Preparation and application of a car-nk cell targeting bcma

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to preparation and application of CAR-NK cells targeting BCMA. The application obtains an antibody sequence with drug potential through hybridoma screening, and the sequence is original. After humanization modification, the antibody still has strong affinity to the antigen. Through CAR-NK preparation, it is found that the cell has stronger tumor killing effect, and plays a good antitumor effect in a MM mouse model in vivo. The BCMA antibody sequence screened by the application and the prepared CAR-NK cells targeting BCMA show good killing effect in an in vitro cell line and an animal experiment, and have the treatment potential of blood tumors such as multiple myeloma.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation and application of CAR-NK cells targeting BCMA. Background Technology

[0002] BCMA is a transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) receptor superfamily. It is mainly expressed on the surface of mature B lymphocytes and plasma cells. It is highly expressed in BCMA-positive hematologic malignancies such as multiple myeloma (MM), mantle cell lymphoma, and diffuse large B-cell lymphoma, as well as some solid tumors (such as ovarian cancer), while it is expressed at low levels or not at all in normal tissues. Therefore, it has become an ideal target for tumor immunotherapy.

[0003] Immunotherapy is one of the important means of cancer treatment. Among them, CAR-T cell therapy has achieved significant efficacy in BCMA-positive multiple myeloma, but it has the following limitations: 1) The preparation cycle is long (14-21 days), which is difficult to meet the needs of emergency patients; 2) It depends on the patient's own T cells. Some patients have insufficient T cell numbers or functional failure due to chemotherapy, so they cannot prepare qualified CAR-T cells; 3) There are serious adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity; 4) In solid tumors, due to the inhibition of the tumor microenvironment, the infiltration and killing efficiency of CAR-T cells is low.

[0004] NK cells, as the core cells of innate immunity, have the following advantages: 1) They can directly kill tumor cells without antigen pre-sensitization and do not depend on MHC molecules, thus avoiding tumor immune escape; 2) They are widely available and can be obtained from peripheral blood (PBMC), umbilical cord blood, and induced pluripotent stem cells (iPSC), making it easy to achieve large-scale allogeneic preparation; 3) They have high safety and are not prone to causing CRS and neurotoxicity; 4) They have natural killing activity against some solid tumors.

[0005] Existing BCMA-targeted CAR-NK cell technology has the following shortcomings: 1) Low affinity of BCMA antibody sequences, resulting in weak targeting and binding ability of CAR-NK cells to BCMA-positive tumors; 2) Simple CAR structural design (mostly containing only CD3ζ signals or single co-stimulatory signals), making it difficult to effectively activate the killing function of NK cells; 3) Unstable in vitro expansion process of NK cells, with risks of contamination due to feeder layer dependence (such as K562 cells), or low expansion efficiency and poor cell viability under serum-free conditions; 4) Low LV vector packaging efficiency and insufficient viral titer lead to low CAR transduction efficiency, affecting the uniformity and efficacy of CAR-NK cells.

[0006] Therefore, developing high-affinity BCMA antibody sequences, optimizing CAR structures, and establishing stable and efficient NK cell expansion and LV vector packaging processes are of great significance for improving the cytotoxic activity, safety, and accessibility of BCMA CAR-NK cells. Summary of the Invention

[0007] The purpose of this invention is to address existing problems by providing a method for preparing and applying CAR-NK cells that target BCMA.

[0008] This invention is achieved through the following technical solution:

[0009] A chimeric antigen receptor (CAR) targeting BCMA, the structure of which is shown in the following formula: L-scFv-CD8 H-CD8 TM-C-CD3ζ

[0010] In the formula,

[0011] Each "-" independently represents a linking peptide or peptide bond;

[0012] L represents the signal peptide sequence;

[0013] scFv is an antigen-binding domain targeting BCMA, comprising the anti-BCMA antibody heavy chain variable region (VH) and the anti-BCMA antibody light chain variable region (VL), as shown in formula A or formula B below:

[0014] VH-VL (A); VL-VH (B)

[0015] The VH and VL are connected by a flexible joint, which is 1-4 consecutive GGGGS sequences.

[0016] CD8 H is the CD8 hinge area;

[0017] CD8™ is the transmembrane region of CD8;

[0018] C is a co-stimulatory signaling molecule, and is a tandem combination of co-stimulatory signaling molecules derived from 2B4 and NKG2D;

[0019] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0020] Furthermore, the amino acid sequence of the variable region VH of the heavy chain of the anti-BCMA antibody is shown in SEQ ID NO.1;

[0021] SEQ ID NO.1:

[0022] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYNDATKYNEKFQGRVTMTTDTSSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGGGTLVTVSS;

[0023] The amino acid sequence of the variable region VL of the light chain of the anti-BCMA antibody is shown in SEQ ID NO.2;

[0024] SEQ ID NO.2:

[0025] DIQMTQSPSSSLSASVGDRVTITCRASQSISDYLHWYQQKPGKAPKLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNGHSFPPTFGQGTKVEIK;

[0026] The amino acid sequence of the linker peptide is shown in SEQ ID NO.3;

[0027] SEQ ID NO.3:

[0028] GGGGSGGGGSGGGGS;

[0029] The amino acid sequence of the signal peptide is shown in SEQ ID NO.4;

[0030] SEQ ID NO.4:

[0031] MALPVRALLLILALLLHAARPAA;

