POLYNUCLEOTIDES ENCODING NK CELL ADAPTERS AND CARs AND MODIFIED

Abstract

The invention discloses polynucleotide for encoding an NK cell adapter and a CAR (chimeric antigen receptor) and a modified cell containing the polynucleotide. The polynucleotide provided by the invention comprises a first polynucleotide for coding an NK cell adapter, a linker and a second polynucleotide for coding a CAR (Chimeric Antigen Receptor), wherein the first polynucleotide comprises a first signal peptide, an anti-CD16 single-domain antibody, an IL-15 (Interleukin-15) and an scFv (Single-chain Factor Vector) for recognizing GPRC5D; the second polynucleotide includes an scFv that recognizes BCMA. A modified cell obtained by introducing the polynucleotide provided by the invention into a host cell through a gene delivery vector has the capability of targeting BCMA < + > tumor cells, and can continuously express and secrete an NK cell adapter capable of targeting GPRC5D < + > tumor cells for a long time, so that an organism and adoptive immune cells can be fully mobilized, a plurality of TAAs can be targeted, and the tumor cell targeting capability is improved. Tumor immune escape is limited, and the tumor treatment efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a polynucleotide encoding an NK cell adapter and a CAR, and a modified cell comprising the polynucleotide encoding the NK cell adapter and the CAR. Background Art

[0002] Multiple myeloma (MM) accounts for 10% of the incidence of hematological malignancies. At present, there is still a lack of a safe and effective treatment method. Conventional chemotherapy can only temporarily relieve half of the patients; while chemotherapy combined with hematopoietic stem cell transplantation can significantly improve the remission rate, but its high recurrence rate remains a major problem. With the emergence of immunotherapies such as monoclonal antibodies, chimeric antigen receptor T cells (CAR-T), immune checkpoint inhibitors, and tumor vaccines, the prognosis of MM patients has been greatly improved. Although some progress has been made in the treatment of MM at present, it is still considered incurable, and the prognosis of relapsed and refractory MM patients is still not ideal. There is still a need to develop new therapeutic drugs and / or treatment methods to improve the clinical treatment effect of MM patients and change the current situation of MM being prone to recurrence and refractory.

[0003] As a branch of immune cell-directed therapy, cell adapters connect two different types of cells through a tumor-associated antigen (TAA) targeting moiety and an effector cell recognition moiety, and selectively attack and kill targeted tumor cells. According to the different cell types, it can be further divided into T cell adapters and NK cell adapters. T cell adapters connect T cells and tumor cells through TAA and TCR components (mainly CD3), avoiding MHC restriction. NK cell adapters target tumor cells and exert killing functions by targeting TAA and NK cell cytotoxic receptor components (including CD16, NKG2D, NKp44, etc.). Summary of the Invention

[0004] The first object of the present invention is to provide a polynucleotide encoding an NK cell adapter and a CAR.

[0005] Another object of the present invention is to provide a gene delivery vector comprising the above polynucleotide.

[0006] Still another object of the present invention is to provide a modified cell comprising the above gene delivery vector, and the modified cell can be used to prepare a drug for treating multiple myeloma.

[0007] According to one aspect of the present invention, there is provided a polynucleotide encoding an NK cell adapter and a CAR, wherein the polynucleotide comprises a first polynucleotide encoding an NK cell adapter, a linker, and a second polynucleotide encoding a CAR.

[0008] In some embodiments, the first polynucleotide encoding an NK cell adapter comprises a first signal peptide, a single-domain antibody against CD16, IL-15, and a single-chain variable fragment (scFv) recognizing G protein-coupled receptor class C group 5 member D (GPRC5D). The sequence of the first polynucleotide comprises a nucleotide sequence encoding the first signal peptide, a nucleotide sequence encoding the single-domain antibody against CD16, a nucleotide sequence encoding IL-15, and a nucleotide sequence encoding the scFv recognizing GPRC5D. Thus, after the polynucleotide is introduced into a host cell through a gene delivery vector, the host cell can continuously express and secrete an NK cell adapter that can target GPRC5D + on tumor cells, triggering an anti-tumor immune response of NK cells against GPRC5D + on tumor cells. At the same time, introducing IL-15 as a bispecific antibody crosslinker into the NK cell adapter can enhance the proliferation, activation, and cytotoxicity of NK cells, overcoming the non-specific mechanism of natural NK cell cytotoxicity.

