CN116535521A - Chimeric antigen receptor of targeted BCMA and application thereof - Google Patents

Chimeric antigen receptor of targeted BCMA and application thereof Download PDF

Info

Publication number
CN116535521A
CN116535521A CN202310214870.8A CN202310214870A CN116535521A CN 116535521 A CN116535521 A CN 116535521A CN 202310214870 A CN202310214870 A CN 202310214870A CN 116535521 A CN116535521 A CN 116535521A
Authority
CN
China
Prior art keywords
bcma
scfv
cells
car
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310214870.8A
Other languages
Chinese (zh)
Inventor
王建勋
李晓瑞
冯娅茹
尚凤琴
余卓营
宋志茹
童建松
刘颖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Cell Valley Biopharmaceutical Co ltd
Original Assignee
Shenzhen Cell Valley Biopharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Cell Valley Biopharmaceutical Co ltd filed Critical Shenzhen Cell Valley Biopharmaceutical Co ltd
Priority to CN202310214870.8A priority Critical patent/CN116535521A/en
Publication of CN116535521A publication Critical patent/CN116535521A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001117—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR] or CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

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 FDA approved for use in the treatment of hematological malignancies, with BCMA-targeted abegma marketed in batch today as the first CAR-T cell product for use in 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 BCMA-targeted chimeric antigen receptor comprising a ScFv structure of human BCMA-targeted, wherein a heavy chain variable region ScFv-VH and a light chain variable region ScFv-VL of ScFv are connected by one or more G4S sequences; 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 SEQ ID No.8 below: 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 below: 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 a human-derived BCMA-targeted ScFv structure of ScFv-VH- (G4S) 3-ScFv-VL, and the amino acid sequence of the heavy chain variable region ScFv-VH is SEQ ID NO.8; the amino acid sequence of the light chain variable region ScFv-VL is 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 structure of ScFv-VH- (G4S) 3-ScFv-VL- (G4S) 4-ScFv-VL- (G4S) 3-ScFv-VH, and the heavy chain variable region ScFv-VH has the amino acid sequence of SEQ ID NO.8; the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.9.
In one embodiment, the receptor comprises a signal peptide and myc tag for detection in tandem upstream in sequence; a ScFv structure of human-derived BCMA comprising a heavy chain variable region and a light chain variable region; CD8 hinge-transmembrane domain; CD28 or 4-1BB synergistic activation domain and cd3ζ 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 invention, the plasmid transient transfection Phoenix-ECO cells of pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 which are successfully constructed by us have the transfection efficiency higher than 50%, the PG13 cell line is transduced by using the transiently transfected retroviral vector particles, the transduction efficiency is higher than 95%, and pPCR detection shows that the retroviral vector particles have higher effect Viral titers, highest titers were all higher than 1X 10 7 copy/mL, indicating successful preparation of PG13 cell lines stably expressing BCMA CAR retroviral vectors. 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 the results showed 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 no differential killing ability from Pan-T for non-human BCMA-expressed 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 fasl under stimulation of 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 second tail vein injection of BCMA CAR31-T, secretion of IFN- γ cytokines was significantly enhanced over BCMA CAR16-T, indicating better activation of BCMA CAR31-T cells; the mice are generally good in condition, and symptoms of cytokine release syndromes such as fever, nausea, vomiting and the like are not generated. During continuous observation of mouse tumor changes, BCMA CAR31-T group tumor signals were significantly attenuated compared to BCMA CAR16-T, showing more potent anti-tumor ability; 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 the 37 th day after tumor inoculation, which indicates 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) (compared to bb2121CAR-T cells, * p < 0.05) resultsA figure;
fig. 9 is a schematic structural diagram of an optimized BCMA CAR;
FIG. 10 shows gel electrophoresis patterns of enzyme digestion assay, wherein A: the fragment was identified by cleavage of pMFG-BCMA CAR31 and pMFG-BCMA CAR 32; b: identifying fragments by enzyme digestion 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 CAR-T cell CD69 expression (n=3) results, 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+ tumor cell stimulated BCMA CAR-T cell surface CD69 expression histogram (P < 0.05, ns: no statistical difference compared to BCMA CAR16-T cells);
Fig. 15 is a graph of the in vitro killing ability (n=3) of BCMA CAR-T cells measured by luciferase bioluminescence (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 mouse xenograft model, a: a live imaging picture of the mouse; b: statistical graphs of mean signal intensity of mouse tumors (ns: no statistical difference);