[0032] The amino acid sequence of the CD8 hinge region is shown in SEQ ID NO.5;

[0033] SEQ ID NO.5:

[0034] KAPRPPTPAPTPEACRGAVHTRGLASD;

[0035] The amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO. 6;

[0036] SEQ ID NO.6:

[0037] FWVLVVVGGVLACYSLLVTVAFIIFWV;

[0038] The amino acid sequence of the NKG2D co-stimulatory signal region is shown in SEQ ID NO.7;

[0039] SEQ ID NO.7:

[0040] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR;

[0041] The amino acid sequence of the 2B4 co-stimulatory signal region is shown in SEQ ID NO. 8;

[0042] SEQ ID NO.8:

[0043] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

[0044] The amino acid sequence of the CD3ζ signal transduction region is shown in SEQ ID NO.9;

[0045] SEQ ID NO.9:

[0046] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0047] The aforementioned amino acid sequence, because one or more codons can encode the same amino acid, a phenomenon known as codon degeneracy, is covered by the claims and, in accordance with codon degeneracy, all corresponding nucleotide sequences encoded, including but not limited to.

[0048] A nucleic acid molecule encoding the CAR molecule as described in claim 1 or 2;

[0049] The 5' end of the nucleic acid molecule contains a promoter sequence;

[0050] The promoter is selected from the group consisting of: MNDU3 promoter, EF-1alpha promoter, CMV promoter, or a combination thereof.

[0051] The vector containing the above-mentioned nucleic acid molecules is selected from any one or combination of the following group: lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV), retroviral vectors, and transposons.

[0052] Furthermore, the vector is a lentiviral vector.

[0053] A CAR-NK cell targeting BCMA, wherein the cell is obtained by transducing the above-mentioned lentiviral vector into NK cells derived from PBMCs, thereby enabling the NK cells to express the above-mentioned CAR molecule.

[0054] A method for preparing CAR-NK cells includes the following steps:

[0055] (1) Screening of BCMA antibodies:

[0056] Balb / C mice were immunized with the BCMA extracellular domain (1-54aa) fusion protein to prepare hybridomas. Specific clones of BCMA HyBen01 were screened by ELISA, and VH and VL sequences were obtained by sequencing. Humanization was performed using the CDR transplantation method.

[0057] (2) Lentiviral vector packaging:

[0058] The BCMA-CAR sequence was placed under the MNDU3 promoter of the third-generation lentiviral packaging construct to construct a lentiviral vector expressing CS1-BCMA-CAR. Lentiviral cells were produced using 293T cells and transduced into NK cells to prepare BCMA-CAR-NK cells.

[0059] The BCMA CAR plasmid and helper plasmids (REV, GAG, PMD2.G) (Gándara C, Affleck V, StollEA. Manufacture of Third-Generation Lentivirus for Preclinical Use, with Process Development Considerations for Translation to Good Manufacturing Practice. Hum Gene Ther Methods. 2018 Feb;29(1):1-15. doi: 10.1089 / hgtb.2017.098. Epub 2018 Jan 24. PMID: 29212357; PMCID: PMC5806069.) were transfected into 293T cells. After culturing for 72 h, the supernatant was collected, centrifuged, filtered and purified to obtain BCMA CAR LV concentrate.

[0060] (3) Isolation and expansion of NK cells derived from PBMCs:

[0061] PBMCs were separated by Ficoll density gradient centrifugation, CD56+CD3-NK cells were obtained by magnetic sorting, and amplified in serum-free medium containing IL-2 500~1000U / mL for 14 days, with an amplification fold of ≥50-fold.

[0062] (4) CAR transduction: BCMA CAR LV concentrate was transduced into expanded NK cells at MOI=5~10, cultured for 48~72h, and identified by flow cytometry to obtain CAR-NK cells.

[0063] The third-generation lentiviral packaging plasmids (quadriplasmid system) include two packaging helper plasmids: pMD2.G, pMDLg / pRRE, and pRSV-Rev, all of which are ampicillin resistant and used to package third-generation lentiviral vector plasmids.

[0064] The third-generation lentiviral packaging system is a highly safe and efficient platform for producing replication-defective lentiviral vectors. This system comprises a transfer vector with self-inactivating long terminal repeats (SIN-LTRs) flanking its transgene cassette, and multiple helper plasmids encoding essential viral proteins: one encoding the Gag-Pol polyprotein, one encoding the Rev protein to facilitate RNA export, and a pseudotype envelope plasmid (e.g., expressing VSV-G for broad tropism). This design eliminates the Tat transcription activator and HIV-1 helper genes (Vif, Vpr, Vpu, Nef), thereby reducing the likelihood of recombination and the generation of replicating viruses, while achieving stable integration and long-term transgene expression in both dividing and non-dividing cells (e.g., hematopoietic stem cells or neurons).

[0065] Furthermore, in step (2), when transfecting 293T cells, the mass ratio of PEI reagent to plasmid is 6:1; the ultracentrifugation conditions are 4℃, 28000rpm, and 1h45min.

[0066] Furthermore, in step (3), NK cell amplification is performed using ImmunoCult. TM The Human NK Cell Expansion Kit culture and maintenance process was as follows: On Day 3, an equal volume of culture medium was added; on Day 7 and Days 10-11, the cells were passaged at an inoculation density of 1 × 10⁻⁶. 5 cells / cm 2 .