[0009] In some embodiments, the first signal peptide is from or comprises a signal peptide of any secreted protein or membrane protein; for example, it can be selected from at least one of the ALB signal peptide, IL-2 signal peptide, GM-CSF signal peptide, immunoglobulin heavy chain signal peptide, and insulin signal peptide.

[0010] In some embodiments, the first signal peptide is the ALB signal peptide.

[0011] In some embodiments, the nucleotide sequence of the first polynucleotide encoding an NK cell adapter has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 1. The first polynucleotide encoding an NK cell adapter whose nucleotide sequence has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 1 can be obtained by at least one of the operations such as codon optimization based on the nucleotide sequence shown in SEQ ID NO: 1, reducing the CpG dinucleotide content, and removing any additional ORF in the sense and antisense directions, and has a nucleotide sequence with at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 1 and can normally encode an amino acid sequence such as the NK cell adapter shown in SEQ ID NO: 4.

[0012] In some embodiments, the second polynucleotide encoding the CAR comprises a scFv that recognizes BCMA. Thus, the polynucleotide-modified cells can directly target BCMA + to exert a killing function on tumor cells.

[0013] In some embodiments, the second polynucleotide encoding the CAR comprises a second signal peptide, a scFv that recognizes BCMA, a CD8α hinge, a CD137 (4-1BB) co-stimulatory domain, and a CD3ζ signaling domain.

[0014] In some embodiments, the second signal peptide is from or comprises a signal peptide of any secreted or membrane protein; for example, it can be selected from at least one of the CD8α signal peptide, the IL-2 signal peptide, and the GM-CSF signal peptide.

[0015] In some embodiments, the second signal peptide is the CD8α signal peptide.

[0016] In some embodiments, the second polynucleotide encoding the CAR may further comprise a Kozak sequence.

[0017] In some embodiments, the nucleotide sequence of the second polynucleotide encoding the CAR has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 2. The second polynucleotide encoding the CAR whose nucleotide sequence has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 2 can be obtained by at least one of operations such as codon optimization, reduction of CpG dinucleotide content, and removal of any additional ORF in the sense and antisense directions, and has a nucleotide sequence with at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 2 and can normally encode an amino acid sequence such as the CAR shown in SEQ ID NO: 5.

[0018] In some embodiments, the linker connecting the NK cell adapter and the CAR can be selected from at least one of P2A, T2A, and E2A.

[0019] In some embodiments, the linker connecting the NK cell adapter and the CAR is T2A.

[0020] In some embodiments, the nucleotide sequence of the polynucleotide has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 3. The polynucleotide whose nucleotide sequence has at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 3 can be obtained by performing at least one of the operations such as codon optimization based on the nucleotide sequence shown in SEQ ID NO: 3, reducing the CpG dinucleotide content, removing any additional ORF in the sense and antisense directions, etc., and has a nucleotide sequence with at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 3 and can normally encode a protein with an amino acid sequence such as that shown in SEQ ID NO: 6.

[0021] According to another aspect of the present invention, there is provided a gene delivery vector comprising the above polynucleotides encoding the NK cell adapter and the CAR.

[0022] In some embodiments, the gene delivery vectors that can introduce the polynucleotides encoding the NK cell adapter and the CAR provided by the present invention into host cells include, but are not limited to, viral vectors, transposons, gene knock-ins, liposomes, gold nanoparticles, exosomes, etc.

[0023] In some embodiments, the gene delivery vector is a viral vector, which includes, but is not limited to, lentiviral vectors, retroviral vectors, etc.

[0024] In some embodiments, the gene delivery vector can be a lentiviral vector, which is mainly prepared by packaging recombinant lentiviral expression plasmids, lentiviral packaging plasmids, and lentiviral envelope plasmids into recombinant lentiviruses.

[0025] In some embodiments, the recombinant lentiviral expression plasmid contains an EF1α promoter and the polynucleotides encoding the NK cell adapter and the CAR provided by the present invention.

