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CN-121203041-B - Preparation and application of BCMA-targeted CAR-NK cells

CN121203041BCN 121203041 BCN121203041 BCN 121203041BCN-121203041-B

Abstract

The invention belongs to the technical field of biology, and particularly relates to preparation and application of BCMA-targeted CAR-NK cells. The invention obtains an antibody sequence with potential of patent medicine through hybridoma screening, and the sequence is original. After humanized transformation, the antibodies still have strong affinity for the antigen. Through the preparation of the CAR-NK, the cell is found to have stronger tumor killing effect and has good anti-tumor effect in the MM mouse model body. The BCMA antibody sequences screened by the invention, prepared BCMA-targeted CAR-NK cells, show good killing effect in vitro cell lines and animal experiments, and have the therapeutic potential of hematological tumors such as multiple myeloma.

Inventors

  • ZHANG JINXIA
  • ZHU HOUSHUN
  • SONG WENCHEN

Assignees

  • 广东海本起源细胞科技有限公司
  • 张锦霞

Dates

Publication Date
20260512
Application Date
20251117

Claims (9)

  1. 1. A Chimeric Antigen Receptor (CAR) targeting BCMA is characterized in that the chimeric antigen receptor CAR has a structure shown in the following formula L-scFv-CD 8H-CD 8 TM-C-CD3 zeta In the formula, Each "-" is independently a connecting peptide or peptide bond; l is a signal peptide sequence, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 4; the scFv is an antigen binding domain of a targeted BCMA and comprises an anti-BCMA antibody heavy chain variable region (VH) and an anti-BCMA antibody light chain variable region (VL), wherein the amino acid sequence of the anti-BCMA antibody heavy chain variable region VH is shown as SEQ ID NO.1, the amino acid sequence of the anti-BCMA antibody light chain variable region VL is shown as SEQ ID NO.2, and the structural formula is shown as the following formula A or formula B: VH-VL (A);VL-VH (B) The VH and VL are connected through a flexible joint, and the flexible joint is 1-4 continuous GGGGS sequences; The amino acid sequence of the connecting peptide is shown as SEQ ID NO. 3; CD 8H is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown as SEQ ID NO. 5; CD8 TM is a CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is shown as SEQ ID NO. 6; C is a costimulatory signal molecule and is a tandem combination of 2B4 and a costimulatory signal molecule derived from NKG2D, the amino acid sequence of the NKG2D costimulatory signal region is shown as SEQ ID NO.7, and the amino acid sequence of the 2B4 costimulatory signal region is shown as SEQ ID NO. 8; CD3 zeta is a cytoplasmic signaling sequence derived from CD3 zeta, and the amino acid sequence of the CD3 zeta signaling region is shown in SEQ ID NO. 9.
  2. 2. A nucleic acid molecule encoding the CAR molecule of claim 1; the 5' end of the nucleic acid molecule comprises a promoter sequence; The promoter is selected from MNDU promoter, EF-1alpha promoter, CMV promoter or combination.
  3. 3. A vector comprising the nucleic acid molecule of claim 2, wherein the vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector (AAV), a retrovirus vector, and a transposon.
  4. 4. The vector of claim 3, wherein the vector is a lentiviral vector.
  5. 5. A BCMA-targeted CAR-NK cell obtained by transducing the lentiviral vector of claim 4 into PBMC-derived NK cells, such that the NK cells express the CAR molecule of claim 1.
  6. 6. A method of making the CAR-NK cell of claim 5, comprising the steps of: (1) Screening of BCMA antibodies: Immunizing Balb/C mice with BCMA extracellular domain 1-54aa fusion protein, preparing hybridomas, screening specific clones BCMA HyBen01 by ELISA, sequencing to obtain VH and VL sequences, and humanizing by a CDR grafting method; (2) Lentiviral vector packaging: the BCMA CAR plasmid and helper plasmids REV, GAG and PMD2.G are transfected into 293T cells, and supernatant is collected after 72 hours of culture, centrifuged, filtered and purified to obtain BCMA CAR LV concentrated solution; (3) Isolation and expansion of PBMC-derived NK cells: Centrifuging and separating PBMC by Ficoll density gradient, magnetically separating to obtain CD56+CD3-NK cells, and amplifying in a serum-free culture medium, wherein the culture medium comprises IL-2 500-1000U/mL, and the amplification factor is more than or equal to 50 times after 14 days of amplification; (4) And (3) CAR transduction, namely transduction of BCMA CAR LV concentrate to amplified NK cells, MOI=5-10, culture for 48-72 h, and flow detection and identification to obtain CAR-NK cells.
  7. 7. The method of claim 6, wherein the mass ratio of PEI reagent to plasmid in step (2) is 6:1 and the ultracentrifugation conditions are 4℃28000rpm for 1h45min.
  8. 8. The method of claim 6, wherein in step (3), NK cells are expanded by ImmunoCult TM Human NK Cell Expansion Kit, and the culture maintenance process comprises the steps of adding equal volume of medium into Day 3, and passaging Day 7 and Day 10-11, wherein the inoculation density is 1×10 5 cells/cm 2 .
  9. 9. Use of a CAR-NK cell as defined in claim 5 or 6 in the manufacture of a medicament for the treatment of multiple myeloma.