[0067] The application of the CAR-NK cells in the preparation of drugs for treating BCMA-positive tumors.

[0068] The present invention has the following advantages over the prior art:

[0069] 1. This invention, through hybridoma screening, yielded an antibody sequence with drug-like potential; the sequence is original. After humanization, the antibody still retains a strong affinity for the antigen. CAR-NK cell preparation revealed enhanced tumor-killing activity in the cells, and it demonstrated excellent anti-tumor effects in MM mouse models.

[0070] 2. The BCMA antibody sequence screened in this invention, and the CAR-NK cells prepared targeting BCMA, have shown good killing effects in in vitro cell lines and animal experiments, and have the potential to treat hematological malignancies such as multiple myeloma. Attached Figure Description

[0071] Figure 1 The structure of the chimeric antigen receptor (CAR) targeting BCMA;

[0072] Figure 2 The killing effect of CAR-NK cells in suspension tumor cell lines;

[0073] Figure 3 The killing effect of CAR-NK cells in adherent tumor cell lines;

[0074] Figure 4 For weight change curves and survival curves;

[0075] Figure 5 These are images taken before drug administration.

[0076] Figure 6 Imaging images taken 14 days after drug administration;

[0077] Figure 7 Imaging images taken 35 days after drug administration;

[0078] Figure 8 Imaging images taken 52 days after drug administration. Detailed Implementation

[0079] To further explain the present invention, the following specific embodiments are described.

[0080] Example 1

[0081] Preparation of CAR-NK

[0082] (1) First, we screened BCMA antibody sequences (BCMA antibody sequences are generated targeting the extracellular domain of BCMA (1-54 amino acids)) using the standard procedure. In short, we prepared hybridomas ourselves after continuously immunizing Balb / C mice with BCMA fusion protein. We used the same antigen for preliminary screening by ELISA. In the first screening, we evaluated the antibody titer of the clone. In the second screening, we evaluated its specificity by assessing the binding of the clone to other antigens. Based on these data, we screened out the BCMA antibody sequence and named its clone BCMAHyBen01), and performed humanization. We used tools such as SWISS-MODEL to perform homology modeling of the original antibody, identified the VH / VL interface and key framework residues supporting CDR, and designed "reversion mutations" more intelligently. We characterized the antibody by antibody affinity activity, flow cytometry detection, protein detection, etc., to prove its affinity activity. Affinity: after optimization, Kd < 5 nM, enhancing targeting efficiency (in vitro killing rate > 80%, Daudi / KMS-11 model). The selected sequences were then used for CAR-NK preparation.

[0083] BCMA scFv-linker-CD8 hinge / TM - 2B4 -NKG2D- CD3ζ (SEQ ID NO.10)

[0084] MALPVRALLLILALLLHAARPAAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYNDATKYNEKFQGRVTMTTDTSSTAYMELSSLRSEDTAVYYCARYNYDGYFD VWGGGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQSISDYLHWYQQKPGKAPKLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNGHSFPPTFGQGT KVEIKLEKAPRPPTPAPTPEACRGAVHTRGLASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0085] (2) The BCMA molecules obtained in (1) were subjected to LV packaging of tetraplasm.

[0086] Observe the cell growth status: Take out the 293T cell culture dish that was divided the day before and cultured overnight from the incubator and observe it under a microscope. If the cell confluence rate reaches 90%~95% and the cell morphology is good with clear and bright edges, then the subsequent operations can be carried out.

[0087] Preparation of the biosafety cabinet: Turn on the biosafety cabinet, wipe the work surface with 75% alcohol, and ventilate for 15 minutes before operating inside.

[0088] Reagent rewarming: After wiping away water droplets from the prepared and dispensed reagents stored in the refrigerator, such as plasmids, DMEM culture medium, PEI transfection reagent, etc., place them in an incubator for preheating for 15-30 minutes, then remove them and place them in a biosafety cabinet for later use.

[0089] Table 1

[0090]

[0091] Adding the virus to prepare the plasmid transfection system: After mixing the plasmid transfection system and letting it stand for 20 minutes, prepare an appropriate volume of serum-free DMEM culture medium according to the number of cell culture dishes to be packaged, and add the plasmid transfection system to the culture medium and mix well.

[0092] Cell medium change: After the virus packaging system is prepared, take out the 293T cell culture dish containing the virus to be packaged from the cell culture incubator and change the medium in the biosafety cabinet to serum-free DMEM medium containing the plasmid transfection system, 30mL / 15cm dish.

[0093] Add serum: After placing the cell culture dishes back in a 37°C, 5% CO2 incubator for 2 hours, add 3 mL of fetal bovine serum to each dish.

[0094] After adding fetal bovine serum, the cell culture dish was placed in a 37°C, 3% CO2 incubator for further culture.

[0095] Observe cell growth status: 72 hours after virus packaging, remove the 293T virus culture dish from the incubator and observe it under a microscope. If the upper layer cells become round and swollen, and some have detached and apoptotic, while the lower layer cells are still attached to the wall and have normal morphology, subsequent operations can be carried out.

[0096] Place the virus culture dish in the biosafety cabinet, take a 25mL pipette and the corresponding number of 250mL centrifuge tubes, label them, and open the tube caps.

[0097] Use a pipette to collect the virus solution from each culture dish into a 250mL centrifuge tube. After all the virus solution in all the virus culture dishes has been collected, cap the centrifuge tube.