[0026] In some embodiments, the lentiviral vector includes, but is not limited to, a three-plasmid lentiviral system and a four-plasmid lentiviral system.

[0027] In some embodiments, the lentiviral vector is prepared using a four-plasmid packaging system; it includes a recombinant lentiviral expression plasmid containing an EF1α promoter and the polynucleotides encoding the NK cell adapter and the CAR provided by the present invention, the lentiviral packaging plasmid pMDLg / pRRE (Kan + ), and pRSV-REV (Kan + ), and the lentiviral envelope plasmid pMD2.G (Kan + ).

[0028] In some embodiments, the preparation of the lentiviral vector includes the following steps: The recombinant lentiviral expression plasmid, lentiviral packaging plasmid, and lentiviral envelope plasmid are co-transfected into lentiviral packaging cells through a transfection reagent, and the lentivirus is packaged and purified to obtain the lentiviral vector.

[0029] In some embodiments, the transfection reagent can be polyetherimide (polysciences, PEI).

[0030] In some embodiments, the lentiviral packaging cells can be HEK293T cells.

[0031] According to another aspect of the present invention, a modified cell containing the above gene delivery vector is provided.

[0032] The modified cell provided by the present invention has the ability to target BCMA + tumor cells. At the same time, it can continuously express and secrete the NK cell adapter for a long time. The NK cell adapter endows NK cells with the ability to target GPRC5D + tumor cells through a bispecific antibody; thus, by combining CAR and the NK cell adapter, the body's own and adoptive immune cells are fully mobilized to target multiple TAAs, limit tumor immune escape, and effectively improve the treatment efficiency of tumors. The modified cell provided by the present invention can be applied to the preparation of drugs for the treatment of multiple myeloma.

[0033] In some embodiments, the host cell can be a prokaryotic cell or a eukaryotic cell; preferably, it can be selected from at least one of mammalian cells, insect cells, and yeast cells.

[0034] In some embodiments, the host cell can be a mammalian cell; among them, the mammalian cell can be selected from at least one of epithelial cells, myeloblasts, fibroblasts, and lymphocytes.

[0035] In some embodiments, the host cell can be a lymphocyte, and among them, the lymphocyte can be a T cell, a B cell, or an NK cell.

[0036] In some embodiments, the modified cell can be prepared by introducing the lentiviral vector into T cells. The specific preparation method can include the following steps: The T cells are infected with the lentiviral vector and polybrene. After 24 h, the medium is replaced, and the cells are harvested after continued culture for 72 - 216 h to obtain the modified cells; among them, the MOI of the lentiviral vector can be 1 - 100, and the final concentration of polybrene can be 1 - 10 μg / mL. Brief Description of the Drawings

[0037] Figure 1Detection results of PCR amplification products of BamH I-EF1α-Xba I fragment by agarose gel electrophoresis; among them, M is 1kb-I DNA marker, and lane 1 and lane 2 are BamH I-EF1α-Xba I fragments; Figure 2 Detection results of PCR amplification products of Xba I-BCMA-BB-Mlu I fragment by agarose gel electrophoresis; among them, M is 1kb-I DNA marker, and lane 1 and lane 2 are Xba I-BCMA-BB-Mlu I fragments; Figure 3 Detection results of PCR amplification products of Mlu I-T2a-engager-Sal I fragment by agarose gel electrophoresis; among them, M is 1kb-I DNA marker, and lane 1 and lane 2 are Mlu I-T2a-engager-Sal I fragments; Figure 4 Plasmid map of pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager recombinant lentiviral expression plasmid; Figure 5 Detection results of digestion products of BCMA-BB in pRRLSIN by agarose gel electrophoresis; among them, M is 1kb-I DNA marker, and lane 1 is the digestion fragment of BCMA-BB in pRRLSIN; Figure 6 Colony PCR identification of pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager recombinant lentiviral expression plasmid; Figure 7 Plasmid map of pRRLSIN.EF1α.CAR(anti-BCMA) recombinant lentiviral expression plasmid; Figure 8 Detection results of engager expression in cell culture supernatant at different time points after lentivirus infection by western blotting; Figure 9 Plasmid map of pRRLSIN.EF1α.CAR(5E5).Kan plasmid; Figure 10 Positive rate of CAR expression in CAR-T cells co-expressing NK cell adapter detected by flow cytometry; Figure 11 For the detection of CD3 - CD56 + Proportion of cells; Figure 12 and 13 is the proportion of GFP + 7-AAD + positive cells in each group of cells; Figure 14 is the concentration of IFN-γ in the cell culture supernatant of each group. Detailed implementation manners