fig. 21 is a graph of results of in vivo imaging (n=6) of a mouse xenograft model, a: a live imaging picture of the mouse; b: mouse tumor mean signal intensity statistic plot (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 invention 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 one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present 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, in order to verify whether ScFv of these 6 BCMA-targeted BCMA can effectively and specifically recognize BCMA-positive tumor cells, we designed and constructed a secondary humanized BCMA CAR structure using CD8 as a transmembrane domain, CD28 and CD3 zeta as intracellular stimulatory domains, see fig. 2; BCMA CAR-T cells were prepared by retroviral vectors. 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);
myc amino acid sequence: EQKLISEEDL (SEQ ID NO. 2);
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:
SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGG GGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG (SEQ ID NO. 20), wherein the sequence is underlined by 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
pUC57-BCMA CAR15, pUC57-BCMA CAR16, pUC57-BCMA CAR17, pUC57-BCMA CAR18, pUC57-BCMA CAR19, pUC57-BCMA CAR20, pMFG-CD8-CD28-CD3 zeta and vector plasmid pMFG strain stored at-80℃were taken out, 50. Mu.L of each was added to 25mL of LB liquid medium (Amp final concentration 50. Mu.g/mL) and shaking was carried out in a shaking table at 37℃at 200rpm for 12-16h to 3 to 4X 10 9 Cell density per mL. 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 and pUC57-BCMA CAR20 plasmids obtained above were subjected to amplification of the target bands by PCR method, and each target fragment was 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.
Purified CAR15-ScFv-CD8-CD28-CD3 zeta, CAR16-ScFv-CD8-CD28-CD3 zeta, CAR17-ScFv-CD8-CD28-CD3 zeta, CAR18-ScFv-CD8-CD28-CD3 zeta, CAR19-ScFv-CD8-CD28-CD3 zeta, CAR20-ScFv-CD8-CD28-CD3 zeta were double digested with XhoI and NotI restriction enzymes and pMFG vector fragments were electrophoresed and recovered.
The target fragment and the carrier fragment are connected, and the XhoI and NotI double enzyme-cut target fragments of CAR15-ScFv-CD8-CD28-CD3 zeta, CAR16-ScFv-CD8-CD28-CD3 zeta, CAR17-ScFv-CD8-CD28-CD3 zeta, CAR18-ScFv-CD8-CD28-CD3 zeta, CAR19-ScFv-CD8-CD28-CD3 zeta, CAR20-ScFv-CD8-CD28-CD3 zeta and the enzyme-cut section of the pMFG carrier are connected, wherein the molar ratio of the target fragment and 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.
Amplifying the target fragment by using a PCR method to obtain the target fragment amplification gel electrophoresis result, amplifying the pUC-BCMA CAR15, pUC-BCMA CAR16, pUC-BCMA CAR17, pUC-BCMA CAR18, pUC-BCMA CAR19 and pUC-BCMA CAR20 plasmids containing the ScFv fragments, wherein the sizes of the plasmids are about 800bp; the plasmid containing CD8-CD28-CD3 zeta fragment is amplified by PCR, the fragment size is about 830bp, and the gel electrophoresis result shows that the size of the target band is 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 plasmid is subjected to XhoI and NotI double digestion identification, the fragment sizes are 7kb and 1.6kb, and the gel electrophoresis result shows that: the constructed plasmid bands of pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 are all 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: first, pMFG-BCMA CAR plasmid was 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)
The human eosinophil packaging cell line Pheonix-ECO, gibbon ape leukemia virus packaging cell line PG13 was purchased from American type culture Collection (American type culture collection, ATCC).
DMEM complete medium: operating in biosafety cabinet, 56mL FBS (10%) and 5.6mL of neomycin diabody (1%) were added per 500mL DMEM (89%); the prepared complete DMEM medium was vacuum filtered using a 500mL filter of 0.22 μm; the bottle body is marked with reagent and put in a refrigerator at 4 ℃ for standby.
Cell cryopreservation solution: FBS, DMSO were pressed in the biosafety cabinet as FBS: DMSO = 9:1 ratio; the prepared cell frozen stock solution is filtered by a 0.22 mu m filter membrane for standby.
Staining buffer: PBS (1×) and FBS were combined as PBS: fbs=9: 1, and placing the mixture at 4 ℃ for standby.
Second, experimental method
Phoenix ECO and PG13 cell culture
1.1. Cell resuscitation
Taking out the cells to be resuscitated from the liquid nitrogen tank, resuspending the cell pellet with DMEM complete medium, transferring the cell suspension to proper culture bottle, adding 37 deg.C and 5% CO 2 Culturing in an incubator.
1.2 passage of cells and liquid exchange
Observing the cell adhesion and growth state under the microscope, if the cell density is less than 80%, but the color of the culture medium turns yellow, the process is neededAnd (5) performing cell liquid exchange treatment. If the cell density is more than 80%, 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. Transferring the cell suspension to a suitable culture flask, placing in 37℃and 5% CO 2 Culturing in an incubator. The cell growth state was observed under an inverted microscope every 24 hours.
1.3 cryopreservation of cells