[0098] Balance the centrifuge and centrifuge at 2500 rpm for 15 minutes.

[0099] Assembly of filter material: Take out a 50mL sterile syringe, pull out the syringe plunger, place the plunger upside down on the safety counter, and then put the syringe head into the 0.22μm needle filter.

[0100] Filtration of viral fluid: Pour the centrifuged viral fluid into a syringe tube, reinstall the plunger into the syringe, gently press down on the plunger to filter the viral fluid, and collect the filtered viral fluid into a new 250mL centrifuge tube.

[0101] Filter replacement: If the filter becomes clogged and filtration can no longer continue, with the syringe tip facing upwards, gently pull out the clogged filter. Open a new 0.22µm filter, then insert the syringe tip into the new filter and continue filtering the virus solution, collecting it into a 250mL centrifuge tube.

[0102] Repeat the above steps until all the virus fluid has been filtered.

[0103] Draw the filtered virus solution into a 50mL syringe and gently inject it into the ultracentrifuge tube until all the filtered virus solution is completely processed, ensuring that each ultracentrifuge tube is filled with virus solution without leaving any gaps.

[0104] Heat and seal the opening of the high-speed centrifuge tube using a heat sealer. After the opening cools, squeeze the centrifuge tube. If no liquid leaks out, the tube is considered a qualified seal. If there is leakage, replace the high-speed centrifuge tube and refill it with virus solution to fill the tube completely.

[0105] Weigh each ultracentrifuge tube using an electronic scale and mark the weight.

[0106] Place centrifuge tubes of equal weight into the corresponding holes of the centrifuge, balancing them in pairs.

[0107] Place the rotor into the centrifuge, turn on the high-speed centrifuge, set the temperature to 4℃, the speed to 28000rpm, and the time to 1 hour and 45 minutes, with the speed increasing and decreasing slowly.

[0108] First, click to start vacuum, then click the start button to begin high-speed centrifugation. Wait until the speed reaches the set speed and stabilizes before leaving the machine.

[0109] After the ultracentrifugation is complete, click to release the vacuum, open the centrifuge cover, and remove the ultracentrifuge tubes.

[0110] Wipe the condensation off the outside of the ultracentrifuge tubes with a lint-free cloth, and place the virus solution after ultracentrifugation in a biosafety cabinet.

[0111] Use a syringe to aspirate the upper layer of liquid from the ultracentrifuge tube. After discarding one-third of the upper layer of liquid, cut the ultracentrifuge tube with sterile scissors.

[0112] After cutting open all the ultracentrifuge tubes, use a pipette to remove all the liquid from the top layer, leaving the virus particles to settle at the bottom of the tube.

[0113] Add VIVO-15 medium to the ultracentrifuge tube to resuspend the virus particles. Gently disperse the virus particles with a pipette tip to avoid generating too many air bubbles.

[0114] Collect the resuspended virus solution from all centrifuge tubes into a new 50 mL centrifuge tube and mix well.

[0115] Assemble the filtration device, connect the 0.22µm needle filter to the 50mL syringe, and filter the collected virus solution through the 0.22µm filter for sterilization. Collect the sterilized virus concentrate into a new 50mL centrifuge tube.

[0116] (3) The LV vector obtained in (2) was used for the preparation of CAR-NK cells. The NK cells were derived from PBMCs and were amplified using in vitro amplification technology. The CAR positivity rate was then detected.

[0117] In vitro expansion technology: Experimental methods for expanding peripheral blood-derived NK cells

[0118] This protocol / method is used to isolate and expand natural killer (NK) cells from peripheral blood. This protocol combines clinical-grade methods (suitable for research or potential clinical applications) and emphasizes feeder-free and serum-free conditions to reduce variability and contamination risks. It is expected to start with 5 × 10⁻⁶ cells. 6 NK cells can be expanded to thousands of times their original yield (e.g., 120-fold expansion within 14 days, with purity >85%). Fresh peripheral blood is used as the starting material. The entire process must be carried out in a 37°C, 5% CO2 incubator.

[0119] Required materials and reagents

[0120] Starting materials: Fresh human peripheral whole blood (20-50 mL), leukocyte isolates or cryopreserved apheresis products (approximately 5 × 10⁻⁶). 7 Total nuclear cells / mL).

[0121] Separation reagent:

[0122] EasySep™ Human NK Cell Isolation Kit (or CliniMACS CD3 / CD19 / CD56 reagent for magnetic bead separation).

[0123] RosetteSep™ NK cell enrichment cocktail (optional, for density gradient separation).

[0124] Ficoll-Paque (used to remove dead cells).

[0125] Culture medium and supplements:

[0126] ImmunoCult™ Human NK Cell Expansion Kit (including ImmunoCult™ NK CellBase Medium, ImmunoCult™ Human NK Cell Expansion Supplement and ImmunoCult™ NKCell Expansion Coating Material).

[0127] Alternative: SCGM or X-VIVO 20 medium, supplemented with 10% heat-inactivated human AB serum or 5% human serum albumin (HSA).

[0128] Cytokines:

[0129] IL-2 (500-1000U / mL).

[0130] IL-15 (10-100 ng / mL, with optional enhanced amplification).

[0131] other:

[0132] PBS + 5% FBS (for washing).