[0038] The present invention will be further described in detail below in conjunction with the implementation manners. The examples are only for explanation and do not limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods without specific conditions noted in the examples are usually carried out under conventional conditions in the art or according to the conditions recommended by the manufacturers.

[0039] Example 1 Preparation of CAR gene-modified cells co-expressing NK cell adaptor 1. Construction of recombinant lentiviral expression plasmid (1) The CAR(anti-BCMA) gene (nucleotide sequence as shown in SEQ ID NO: 2) was artificially synthesized by General Biosystems (Anhui) Co., Ltd. and cloned between the Xba I and Mlu I restriction enzyme sites of the pRRLSIN plasmid (provided by Guangzhou Anjie Biomedical Technology Co., Ltd.), and the constructed vector was named BCMA-BB in pRRLSIN.

[0040] (2) The T2A-engager gene (containing the T2A sequence and the nucleotide sequence shown in SEQ ID NO: 1, specific sequence as shown in SEQ ID NO: 7) was artificially synthesized by General Biosystems (Anhui) Co., Ltd. and cloned between the Mlu I and Sal I restriction enzyme sites of the pGEM-4Z plasmid (provided by Guangzhou Anjie Biomedical Technology Co., Ltd.), and the constructed vector was named T2a-engager in pGEM.

[0041] (3) Using pRRLSIN.EF1α.CAR(5E5).Kan (provided by Guangzhou Anjie Biomedical Technology Co., Ltd., its map is as Figure 9 shown) as a template, primers were designed and the EF1α sequence was amplified by PCR; among them, the primer sequences are as follows: EF1α-F(BamH I): 5’-CGGGATCCGCTCCGGTGCCCGTCAGT-3’ (SEQ ID NO: 8) EF1α-R(Xba I): 5’-CTAGTCTAGATCACGACACCTGAAATGGAAG-3’ (SEQ ID NO: 9) PCR reaction parameters: Pre-denaturation: 94°C, 2 min; Denaturation: 94°C, 15 s; Annealing: 55°C, 30 s; Extension: 68°C, 70 s; 32 cycles; Extension at 68°C for 8 min; 4°C, forever.

[0042] The PCR amplification products of the BamH I-EF1α-Xba I fragment were identified and separated by 1% agarose gel electrophoresis. The identification results are as Figure 1 shown, where lane 1 and lane 2 are the BamH I-EF1α-Xba I fragment, with a size of approximately 1.1 kb.

[0043] The BamH I-EF1α-Xba I amplified fragment was recovered and purified using a DNA gel extraction kit.

[0044] (4)Using BCMA-BB in pRRLSIN as a template, primers were designed and the BCMA-BB sequence was amplified by PCR; among them, the primer sequences are as follows: Xba I-BCMA-BB F: 5’-CTAGTCTAGAGCCACCATGGCCTTACCAGT-3’ (SEQ ID NO: 10) BCMA-BB-Mlu I R: 5’-CGACGCGTGCGAGGGGGCAGGGCCTGCAT-3’ (SEQ ID NO: 11) PCR reaction parameters: Pre-denaturation: 94°C, 2 min; Denaturation: 94°C, 15 s; Annealing: 55°C, 30 s; Extension: 68°C, 100 s; 32 cycles; Extension at 68°C for 8 min; 4°C, forever.

[0045] The PCR amplification products of the Xba I-BCMA-BB-Mlu I fragment were identified and separated by 1% agarose gel electrophoresis. The identification results are as Figure 2 shown, where lane 1 and lane 2 are the Xba I-BCMA-BB-Mlu I fragment, with a size of approximately 1.5 kb.