The cells were digested by adding an appropriate volume of trypsin, digested at 37℃for about 5min, and the digested condition was observed under a mirror after taking out 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 by a pipette and discarded, taking care not to contact the cell pellet, and the cells were resuspended in cell cryopreservation solution (typically 5X 10 cells cryopreservation concentration 6 -1×10 7 /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 and incubated at 37℃for about 5min to accelerate digestion of cells. 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.
According to 1X 10 6 Single cell suspensions required for seeding into six well cell culture plates were calculated and formulated per well, 2.5 mL/well. Will be diluted to 1X 10 6 Single cell suspensions 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 5% CO2 incubator at 37℃and incubated overnight.
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℃incubator containing 5% CO2 for cultivation.
2.3 harvesting of the supernatant of the aviphilic retroviral vector
After 24h transfection, six-well plates were removed from the incubator, the culture supernatant carefully removed, and fresh DMEM complete medium, 2.5 mL/well, was slowly added; six-well plates were moved to 32℃with 5% CO 2 Culturing in an incubator. After 48h of transfection, the supernatant was carefully collected into a centrifuge tube; slowly adding new DMEM complete medium, 2.5 mL/hole; moving the six-hole culture plate to 32 ℃ and culturing in a 5% CO2 incubator; filtering the collected culture supernatant with a low adsorption virus filter membrane of 0.45 μm, and packaging and storing at-80deg.C for use. After 72 hours of transfection, taking out the six-hole culture plate and putting the six-hole culture plate into a biological safety cabinet; carefully collecting the supernatant into a centrifuge tube; filtering the collected culture supernatant with a low adsorption virus filter membrane of 0.45 μm, and packaging and storing at-80deg.C for use.
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, 900L PBS was added to wash the cells. 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. Grouping: PG13 cell control group, BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19, and BCMA CAR20 experimental group. The supernatant of the avidity retroviral vector collected after 72h transfection with Phoenix-ECO was removed from-80℃and re-thawed at room temperature, and 1mL was added to each well of a 12-well plate. The culture plates were centrifuged at 30℃for 1 hour in 12-well cells to which the supernatant of the philic retrovirus vector was added. PG13 cells were removed from the 37℃incubator, the cell state was observed under a microscope, trypsinization was terminated, and a pipette was blown into a single cell suspension. Cell count, 1X 10 cells per well 6 Individual cells were transferred to a centrifuge tube and centrifuged 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 harvested and designated as H0, and fresh DMEM complete medium was added and the plates were transferred to 32℃with 5% CO 2 Culturing in incubator, and extracting the virus vector supernatant 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 further 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 detection of BCMA CAR retroviral vector titres, detection of BCMA CAR amphotropic retroviruses produced by the above-collected PG13 cell line Viral titer of the viral vector supernatant 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, pMFG-BCMA CAR15 transfection efficiency was 56.70, pMFG-BCMA CAR16 transfection efficiency was 64.91, pMFG-BCMA CAR17 transfection efficiency was 59.11, pMFG-BCMA CAR18 transfection efficiency was 57.88, pMFG-BCMA CAR19 transfection efficiency was 59.85, pMFG-BCMA CAR20 transfection efficiency was 57.95%, 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 that amphotropic retroviral vector particles were successfully prepared and PG13 cell lines stably producing amphotropic retroviral vector particles were successfully constructed.
3. Successful preparation of BCMA CAR amphotropic retroviral vectors
Expanding and culturing the PG13 cell line transduced by BCMA CAR amphotropic retroviral vector into a T75 culture flask, and continuously harvesting amphotropic retroviral vector particles for 5 days for qPCR detection, wherein the result is that: BCMA CAR amphotropic retroviral vector titres up to BCMA CAR 15H 2:1.40×10 7 ±1.13×10 6 copy/mL, BCMA CAR 16H 1: 1.33X10 7 ±7.07×10 4 Copying/mL，BCMA CAR17 H2：3.09×10 7 ±1.70×10 6 copy/mL, BCMA CAR 18H 2:2.44×10 7 ±2.55×10 6 copy/mL, BCMA CAR 19H 2:1.95×10 7 ±2.36×10 4 copy/mL, BCMA CAR 20H 2: 2.68X10 7 ±4.24×10 6 copy/mL, all higher than 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 fig. 5: first, human peripheral blood PBMC is 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 by a centrifugation mode, BCMA CAR-T cells are constructed, and the expression of a cell surface Myc label is detected by a flow cytometry method, 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.
