAR16-T group, showing a longer lasting antitumor ability.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.

Claims (14)

1. A chimeric antigen receptor targeting BCMA is characterized in that the receptor comprises a ScFv structure of human-derived target BCMA, a heavy chain variable region ScFv-VH and a light chain variable region ScFv-VL of the ScFv are connected through one or more G4S sequences, the amino acid sequence of the heavy chain variable region ScFv-VH is SEQ ID NO.8：SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSS; and the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.9：QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG.

2. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the ScFv structure of the receptor comprising human-derived BCMA-targeted ScFv is ScFv-VH- (G4S) n-ScFv-VL, wherein n is an integer of 1 or more.

3. The BCMA-targeted chimeric antigen receptor according to claim 2, wherein n is 3 or 4.

4. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the receptor comprises human BCMA-targeted ScFv structure ScFv-VH- (G4S) 3-ScFv-VL, heavy chain variable region ScFv-VH has the amino acid sequence of SEQ ID No.8, and light chain variable region ScFv-VL has the amino acid sequence of SEQ ID No.9.

5. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the receptor comprises a human BCMA-targeted ScFv structure of ScFv-VH- (G4S) n-ScFv-VL- (G4S) n-ScFv-VH, wherein n is an integer of 1 or more.

6. The BCMA-targeted chimeric antigen receptor according to claim 5, wherein n is 3 or 4.

7. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the receptor comprises human BCMA-targeted ScFv structure ScFv-VH- (G4S) 3-ScFv-VL- (G4S) 4-ScFv-VL- (G4S) 3-ScFv-VH, heavy chain variable region ScFv-VH has the amino acid sequence of SEQ ID No.8, and light chain variable region ScFv-VL has the amino acid sequence of SEQ ID No.9.

8. The chimeric antigen receptor according to any one of claims 1-7, wherein the receptor comprises an upstream signal peptide and a myc tag for detection in tandem, a ScFv structure of human-derived BCMA comprising a heavy chain variable region and a light chain variable region, a CD8 hinge-transmembrane domain, a CD28 or 4-1BB co-activation domain and a CD3 zeta intracellular signaling domain.

9. BCMA-targeted chimeric antigen receptor T cell characterized in that it expresses the chimeric antigen receptor according to any one of claims 1 to 8.

10. A medicament for the treatment of tumors, characterized in that it contains chimeric antigen receptor T cells according to claim 9, said tumors being surface BCMA positive tumors.

11. Use of a chimeric antigen receptor according to any one of claims 1-8 for the preparation of chimeric antigen receptor T cells.

12. Use of a chimeric antigen receptor according to any one of claims 1-8 for the preparation of a medicament for the treatment of a tumor, said tumor being a surface BCMA positive tumor.

13. The use of claim 12, wherein the tumor is multiple myeloma.

14. The use of a chimeric antigen receptor according to any one of claims 1-8, wherein the chimeric antigen receptor T cell is prepared by inserting a gene fragment encoding the chimeric antigen receptor into a viral expression vector, packaging into viral vector particles, and infecting human T cells.