[0046] The Xba I-BCMA-BB-Mlu I amplified fragment was recovered and purified using a DNA gel extraction kit.

[0047] (5)Using T2a-engager in pGEM as a template, primers were designed and the T2a-engager sequence was amplified by PCR; among them, the primer sequences are as follows: Mlu I-cam16 F: 5’-CGACGCGTGGAAGCGGAGAGGGCAGA-3’ (SEQ ID NO: 12) GPRC5D-Sal I R: 5’-TGCGGTCGACTCACTTGATCTCCAGCTTGGT-3’ (SEQ ID NO: 13) PCR reaction parameters: Pre-denaturation: 94°C, 2 min; Denaturation: 94°C, 15 s; Annealing: 55°C, 30 s; Extension: 68°C, 100 s; Cycle 32 times; Extension at 68°C for 5 min; 4°C, forever.

[0048] Identify and separate the PCR amplification product of the Mlu I-T2a-engager-Sal I fragment by 1% agarose gel electrophoresis. The identification results are as Figure 3 shown, where lane 1 and lane 2 are the Mlu I-T2a-engager-Sal I fragments, with a size of approximately 1.5 kb.

[0049] Recover and purify the Mlu I-T2a-engager-Sal I amplification fragment using a DNA gel extraction kit.

[0050] (6) Insert the EF1α sequence, BCMA-BB sequence, and T2a-engager sequence between the BamH I and Sal I restriction sites of BCMA-BB in pRRLSIN to construct a recombinant lentiviral expression plasmid, named pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager plasmid. Its plasmid map is as Figure 4 shown. The specific process is as follows: S1: Double-digest the BamH I-EF1α-Xba I amplification fragment with BamH I and Xba I, double-digest the Xba I-BCMA-BB-Mlu I amplification fragment with Xba I and Mlu I, and double-digest the Mlu I-T2a-engager-Sal I amplification fragment with MluI and SalI. The digested products are directly recovered; S2: Double-digest the vector BCMA-BB in pRRLSIN with BamH I and Sal I. After identifying the digested product by 1% agarose gel electrophoresis, cut the gel and recover it. The identification results are as Figure 5 shown, where lane 1 is the digested fragment of the vector BCMA-BB in pRRLSIN, with a size of approximately 6.0 kb; S3: The recovered digested products were ligated with T4 DNA ligase. The ligation products were transformed into TransStbl3 chemically competent cells (provided by Beijing Quanshijin Biotechnology Co., Ltd.) and streaked on plates for culture. S4: Twelve single colonies were picked and identified by colony PCR using EF1α-F (BamHI) / EF1α-R (XbaI) as primers. Colony PCR reaction parameters: pre-denaturation: 94°C, 5 min; denaturation: 94°C, 30 s; annealing: 55°C, 30 s; extension: 72°C, 70 s; 32 cycles; extension at 72°C for 10 min; 4°C, forever.

[0051] The results were as Figure 6 shown. A target band was amplified in lane 10 of the clone and the band was obvious, indicating that it might be a positive clone. S5: The positive clone was cultured in a shaker and the plasmid was extracted using a UE plasmid miniprep kit (Suzhou Youyiland Biotech Co., Ltd.). The plasmid was sequenced, and the sequencing primers were EF1α-F (BamHI) / WPRE R. The sequencing results showed that the sequences were all correct, indicating that the recombinant lentiviral expression plasmid had been successfully constructed and named pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager plasmid. Among them, the sequence of primer WPRE R was as follows: 5’-CATAGCGTAAAAGGAGCAACA-3’ (SEQ ID NO: 14) S6: The plasmid was extracted using an endo-free plasmid maxi kit (Endo-free Plasmid Maxi Kit, Omega) and stored in a -20°C refrigerator.

[0052] 2. Lentivirus packaging and purification (1) The cryopreserved HEK293T cells were revived and passaged with DMEM complete medium (DMEM medium + 10% FBS). The HEK293T cells were seeded into a 10-layer cell factory, DMEM complete medium was added, and the cells were cultured overnight to reach a confluence of 80 - 90% for plasmid transfection.