[0133] Antibodies: CD56-PE, CD3-FITC (used for flow cytometry identification of NK cells: CD56+CD3-).

[0134] Equipment: centrifuge, magnetic separator, G-Rex100 culture flask (or T-75 cm² culture dish), flow cytometer, CellSaver (for harvesting).

[0135] 1. NK cell isolation (Day 0, approximately 1-2 hours)

[0136] 1.1 Isolation of peripheral blood mononuclear cells (PBMCs) from peripheral blood: Take 20-50 mL of whole blood, dilute with an equal volume of PBS, and centrifuge using a Ficoll density gradient (400 g, 30 min, 20°C, without brakes). Collect the PBMC layer and wash 3 times (PBS + 5% FBS, 300 g, 10 min).

[0137] 1.2 Negative selection enrichment using specific magnetic beads:

[0138] Add EasySep™ NK cell separation reagent and incubate for 15 minutes according to the instructions.

[0139] Place them in a magnetic separator and wash repeatedly to remove non-NK cells (CD3+ T cells, CD19+ B cells, etc.).

[0140] Expected purity: >90% CD56+CD3-NK cells; Initial yield: 4-5 × 10⁻⁵ 6 NK cells (from 50 mL of blood).

[0141] 1.3 Verification using flow cytometry: Stain CD56 and CD3 to confirm the NK cell ratio. Remove dead cells (Ficoll gradient).

[0142] 2. NK cell expansion (Day 0-14, approximately 2 weeks)

[0143] 2.1 Pre-coating culture surface (Day 0): Coat G-Rex100 bottles or 96-well plates with ImmunoCult™ NK Cell Expansion Coating Material (according to instructions, 37°C, 1h), then discard the coating solution.

[0144] 2.2 Cell Seeding (Day 0): Isolated NK cells (initially 3-5 × 10⁻⁵) were seeded. 6 Cells were resuspended in ImmunoCult™ NK Cell Expansion Medium (containing IL-2 500 U / mL) at 1–2 × 10⁻⁶ ppm. 5 cells / cm 2 Density inoculation. Total volume: 10-20 mL / vial.

[0145] 2.3 Cultivation and Maintenance:

[0146] Day 3: Add fresh culture medium (equal volume), without replacing the old medium.

[0147] Day 7: Harvest cells (300g, 5min), count, resuspend on fresh coated surface, and re-seed (density 1×10⁻⁶). 5 cells / cm 2 ).

[0148] Days 10-11: Repeat harvesting and re-inoculation.

[0149] Day 14: Final harvest, collect expanded cells using CellSaver or centrifugation.

[0150] 2.4 Optional feeder layer enhancement (for higher yields): Adding irradiated (100 Gy) K562-41BBL-mbIL-15 feeder cells (NK: feeder layer = 1:10) and culturing in X-VIVO 20+IL-15 can shorten the time to 10 days.

[0151] Timeline:

[0152] Day 0: Isolation + Inoculation.

[0153] Day 3: Supplement culture medium.

[0154] Days 7 & 10: Passing on the torch.

[0155] Day 14: Harvest (Total duration: 14 days).

[0156] Expected output: from 5×10 6 Initiating NK cells, expansion >50-100 times (reaching 2-5 × 10⁻⁶) within 14 days 8 Cells), purity >85% CD56+CD3-. Using a feeder layer can achieve >200x growth.

[0157] 3. Quality control and functional verification

[0158] 3.1 Phenotypic analysis: The CD56+CD3- ratio and activity (>90%) were detected by flow cytometry every 3-4 days.

[0159] 3.2 Functional Testing (Day 14):

[0160] Cytotoxicity: Apoptosis was detected by flow cytometry or Incucyte assay after co-culturing with K562 target cells (E:T = 1:1 to 10:1, 4 h, 37°C) (% kill rate >40%).

[0161] Degranulation and cytokines: PMA / ionomycin or K562 was stimulated (4h), and the expression of CD107a, IFN-γ, and TNF-α was detected.

[0162] 3.3 Purity / Contamination Check: Sterility test, nuclease treatment to remove DNA residue.

[0163] (3) The CAR-NK cells from step (2) were subjected to in vitro tumor cell killing experiments to demonstrate their cell killing effect; at the same time, a suitable mouse model was selected, and the MM1S cell line was used to model the model. NK or CAR-NK was reinfused into the mouse once in vivo. By comparing with the control group, the good anti-tumor activity of CAR-NK was demonstrated.

[0164] Example 2

[0165] Flow cytometry assay of CAR-NK cells killing suspension tumor cell lines in vitro (suspension tumor cell lines: U266, K562, MOLT4)

[0166] Preparation before the experiment:

[0167] ① Preparation of CFSE stock solution (5mM)

[0168] Take one vial of CFSE dry powder (biolegend / 423801), bring it to room temperature, add 36 μL of DMSO with a pipette, mix well by pipetting, let stand for 10 min, dispense 5 μL / vial, store at -20℃, label with name and preparation date, shelf life 3 months.

[0169] ② Preparation of CFSE working solution (10μM)

[0170] Take one vial of CFSE stock solution and thaw it at room temperature. Take one 1.5mL centrifuge tube, add 500mL of culture medium 1, add 1μL of the thawed CFSE stock solution, mix well, and store in the dark for later use. Prepare fresh before use.