Description

Preparation and application of BCMA-targeted CAR-NK cells Technical Field The invention belongs to the technical field of biology, and particularly relates to preparation and application of BCMA-targeted CAR-NK cells. Background BCMA is a transmembrane glycoprotein, belongs to a Tumor Necrosis Factor (TNF) receptor superfamily member, is mainly expressed on the surfaces of mature B lymphocytes and plasma cells, is highly expressed in BCMA positive blood system tumors such as Multiple Myeloma (MM), mantle cell lymphoma, diffuse large B cell lymphoma and the like and partial solid tumors (such as ovarian cancer), and is low expressed or not expressed in normal tissues, thus becoming an ideal target point for tumor immunotherapy. Immune cell therapy is one of the important means of current tumor therapy, wherein CAR-T cell therapy has obvious curative effect in BCMA positive multiple myeloma, but has the following limitations that 1) the preparation period is long (14-21 days are needed) and is difficult to meet the requirements of emergency patients, 2) patients depend on autologous T cells, partial patients cannot prepare qualified CAR-T cells due to insufficient T cell number or failure caused by chemotherapy, 3) serious adverse reactions such as Cytokine Release Syndrome (CRS), neurotoxicity and the like exist, and 4) the CAR-T cell infiltration and killing efficiency are low due to tumor microenvironment inhibition in solid tumors. NK cells are used as core cells for natural immunity, and have the advantages of 1) capability of directly killing tumor cells without antigen pre-sensitization and avoiding tumor immune escape without depending on MHC molecules, 2) wide sources, capability of being obtained from Peripheral Blood (PBMC), umbilical cord blood and Induced Pluripotent Stem Cells (iPSC), easiness in realizing variant large-scale preparation, 3) high safety, difficulty in initiating CRS and neurotoxicity, and 4) natural killing activity on part of solid tumors. The existing BCMA targeted CAR-NK cell technology has the following defects that 1) the BCMA antibody sequence has low affinity, so that the targeting binding capacity of CAR-NK cells to BCMA positive tumors is weak, 2) the CAR structure is single in design (contains only CD3 zeta signals or single co-stimulatory signals), the killing function of NK cells is difficult to activate effectively, 3) the NK cells in-vitro amplification process is unstable, the pollution risk caused by feeder layer dependence (such as K562 cells) exists, or the amplification efficiency is low and the cell activity is poor under serum-free conditions, 4) the LV carrier packaging efficiency is low, the CAR transduction efficiency is low due to insufficient virus titer, and the uniformity and the curative effect of the CAR-NK cells are influenced. Therefore, the development of high-affinity BCMA antibody sequences, the optimization of CAR structures and the establishment of stable and efficient NK cell amplification and LV carrier packaging processes are of great significance in improving the killing activity, safety and accessibility of BCMA CAR-NK cells. Disclosure of Invention The invention aims at solving the existing problems and provides preparation and application of BCMA-targeted CAR-NK cells. The invention is realized by the following technical scheme: A Chimeric Antigen Receptor (CAR) targeting BCMA, the chimeric antigen receptor CAR having the structure of L-scFv-CD 8H-CD 8 TM-C-CD3 zeta In the formula, Each "-" is independently a connecting peptide or peptide bond; l is a signal peptide sequence; scFv is an antigen binding domain targeting BCMA, comprising an anti-BCMA antibody heavy chain variable region (VH) and an anti-BCMA antibody light chain variable region (VL), represented by structural formula a or formula B: VH-VL (A);VL-VH (B) The VH and VL are connected through a flexible joint, and the flexible joint is 1-4 continuous GGGGS sequences; CD 8H is the CD8 hinge region; CD8 TM is the CD8 transmembrane region; c is a costimulatory signaling molecule, and is a tandem combination of 2B4 and NKG 2D-derived costimulatory signaling molecules; cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ. Further, the amino acid sequence of the anti-BCMA antibody heavy chain variable region VH is shown as SEQ ID NO. 1; SEQ ID NO.1: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYNDATKYNEKFQGRVTMTTDTSTSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGGGTLVTVSS; the amino acid sequence of the variable region VL of the anti-BCMA antibody is shown as SEQ ID NO. 2; SEQ ID NO.2: DIQMTQSPSSLSASVGDRVTITCRASQSISDYLHWYQQKPGKAPKLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNGHSFPPTFGQGTKVEIK; The amino acid sequence of the connecting peptide is shown as SEQ ID NO. 3; SEQ ID NO.3: GGGGSGGGGSGGGGS; The amino acid sequence of the signal peptide is shown as SEQ ID NO. 4; SEQ ID NO.4: MALPVRALLLILALLLHAARPAA; The amino acid sequence of the CD8 hinge region is shown as SEQ ID NO. 5; SEQ