[0053] (2) The pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager plasmid, the lentiviral packaging plasmid pMDLg / pRRE (Kan + ), and pRSV-REV (Kan + ), and the lentiviral envelope plasmid pMD2.G (Kan + ) were added to serum-free DMEM, mixed well and left standing for 5 min to obtain a plasmid mixture. PEI was added to serum-free DMEM, mixed well and left standing for 5 min to obtain a transfection reagent. Add the transfection reagent to the plasmid mixture, mix well, and let stand for 20 min to form a DNA-PEI complex; (3) Add the DNA-PEI complex to 1 L of DMEM medium containing 5% FBS, mix well, replace the culture medium in the 10-layer cell factory in step (1), and culture in an incubator at 37 °C and 5% CO2; (4) Collect the culture supernatant at 48 h and 72 h after transfection and store it in a refrigerator at 2-8 °C; (5) Mix the collected culture supernatants and use a capsule filter (Sartorius) to remove cells and cell debris; then concentrate the clarified and filtered lentivirus supernatant 10-15 times through a Sartorius tangential flow filtration system (KROSFLO ® KR2I). After filtering the concentrated solution through a 0.45 μm filter membrane, perform chromatography purification; filter the purified lentivirus through a 0.22 μm filter (Sartorius) to remove bacteria, aliquot, and store in a refrigerator at -80 °C.

[0054] The obtained lentivirus is the pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager lentiviral expression vector, denoted as pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager lentivirus.

[0055] 3. CAR gene-modified cells co-expressing NK cell adaptors (1) Transfer the collected blood sample to a centrifuge tube, centrifuge at 1100 g for 10 min, collect the lower-layer cells and dilute them with 0.9% saline. Add the diluted cells on top of the lymphocyte separation medium and centrifuge at 800 g for 20 min. Aspirate the "buffy coat" to obtain PBMC. Wash the cells with 0.9% saline and count. Take the required amount of magnetic beads, vortex for 30 s to resuspend the magnetic beads; add PBS to wash the magnetic beads and discard the supernatant. Resuspend the magnetic beads and cell mixture with T cell expansion medium (KBM581 medium containing 1000 IU / mL IL2) and adjust the cell density to 1.0×10 6 cells / mL and culture in an incubator at 37 °C and 5% CO2.

[0056] (2) After culturing for 48 h, add pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager lentivirus (MOI = 10) and polybrene (final concentration 6 μg / mL), centrifuge for infection (500 g, 1.5 h), and then culture in an incubator at 37 °C and 5% CO2.

[0057] (3)After 24 h of lentivirus infection, change the medium, add KBM581 medium containing 1000 IU / mL IL2, and continue to amplify and culture in an incubator at 37 °C and 5% CO2.

[0058] (4)Harvest the cells 72 h after lentivirus infection. Among them, the harvested cells are CAR gene-modified cells co-expressing the NK cell adaptor.

[0059] Experimental Example 1 (1)Refer to the method of Example 1 to prepare a recombinant lentiviral expression plasmid without the T2a-engager sequence, named pRRLSIN.EF1α.CAR(anti-BCMA) plasmid. Its plasmid map is as Figure 7 shown. Co-transfect HEK293T cells with the pRRLSIN.EF1α.CAR(anti-BCMA) plasmid, lentiviral packaging plasmid, and lentiviral envelope plasmid, package the lentivirus and purify it. The obtained lentivirus is the pRRLSIN.EF1α.CAR(anti-BCMA) lentiviral expression vector, denoted as pRRLSIN.EF1α.CAR(anti-BCMA) lentivirus.

[0060] (2)Refer to the method of Example 1 to infect T cells with pRRLSIN.EF1α.CAR(anti-BCMA) lentivirus and pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager lentivirus respectively. After 24 h of lentivirus infection, change the medium, add KBM581 medium containing 1000 IU / mL IL2, and continue to amplify and culture in an incubator at 37 °C and 5% CO2. Collect the culture supernatants at 5 d, 7 d, and 9 d after lentivirus infection respectively.

[0061] (3)Detect the expression of engager (i.e., NK cell adaptor) at different times after lentivirus infection by western blotting.