[0171] ③ Preparation of medium 1: RPMI 1640 medium with 0.5% FBS

[0172] Use a pipette to draw 3980 mL of 1640 culture medium, add 20 mL of FBS, mix thoroughly by pipetting, and label the culture medium with the name and preparation date. Prepare and use immediately.

[0173] ④ Medium 2: Prepare RPMI 1640 medium with 10% FBS.

[0174] Use an electric pipette to draw 18 mL of 1640 culture medium, add 2 mL of FBS, mix thoroughly by pipetting, and label the culture medium with the name and preparation date. Prepare and use immediately.

[0175] ⑤ Culture medium 3: Prepared with RPMI 1640 medium containing 2 IU / mL IL-2.

[0176] Use a pipette to draw 2 mL of 1640 culture medium, add 1.6 mL of IL-2, mix thoroughly with an electric pipette, and label the culture medium with the name and preparation date. Prepare and use immediately.

[0177] Target cell preparation

[0178] ① Target cell count: Use an electric pipette to homogenize the suspension of tumor cells in the logarithmic growth phase and take a sample once to estimate the number of cells.

[0179] ② Adjust target cell density: Based on the counting results, take 2E6 (the number can be adjusted according to the actual sample volume, but the stained cell density must remain unchanged) of tumor cells (enough for 10 samples to be tested), centrifuge at 300rcf for 5 min, discard the supernatant, resuspend the tumor cells in 1 mL of culture medium 1 to ensure uniform density, take 500 mL of cell suspension into a new 15 mL centrifuge tube 2, and leave the remaining cells at room temperature for later use;

[0180] ③ CFSE staining: Take 500 mL of CFSE working solution (10 μM), add it to a 15 mL centrifuge tube 2, vortex and mix thoroughly with tumor cells (final cell concentration 1E6 cells / mL, final CFSE concentration 5 μM), place in a 37℃ incubator to stain for 15 min in the dark, and take it out every 5 min to vortex and mix.

[0181] ④ Stop staining & wash: Add 5 mL of culture medium 2 to a 15 mL centrifuge tube 2, vortex to mix, centrifuge at 200 rcf for 5 min, and wash twice with culture medium 2.

[0182] ⑤ Counting: Discard the supernatant, resuspend the cells in 1 mL of culture medium 2, mix well, take about 100 mL of cell suspension and count 3 times, take the average value, take the previously set aside unstained tumor cells, mix well, take the cell suspension and count once, and adjust the density of stained and unstained cells to 2E5 cells / mL with culture medium 2 according to the counting results.

[0183] ⑥ Target cell plating: Add the corresponding target cell suspension to a 96-well U-shaped plate. Each of the three experimental groups and the negative control group has three replicates. Control group 1 (flow cytometry blank tube), control group 2 (CFSE single-stained tube) and control group 3 (PI single-stained tube) have no replicates.

[0184] Effector cell purity detection

[0185] ① Take out 1.5 EP tubes and label them as NC (Negative control), PerCP / Cy5.5-CD3 single staining tube, BV785-CD56 single staining tube, and fully stained tube (PerCP / Cy5.5-CD3+BV785-CD56). Take 1E6 cells / 100μL NK cells and add them to the EP tubes respectively.

[0186] ② Cell washing: Add 1 mL of PBS to each tube, mix the cells with a vortex mixer, and centrifuge once at 4°C, 500 g, for 5 min.

[0187] ③ Cell blocking: Discard the supernatant, resuspend the cells in 200 μL of blocking solution, mix well, and incubate at room temperature for 10 min in the dark.

[0188] ④ Antibody incubation: Add the corresponding antibody according to the label on the EP tube, mix well, and label at room temperature for 20 minutes in the dark (mix once every 10 minutes).

[0189] ⑤ After antibody labeling, add PBS to each tube of cells, centrifuge at 500g for 5 min at 4℃, and wash twice.

[0190] ⑥ Add 200 μL PBS and perform flow cytometry analysis.

[0191] Effect / target cell co-culture

[0192] ① Collect NK cells: Mix the cells, take the expected number of samples once, and transfer about 2E6 NK cell suspensions into 15mL centrifuge tubes 3. Centrifuge at 500rcf at room temperature for 5min and discard the supernatant.

[0193] ② Resuspension counting: Resuspend the cells in 1 mL of culture medium, mix well, take 100 mL of cell suspension and count 3 times, and take the average value;

[0194] ③ Adjust cell density: Based on the counting results and CD56+ cell purity, dilute the cell suspension with culture medium 3 to 8E5 CD56+ cells / mL (effect cell 1), and mix the cells thoroughly;

[0195] ④ Take 1 mL of effector cell 1, add 1 mL of culture medium 3 to dilute the cell suspension to 4E5 CD56+ cells / mL (effector cell 2), and mix the cells thoroughly;

[0196] ⑤ Take 1 mL of effector cell 2, add 1 mL of culture medium 3 to dilute the cell suspension to 2E5 CD56+ cells / mL (effector cell 3), and mix the cells thoroughly;

[0197] ⑥ Plate preparation; add the effector cell suspension to the corresponding wells;

[0198] ⑦ Incubation: Place the 96-well plate in a centrifuge and centrifuge at 200 rcf at room temperature for 2 min to ensure sufficient contact between the effector and target cells. Incubate at 37°C for 3.5 h.

[0199] ⑧ Transfer: After incubation, remove the 96-well plate and transfer the cells from each well into flow cytometry tubes.