Grouping: t cell non-transduced control (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 (currently marketed BCMA CAR, except for ScFv, the rest of the structure is the same as that 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. The amphotropic retroviral vector supernatant collected from the PG13 cell line was removed from-80℃and reconstituted at room temperature1mL of the medium was added to each well of the 12-well plate. The culture plates were centrifuged at 30℃for 1 hour in 12-well cells to which the amphotropic retrovirus vector supernatant was added. T cells were taken out from the 37℃incubator, the cell state was observed under a microscope, and a pipette was blown into a single cell suspension. Cell count, 1X 10 cells per well 6 The cell suspensions were transferred to a centrifuge tube 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 in which the cells were resuspended. Placing 12-well culture plate at 37deg.C, 5% CO 2 Culturing in an incubator 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. Placing 12-well culture plate at 37deg.C, 5% CO 2 Culturing in an incubator.
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 absence of light, 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%, so that 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 of about 61kDa in size and is the substrate ATP-Mg 2+ Can catalyze the oxidation of fluorescein, and chemical energy in the oxidation process is converted into electron transition to generate light energy, so that the product molecule oxidized fluorescein is formed. 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. Grouping: 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. And (3) paving: blowing RPMI-gfp-luc into single cell suspension and counting; when the cell growth state is good, the cells are diluted to 4×10 4 50. Mu.L/well was inoculated into 96 Kong Quanbai 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). 37 ℃,5% CO 2 Culturing in an incubator for 12h.
4. Each of whichAdding ONE-Glo with the same volume as the culture medium into the hole TM The luciferase detection reagent was thoroughly mixed.
5. Analysis was performed using the following formula: cell lysis ratio = 1- (experimental lysis-placebo lysis)/(maximum release Kong Liejie-placebo lysis) ×100%. Each experiment was repeated three times.
6. And (3) data processing: statistical analysis was performed using GraphPad Prism 8 software, the metering data were expressed as (x±s), the comparison between the two groups was performed using a 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. Grouping: 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. And (3) paving: blowing K562-hBCMA-gfp into single cell suspension and counting; when the cell growth state is good, the cells are diluted to 4×10 4 100. Mu.L/well was plated in 96-well plates.
3. Effector cells were mixed with target cells (tumor cell group was set as blank) at different potency target ratios (1:4, 1:2,1:1,2:1, 4:1), 37 ℃,5% CO 2 Culturing in an 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 hole, and the mixture is 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 at 4℃for 40min in the dark.
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: pan-T group, positive control group (bb 2121 CAR-T group), BCMA CAR16-T group.
2. And (3) paving: blowing RPMI-gfp-luc into single cell suspension and counting; when the cell growth state is good, the cells are diluted to 4×10 4 100. Mu.L/well was plated in 96-well plates.
3. Pan-T, bb2121 CAR-T and BCMA CAR16-T cells were mixed with target cells at different effective target ratios (1:4, 1:2,1:1, 2:1) respectively (tumor cell group was set as blank), 37℃and 5% CO 2 Culturing in an 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
K562-hBCMA-gfp, RPMI-gfp-luc, K562-cBCMA, K562 tumor cells were stained with BV421anti-human BCMA antibody, and surface BCMA expression was flow-detected, which showed that: K562-hBCMA-gfp and RPMI-gfp-luc cell surface BCMA are expressed in different degrees, wherein the K562-hBCMA-gfp surface BCMA is expressed as 85.5 percent and the RPMI-gfp-luc surface BCMA is expressed as 58.5 percent, which can be used as target cells in the research; the surfaces of K562-cBCMA and K562 cells do not substantially express BCMA, and can serve 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. After 12 hours, the apoptosis rate of the target cells is detected by a luciferase bioluminescence method, and the result shows that: compared with Pan-T group, BCMA CAR16-T group and BCMA CAR17-T group have enhanced killing ability to tumor cells, and are positively correlated with the effective target ratio, BCMA CAR16-T shows the strongest killing effect to 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. After 12h, the apoptosis rate of the target cells is detected in a flow manner, and the result is shown in the figure: compared with Pan-T group, BCMA CAR16-T group and BCMA CAR17-T group have enhanced killing ability to tumor cells, and the killing ability is positively correlated with the effective target ratio, BCMA CAR16-T shows the strongest killing effect to tumor cells, three independent repeated experiments show the same result, and the detection result is consistent with that of 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 the most remarkable killing effect on tumor cells and comparing the killing effect with positive bb2121 CAR-T cells, the result is shown in fig. 8 as follows: although BCMA CAR16-T has a significantly more pronounced killing effect on tumor cells than Pan-T group, the effect was not as statistically significant as the positive control group bb2121 CAR0-T, the difference. 