[0062] The results are as Figure 8As shown, M is the protein marker (in the figure, the 70kD band is not shown). Lane 1-3 are the detection results of the expression of engager in the culture supernatants of T cells infected with lentivirus at 5d, 7d, and 9d respectively. Among them, the virus infected in the upper row is the pRRLSIN.EF1α.CAR(anti-BCMA) lentivirus, and the virus infected in the lower row is the pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager lentivirus. Lane 4 is the detection result of the expression of engager in the culture supernatant of normal T cells. The results show that after infecting with the pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager lentivirus, bands of the expected size can be detected in cells at all time points, and the protein expression level increases with time, indicating that the CAR gene-modified cells co-expressing the NK cell engager of the present invention can express the NK cell engager and continuously secrete it extracellularly, which plays a very important role in fully mobilizing the body's immune response.

[0063] Experimental Example 2 Anti-tumor effect of CAR-T cells co-expressing NK cell engager in vitro 1. Preparation of CAR-T cells co-expressing NK cell engager Refer to the method in Example 1 to prepare CAR-T cells co-expressing NK cell engager. The steps are as follows: (1) Take monocytes, adjust the cell density to 1.5×10 6 cells / mL, use KBM581 medium containing 1000 IU / mL IL2 and 5% - 10% plasma, add CD3 / CD28 magnetic beads for activation, and place them in a 37°C, 5% CO2 incubator for culture.

[0064] (2) After activation for 24 - 96 h, collect T cells, wash the cells twice and count, adjust the cell density to 1×10 6 cells / mL, add pRRLSIN.EF1α.CAR(anti-BCMA)-T2a-engager lentivirus (MOI = 50) and transfection enhancer polybrene (8 μg / mL). At the same time, set up a group of T cells without adding lentivirus as a control. After centrifugal infection (500g, 60 min), place the cells in a 37°C, 5% CO2 incubator for culture.

[0065] (3) Replace the medium 24 h after infection, add KBM581 medium containing 1000 IU / mL IL2, and continue to culture the cells in a 37°C, 5% CO2 incubator.

[0066] On days 3 to 7 after infection, cells were labeled successively with Biotin-Goat Anti-Mouse IgG, F(ab')2 (purchased from Jackson Immunoresearch, catalog number 115-065-072) and PE-Streptavidin (purchased from BD Biosciences, catalog number 554061), and then the positive rate of CAR was detected by flow cytometry.

[0067] The results were as Figure 10 shown. Cells were collected on day 3 after infection, and the expression of CAR on CAR-T cells was detected by flow cytometry, and the positive rate reached about 50%, indicating the successful preparation of CAR gene-modified CAR-T cells co-expressing NK cell adaptors.

[0068] 2. Preparation of NK cells Culture flasks were pre-coated with anti-human CD16 monoclonal antibody overnight. Mononuclear cells were taken and CD56 + cells were enriched by CD56 magnetic bead sorting. The cell density was adjusted to 1.5×10 6 cells / mL, and the sorted CD56 + cells were cultured using KBM581 medium containing 1000 IU / mL IL2, 60 ng / mL IL15, and 5% - 10% plasma. The cells were seeded into the pre-coated culture flasks and cultured in an incubator at 37°C and 5% CO2. The purity of NK cells was detected by flow cytometry on days 7 to 10 after culture.

[0069] The results were as Figure 11 shown. The proportion of CD3 - CD56 + cells in NK cells reached 97.2% after 10 days of culture.

[0070] 3. In vitro killing effect of CAR-T cells co-expressing NK cell adaptors on RPMI8226-Luc-GFP cells The killing effect of CAR gene-modified human immune cells co-expressing NK cell adaptors on human multiple myeloma cells RPMI8226-Luc-GFP double-labeled with Luciferase and GFP was studied. The steps were as follows: (1) Using RPMI8226-Luc-GFP cells as target cells, the cells were centrifuged and washed 3 times with RPMI 1640 culture medium at 300g for 5 min; the prepared T cells, CAR-T cells co-expressing NK cell adaptors (CAR-T), and NK cells were collected as effector cells, and the cells were centrifuged and washed 3 times with RPMI 1640 culture medium at 300g for 5 min.