[0200] ⑨ Staining: Add 5 μL of PI staining solution to each of the three experimental groups, the negative control group, and control group 3. Vortex mix well, incubate in the dark for 5 min, and then load the solution onto the instrument.

[0201] On-machine testing

[0202] Create an experiment, enter the sample name, set the flow rate (to medium speed), and set the stopping conditions (collect 10,000 CFSE-positive cells). Figure 2 Establish gate logic, set relevant gate strategies, create graphs and gates, draw relevant peak plots or scatter plots, and collect data;

[0203] Criteria for FITC-CFSE positivity: In control group 1, the FITC-CFSE positivity rate was 0.5% to 1%.

[0204] PI positive area: In control group 2, the positive rate of PC5.5-PI was 0.5-1%.

[0205] Figure 2 The study demonstrated the antitumor activity of CAR-NK cells in suspension tumor cell lines. The killing activity was positively correlated with the effector-to-target ratio (effector cells:tumor cell lines).

[0206] Example 3

[0207] RTCA assay for CAR-NK cell line killing in vitro (adherent tumor cell lines: SKOV3, OVCAR3, A375)

[0208] RTCA detection experiment

[0209] (1) Adherent tumor cells were digested into single-cell suspensions using trypsin containing EDTA.

[0210] (2) Centrifuge at 140g for 5 minutes. After centrifugation, resuspend the cells in complete culture medium, take 20μL of the cell suspension for cell counting, and adjust the density of the cell suspension to 1.6×10⁻⁶. 5 Cells / mL.

[0211] (3) Add 50 μL of complete culture medium to the well of E-Plate 16 and place it on the RTCA Station.

[0212] (4) The RTCA system will automatically perform a scan ("Scan Plate") to check for good contact (the "Message" page will display "Connection OK").

[0213] (5) Start testing the baseline (Background) ≥ confirm that the selected hole is in normal contact.

[0214] (6) Take out E-Plate 16 and add 100 μL of tumor cell suspension to the well. Place E-Plate 16 on a clean bench for 20 minutes to allow the cells to settle to the bottom of the plate.

[0215] (7) Place E-Plate 16 back into the RTCA Station.

[0216] (8) After the system automatically scans the “Scan Plate”, Step 2 begins to perform real-time dynamic monitoring of cell proliferation.

[0217] (9) After about 12 hours, when the system's cell index is around 2.0, you can start preparing to add NK cells.

[0218] (10) Use complete culture medium to dilute NK cell suspension (or an equal volume of culture medium: blank control) to 50 volumes according to different effector-target ratios.

[0219] 11. Add NK cells to the corresponding target cell culture wells, and set up control wells containing only target cells and only NK cells;

[0220] 12. The E-Plate 16 was returned to the RTCA Station for real-time monitoring to observe the real-time killing effect of NK cells on OVCAR3 cells, SKOV cells, and A375 cells.

[0221] ⒀ Result calculation method: (average cell index of control group - average cell index of experimental group) / average cell index of control group × 100%.

[0222] Figure 3The study demonstrated the antitumor activity of CAR-NK cells in adherent tumor cell lines. The killing activity was positively correlated with the effector-to-target ratio (effector cells:tumor cell lines).

[0223] Example 4

[0224] CAR-NK for the treatment of MM1S-induced myeloma mouse models

[0225] Tumor cell lines

[0226] Tumor cell line: MM.1S-luciferase / e-GFP, a human multiple myeloma cell line carrying the luciferase and e-GFP genes.

[0227] Culture medium conditions: RPMI 1640 medium containing 10% fetal bovine serum, 1× penicillin-streptomycin mixture, and 0.2 μg / mL puromycin.

[0228] experimental animals

[0229] Animal species: NOD.Cg-PrkdcscidIL2rgtm1Sug / JicCrl (abbreviated as NOG mouse)

[0230] Animal rating: SPF

[0231] Animal age: 5-6 weeks old at the time of receipt; the actual age in weeks will be listed in the summary report.

[0232] Animal weight: 16~20g upon receipt; actual weight will be listed in the summary report.

[0233] Number and sex of animals: 65 female animals were purchased.

[0234] Tumor inoculation and grouping

[0235] Tumor cells were routinely resuscitated and expanded, passaged at least twice, and then harvested in logarithmic growth phase. The cells were resuspended in serum-free medium to a concentration of 1.0 × 10⁻⁶. 7 Cells were prepared at a density of 10 cells / mL and then sent to the animal facility for seeding. Cells must be prepared and used immediately. The low-dose group received 2.0 × 10⁶ cells per animal. 6 1.0 × 10 cells 7 (0.2 mL / mL), high-dose group: 1.0 × 10⁻⁶ cells / mL per animal. 7 Cells were inoculated into all animals under aseptic conditions via the tail vein.

[0236] Approximately 14 days after inoculation, tumor-bearing mice were selected based on fluorescence signals and body weight, and then randomly divided into groups of 10 mice each.

[0237] Administration method

[0238] All animals were administered the drug via a single tail vein injection, with each injection lasting at least 40 seconds. The day of administration was designated as D1, the following day as D2, and so on.

[0239] detection indicators

[0240] (1) General clinical observation

[0241] During all animal trials, animals were observed at least twice daily (once in the morning and once in the afternoon), and any observed animal deaths or abnormal clinical manifestations were recorded. Survival curves were plotted at the trial endpoint.