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 regions, the three ScFv regions are connected, and the rest structures are 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 with XhoI and NgoMIV, the vector pMFG fragment was about 7kb in size, the target fragment BCMA CAR31 fragment was about 1.7kb in size, the BCMA CAR32 fragment was about 1.7kb in size, and the BCMA CAR33 fragment was about 2.5kb in size, and the electrophoresis results showed 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 were subjected to plasmid extraction and then identified by double digestion (XhoI/NgoMIV), the objective fragment BCMA CAR31 fragment size was about 1.7kb, BCMA CAR32 fragment size was about 1.7kb, BCMA CAR33 fragment size was about 2.5kb, the vector pMFG fragment size was about 7kb, and the electrophoresis results were shown in FIG. 10: the enzyme digestion identification of the pMFG-BCMA CAR31 plasmid (1) (2) (3) (4) (5) is correct; the enzyme digestion identification of the pMFG-BCMA CAR32 plasmid (1) (4) (5) is correct; the pMFG-BCMA CAR33 plasmid was digested and identified correctly (2) (3) (5). 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: the 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: firstly, the optimized pMFG-BCMA CAR plasmid is transiently transfected into Phoenix ECO cells, and BCMA CAR avirulent retrovirus 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
Plasmid transfection of pMFG-BCMA CAR31, pMFG-BCMA CAR32, pMFG-BCMA CAR33 into Phoenix-ECO cells, detection of transfection efficiency by flow cytometry after 72 h: the transfection efficiency of pMFG-BCMA CAR31 was 59%, the transfection efficiency of pMFG-BCMA CAR32 was 58.07%, the transfection efficiency of 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 result is: BCMA CAR31 transduction efficiency was 93.66%, BCMA CAR32 transduction efficiency was 95.35%, and BCMA CAR33 transduction efficiency was 98.24%, 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 with BCMA CAR31, BCMA CAR32 and BCMA CAR33 avirulent retroviral vector were expanded into T75 flasks and qPCR assays were performed with continuous harvest of 5 days of amphotropic retroviral vector particles, with the following results: BCMA CAR31 retrovirus Vector titers were up to BCMA CAR 31H 4:2.29×10 7 ±1.06×10 6 copy/mL, BCMA CAR 32H 3:1.64×10 7 ±1.12×10 6 copy/mL, BCMA CAR 33H 4:2.40×10 7 ±4.53×10 6 copy/mL, all higher than 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 fig. 5: firstly, separating out human peripheral blood PBMC by using a density gradient centrifugation method, activating primary T cells under the stimulation of a CD3 monoclonal antibody and a cytokine IL-2, transducing BCMA CARs by the activated T cells in a centrifugal way, constructing BCMA CARs-T cells, and detecting the expression of a cell surface Myc tag by using a flow cytometry method so as to detect whether the BCMA CARs are expressed on the surface of the T cells; further, the number of copies of BCMA CAR retroviral vector in each human primary T cell genome was detected by qPCR to determine whether it was successfully integrated into the human primary T cell genome. The following results were obtained using a similar method as before.
Successful transduction of human primary T cells by BCMA CAR retroviral vectors
BCMA CAR amphotropic retroviral vectors were transduced into human primary T cells and the transduction efficiency was examined after 48h and the results are shown in figure 11: BCMA CAR16-T transduction efficiency was 61.10% ± 3.65%, BCMA CAR31-T transduction efficiency was 61.97% ± 2.98%, BCMA CAR32-T transduction efficiency was 62.20% ± 1.59%, BCMA CAR33-T transduction efficiency was 57.80% ± 3.02%, and the results indicate that transduced human primary T cells successfully expressed BCMA CAR, and there was no statistical difference in transduction efficiency between the three groups compared to control group BCMA CAR16-T, we successfully prepared BCMA CAR-T cells.
BCMA CARs successfully integrate into the human primary T cell genome. BCMA CAR amphotropic retroviral vector transduces human primary T cells and after 48h the copy number integrated into the T cell genome is detected, showing that: BCMA CAR16-T integration into genomic copy number 0.44±0.05 copies/T cell, BCMA CAR31-T integration into genomic copy number 0.48±0.02 copies/T cell, BCMA CAR32-T integration into genomic copy number 0.43±0.04 copies/T cell, BCMA CAR33-T integration into genomic copy number 0.32±0.04 copies/T cell, and the results indicate that BCMA CAR was all successfully integrated into 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. Using different tumor cells expressing human BCMA antigen as target cells, using target cells not expressing human BCMA antigen as control, monitoring the survival of the tumor cells through luciferase bioluminescence experiment and incucyte real-time dynamic living cell imaging, and detecting the apoptosis of the tumor cells by using flow cytometry to verify the killing specificity and effectiveness of BCMA CAR-T cells on the target cells; detecting BCMA CAR-T cell secretion cytokine levels by CBA experiments; the proliferative capacity of BCMA CAR-T was tested by CFSE experiments. 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