[0071] (2) Resuspend effector cells and target cells with RPMI 1640 culture medium, count them respectively, prepare co-incubation wells with an effector-to-target ratio of 1. The groups are as follows: target cell group, target cell + T, target cell + NK, target cell + T + NK, target cell + CAR-T, target cell + CAR-T + NK. Additionally, set up control wells for compensation adjustment.

[0072] (3) Gently tap and mix the cell culture plate, and place it in an incubator at 37°C with 5% CO2. After 15 h, take out the cell culture plate from the incubator, collect the cells in each well, and make a single-cell suspension. Centrifuge and wash the cells in each well 3 times with PBS at 300g for 5 min.

[0073] (4) Add 5 μL of 0.05 mg / mL 7-AAD (7-Amino-Actinomycin D) to the cell wells and incubate in the dark at room temperature for 5 min. Detect the proportion of GFP + 7-AAD + cells in each group by flow cytometry.

[0074] The results are as Figure 12 and 13 shown. Compared with the T cell group, NK cell group, and T + NK cell group, the proportion of GFP + 7-AAD + cells in the CAR-T cell group and CAR-T + NK cell group increased, indicating an increased killing effect on target cells RPMI8226-Luc-GFP. Compared with the CAR-T cell group, the proportion of GFP + 7-AAD + cells in the CAR-T + NK cell group was further increased. Compared with T and NK alone, T + NK did not increase the proportion of GFP + 7-AAD + cells, indicating that co-expression of the NK cell adaptor can increase the killing of target cells by CAR gene-modified immune cells through NK-mediated cytotoxicity.

[0075] (5) Collect the culture supernatant of the co-incubation wells and detect the concentration of IFN-γ by ELISA.

[0076] The results are as Figure 14 shown. The IFN-γ release levels in the CAR-T cell group and CAR-T + NK cell group were higher than those in other groups, and the CAR-T + NK cell group was higher than the CAR-T group, which was consistent with the cell killing results.

[0077] The above results indicate that CAR-T cells targeting BCMA have significant anti-tumor effects on multiple myeloma cells, and the co-expressed NK cell adaptor can enhance the anti-tumor effects of CAR gene modification through NK-mediated cytotoxicity.

[0078] The above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A polynucleotide encoding an NK cell adaptor and a CAR, characterized in that, The polynucleotide comprises a first polynucleotide encoding an NK cell adapter, a linker, and a second polynucleotide encoding a CAR; wherein the nucleotide sequence of the first polynucleotide encoding the NK cell adapter is as shown in SEQ ID NO: 1; the nucleotide sequence of the second polynucleotide encoding the CAR is as shown in SEQ ID NO:

2.

2. The polynucleotide encoding the NK cell adaptor and CAR according to claim 1, wherein, The linker is selected from at least one of P2A, T2A, and E2A.

3. The polynucleotide encoding the NK cell adaptor and the CAR according to claim 2, wherein, The linker is T2A, and the nucleotide sequence of the polynucleotide encoding the NK cell adapter and the CAR is as shown in SEQ ID NO:

3.

4. A gene delivery vector comprising the polynucleotide encoding the NK cell adapter and the CAR according to any one of claims 1 to 3.

5. The gene delivery vector according to claim 4, characterized in that, The gene delivery vector is selected from at least one of a viral vector, a transposon, gene knock-in, liposome, gold nanoparticle, and exosome.

6. The gene delivery vector according to claim 5, wherein The gene delivery vector is a lentiviral vector, which is mainly prepared by packaging a recombinant lentiviral expression plasmid, a lentiviral packaging plasmid, and a lentiviral envelope plasmid into recombinant lentivirus; the recombinant lentiviral expression plasmid contains an EF1α promoter and the polynucleotide encoding the NK cell adapter and the CAR according to any one of claims 1 to 3.

7. A modified cell comprising the gene delivery vector according to any one of claims 4 to 6.

8. The modified cell according to claim 7, wherein The host cell of the modified cell is selected from at least one of mammalian cells, insect cells, and yeast cells.

9. Use of the modified cell according to claim 7 or 8 in the preparation of a medicament for treating multiple myeloma.

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