[0242] (2) Weight

[0243] Animals were weighed once before receiving and before vaccination, once a week after vaccination, and twice a week after administration of medication.

[0244] like Figure 4 As shown, the left side is the weight change curve. Overall, the weight loss in the treatment group was less, and it showed a dose-dependent effect (high dose showed the best results). The right side is the survival curve, i.e., the mortality of the mice. All untreated mice died after 42 days, while the treatment groups had survival rates of 60% and 70%, respectively (two mice in the high-dose group were euthanized on day 52).

[0245] (3) In vivo imaging

[0246] Administer once before administration and once weekly after administration. All animals were intraperitoneally injected with 150 mg / kg (15 mg / mL, 10 mL / kg) of D-fluorescein potassium. Chemiluminescence signals were captured using a small animal imaging system 10–15 minutes after injection under isoflurane anesthesia.

[0247] Imaging images of animals before administration and at different times after treatment ( Figure 5-8 The control group received no treatment, while the low-dose group received an injection of 2.0 × 10⁻⁶. 6 There were two CAR-NK cell therapy groups, with the high-dose group receiving 1.0 × 10⁷ cells. Imaging showed that a larger shadow area indicated a more severe tumor. Before treatment, the shadow areas were uniform; after treatment, the shadow areas in the treatment group were smaller than those in the control group, and this difference was concentration-dependent.

[0248] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor (CAR) targeting BCMA, characterized in that, The structure of the chimeric antigen receptor CAR is shown in the following formula: L-scFv-CD8 H-CD8 TM-C-CD3ζ In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the signal peptide sequence, and the amino acid sequence of the signal peptide is shown in SEQ ID NO.4; 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). The amino acid sequence of the anti-BCMA antibody heavy chain variable region VH is shown in SEQ ID NO.1, and the amino acid sequence of the anti-BCMA antibody light chain variable region VL is shown in SEQ ID NO.

2. The structural formula is shown in formula A or formula B below: VH-VL (A); VL-VH (B) The VH and VL are connected by a flexible joint, which is 1-4 consecutive GGGGS sequences. The amino acid sequence of the linker peptide is shown in SEQ ID NO.3; CD8 H is the CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO.5; CD8™ is the CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.6; C is a co-stimulatory signal molecule, and is a tandem combination of co-stimulatory signal molecules derived from 2B4 and NKG2D. The amino acid sequence of the NKG2D co-stimulatory signal region is shown in SEQ ID NO.7, and the amino acid sequence of the 2B4 co-stimulatory signal region is shown in SEQ ID NO.

8. CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ, and the amino acid sequence of the CD3ζ signal transduction region is shown in SEQ ID NO.

9.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the CAR molecule as described in claim 1; The 5' end of the nucleic acid molecule contains a promoter sequence; The promoter is selected from the group consisting of: MNDU3 promoter, EF-1alpha promoter, CMV promoter, or a combination thereof.

3. A carrier comprising the nucleic acid molecule of claim 2, characterized in that, The vector is selected from any one or combination of the following group: adenovirus vector, adeno-associated virus vector (AAV), retrovirus vector, and transposon.

4. The carrier according to claim 3, characterized in that, The vector is a lentiviral vector.

5. A CAR-NK cell targeting BCMA, characterized in that, The cells are obtained by transducing the lentiviral vector of claim 4 into NK cells derived from PBMCs, thereby causing the NK cells to express the CAR molecule of claim 1.

6. A method for preparing CAR-NK cells as described in claim 5, characterized in that, Includes the following steps: (1) Screening of BCMA antibodies: Balb / C mice were immunized with the BCMA extracellular domain 1-54aa fusion protein to prepare hybridomas. Specific clones of BCMA HyBen01 were screened by ELISA, and VH and VL sequences were obtained by sequencing. Humanization was performed by CDR transplantation. (2) Lentiviral vector packaging: BCMA CAR plasmid and helper plasmids REV, GAG, and PMD2.G were transfected into 293T cells. After culturing for 72 hours, the supernatant was collected, centrifuged, filtered, and purified to obtain BCMA CAR LV concentrate. (3) Isolation and expansion of NK cells derived from PBMCs: PBMCs were separated by Ficoll density gradient centrifugation, CD56+CD3-NK cells were obtained by magnetic sorting, and amplified in serum-free medium containing IL-2 500~1000U / mL for 14 days, with an amplification fold of ≥50-fold. (4) CAR transduction: BCMA CAR LV concentrate was transduced into expanded NK cells at MOI=5~10, cultured for 48~72h, and identified by flow cytometry to obtain CAR-NK cells.

7. The preparation method according to claim 6, characterized in that, In step (2), when transfecting 293T cells, the mass ratio of PEI reagent to plasmid is 6:1; the ultracentrifugation conditions are 4℃, 28000rpm, 1h45min.

8. The preparation method according to claim 6, characterized in that, In step (3), NK cell expansion was performed using ImmunoCult. TM The Human NK Cell Expansion Kit culture and maintenance process is as follows: Add an equal volume of culture medium on Day 3; subculture on Day 7 and Days 10-11, with an inoculation density of 1×10⁻⁶. 5 cells / cm 2 .

9. The use of CAR-NK cells as described in claim 5 or 6 in the preparation of a medicament for treating multiple myeloma.

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