BCMA CAR16-T, BCMA CAR31-T, BCMA CAR32-T, BCMA CAR33-T were targeted to K562-hbma-gfp cells at an effective target ratio of 1:1 co-incubation, continuously monitoring tumor cell fluorescence change by real-time fluorescence, recording every 2h, continuously monitoring for 48h, and the results are shown in fig. 13: the killing effect of BCMA CAR31-T on tumor cells is stronger than that of 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
Flow cytometry detected BCMA CAR-T cell surface CD69 expression, as shown in fig. 14: in the absence of tumor cell stimulation, BCMA CAR31-T has no statistical difference in surface CD69 expression compared with BCMA CAR16-T, the BCMA CAR31-T has higher expression than the BCMA CAR16-T cell surface CD69 under the stimulation of BCMA-expressing K562-hBCMA-gfp cells, 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 to target ratios for 12h for chemiluminescent signal intensity detection, experimental results are shown in fig. 15: compared with BCMA CAR16-T, BCMA CAR31-T shows stronger capability of killing tumor cells in vitro under each effect target ratio, and the difference has statistical significance.
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, BCMA CAR31-T exhibited no differential killing capacity against non-human BCMA expressing K562-cBCMA and BCMA negative K562 cells compared to Pan-T, indicating BCMA antigen specificity of BCMA CAR31-T cells against 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 was harvested and cytokines such as TNF- α, IFN- γ, IL-6, IL-17A, aFasL were measured using CBA kit. The results are shown in FIG. 17: the cytokines TNF- α, IFN- γ, IL-6, IL-17A, aFasL secreted by BCMA CAR31-T cells were all significantly increased upon stimulation by BCMA positive tumor cells as compared to BCMA CAR16-T 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: after 72h of cell culture, the CFSE green fluorescent signal of BCMA CAR16-T cells was significantly reduced compared to BCMA CAR31-T group cells, indicating that the proliferation rate of the BCMA CAR31-T cells was faster.
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
2X 10 intravenous injection to tail of NPG mice 6 The results of in vivo imaging of mice after 10 days with RPMI-gfp-luc cells are shown in FIG. 20: tumor signals were seen in all mice, and the uniformity of the neoplasia showed successful construction of the mouse xenograft model. 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 BCMA CAR-T injection, mice were imaged in vivo once a week, and mice were tested for tumor signal intensity changes, as shown in fig. 21: after the first injection of BCMA CAR31-T, the tumor signals of mice have no statistical difference with BCMA CAR16-T, and the imaging of mice after the second injection shows that the tumor signals of BCMA CAR31-T group are obviously weakened compared with BCMA CAR16-T, and the difference has statistical significance, so the result shows that the BCMA CAR31-T has effective in vivo anti-tumor capability.
Increased secretion of IFN-gamma by BCMA CAR-T
Mouse peripheral blood was collected 48h after two BCMA CAR-T cell injections, serum was isolated, and the serum was assayed for human IFN- γ secretion levels by ELISA, as follows: after the first BCMA CAR-T injection, the IFN-gamma secretion level of BCMA CAR31-T is lower than 180.29 +/-31.63 pg/mL, no difference exists between the BCMA CAR16-T (157.74 +/-45.51 pg/mL), 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
Blood was collected on day 24, day 31 and day 37 after inoculation of tumor cells in mice, and injected CD3 in peripheral blood of mice was detected + T cells, the results are shown below: CD3 in mouse peripheral blood per 100. Mu.L 24 days after tumor cell inoculation + The T cell content is that model groups (-1474.00 +/-3670.66), pan-T groups (-288.33 +/-8110.37), BCMA CAR16-T groups (11800.00 +/-7019.97), BCMA CAR31-T groups (29850.00 +/-15939.23), the content of the BCMA CAR31-T groups in the peripheral blood of the mice is obviously higher than that of the BCMA CAR16-T groups, and the difference has statistical significance; CD3 in mouse peripheral blood per 100. Mu.L on day 31 after tumor cell inoculation + The T cell content is that model groups (-4996.00 +/-2067.62), pan-T groups (-983.33 +/-1922.31), BCMA CAR16-T groups (8000.00 +/-7186.93), BCMA CAR31-T groups (23800.00 +/-11440.80), the content of the BCMA CAR31-T groups in the peripheral blood of the mice is obviously higher than that of the BCMA CAR16-T groups, and the difference has statistical significance; CD3 in mouse peripheral blood per 100. Mu.L on day 37 after tumor cell inoculation + The T cell content is that of model groups (1783.33 +/-8431.47), pan-T groups (-1750.00 +/-3573.65), BCMA CAR16-T groups (11400.00 +/-9017.98), BCMA CAR31-T groups (54866.67 +/-18619.20), and the content of BCMA CAR31-T groups in the peripheral blood of mice is significantly higher than that of BCMA CAR16-T groups, and the difference is statisticallyMeaning. 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 (12)

1. A BCMA-targeted chimeric antigen receptor comprising a ScFv structure of human-derived BCMA, wherein the heavy chain variable region ScFv-VH and the light chain variable region ScFv-VL of ScFv are linked by one or more G4S sequences; 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 SEQ ID No.8 below: SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSS;
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 below: QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG.
2. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the receptor comprises a human BCMA-targeted ScFv structure ScFv-VH- (G4S) n-ScFv-VL, wherein n is an integer greater than or equal to 1, preferably 3 or 4.
3. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the receptor comprises a human BCMA-targeted ScFv structure ScFv-VH- (G4S) 3-ScFv-VL, and the heavy chain variable region ScFv-VH has the amino acid sequence of SEQ ID No.8; the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.9.
4. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the receptor comprises a human BCMA-targeted ScFv structure ScFv-VH- (G4S) n-ScFv-VL- (G4S) n-ScFv-VH, wherein n is an integer equal to or greater than 1, preferably 3 or 4.
5. The BCMA-targeted chimeric antigen receptor according to claim 1, wherein the receptor comprises human-derived BCMA-targeted ScFv structure ScFv-VH- (G4S) 3-ScFv-VL- (G4S) 4-ScFv-VL- (G4S) 3-ScFv-VH, and the heavy chain variable region ScFv-VH has the amino acid sequence of SEQ ID No.8; the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.9.
6. The chimeric antigen receptor according to any one of claims 1-5, wherein the receptor comprises a signal peptide and myc tag for detection in tandem in sequence upstream; a ScFv structure of human-derived BCMA comprising a heavy chain variable region and a light chain variable region; CD8 hinge-transmembrane domain; CD28 or 4-1BB synergistic activation domain and cd3ζ intracellular signaling domain.
7. A chimeric antigen receptor T cell targeted to BCMA, characterized in that it expresses the chimeric antigen receptor according to any one of claims 1 to 6.
8. A medicament for the treatment of tumors, characterized in that it contains the chimeric antigen receptor T cells according to claim 7.
9. The use of a chimeric antigen receptor according to any one of claims 1-6 in the preparation of chimeric antigen receptor T cells and their use in tumor therapy.
10. The use of claim 9, wherein the tumor is a surface BCMA positive tumor.
11. The use of claim 9, wherein the tumor is multiple myeloma.
12. The use of a chimeric antigen receptor according to any one of claims 1 to 6, wherein the chimeric antigen receptor T cells are 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, for use in surface BCMA positive tumor treatment.
CN202310214870.8A 2023-03-08 2023-03-08 Chimeric antigen receptor of targeted BCMA and application thereof Pending CN116535521A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310214870.8A CN116535521A (en) 2023-03-08 2023-03-08 Chimeric antigen receptor of targeted BCMA and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310214870.8A CN116535521A (en) 2023-03-08 2023-03-08 Chimeric antigen receptor of targeted BCMA and application thereof

Publications (1)

Publication Number Publication Date
CN116535521A true CN116535521A (en) 2023-08-04

Family

ID=87454884

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310214870.8A Pending CN116535521A (en) 2023-03-08 2023-03-08 Chimeric antigen receptor of targeted BCMA and application thereof

Country Status (1)

Country Link
CN (1) CN116535521A (en)

Similar Documents

Publication Publication Date Title
US11932872B2 (en) Dual chimeric antigen receptor-t cell which can be regulated, construction method therefor and use thereof
CN110684121B (en) Chimeric antigen receptor of targeted HER2 combined expression PD1-MICA fusion protein, expression vector and application thereof
CN105296431A (en) Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof
CN112584849A (en) Therapeutic agents comprising nucleic acids and CAR-modified immune cells and uses thereof
CN110684120B (en) Chimeric antigen receptor targeting GPC3 and application thereof
CN110055269B (en) Human mesothelin chimeric antigen receptor, T cell thereof, preparation method and application thereof
CN109320615A (en) Target the Chimeric antigen receptor and application thereof of novel B CMA
CN107793483B (en) Chimeric antigen receptor and its gene and recombinant expression carrier, CARMSLN-NKT cell and its preparation method and application
CN109517798B (en) NK (natural killer) cell of chimeric CEA antigen receptor as well as preparation method and application of NK cell
CN105950662B (en) A kind of replication defective recombinant slow virus CAR-T transgene carrier targeting CD22 and its construction method and application
CN112812185B (en) Monoclonal antibody for resisting B cell maturation antigen and application thereof
WO2023217145A1 (en) Use of antigen short peptide in screening of drug for treating hpv-related disease, and tcr screened by antigen short peptide
CN110093376B (en) Construction method of LRFFT1 cells
CN116143943B (en) Targeting BAFFR chimeric antigen receptor, CAR-T cell and application
CN116478931A (en) High-affinity anti-tumor NK cell and preparation method and application thereof
CN116535521A (en) Chimeric antigen receptor of targeted BCMA and application thereof
US11718827B2 (en) LRFFT2 cell
CN110669144A (en) Chimeric antigen receptor of targeting B cell mature antigen and application thereof
CN117024605B (en) Chimeric antigen receptor, microglia expressing chimeric antigen receptor and application thereof
CN110093374B (en) Construction method of MRFFT1 cell
CN114369621B (en) Gene biological agent and preparation method and application thereof
CN113004423B (en) CAR-T cell for specifically targeting and activating hepatic stellate cell and preparation method and application thereof
CN113046320B (en) CAR molecule with extracellular segment of V delta1 (GTM) V gamma4, CAR-T cell expressing same and application thereof
CN117003855B (en) T cell receptor and application thereof
CN107557338A (en) Specific recognition NY ESO 1 T cell and its united application with cell factor

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination