Dual-targeting chimeric antigen receptor, gene encoding same, recombinant expression vector, natural killer cell, and use

Abstract

The present invention relates to the technical field of tumor immunotherapy. Provided are a dual-targeting chimeric antigen receptor, a gene encoding same, a recombinant expression vector, a natural killer cell, and the use. The dual-targeting chimeric antigen receptor comprises a dual CAR molecule encoding sequence, which consists of the following sequentially linked sequences: an extracellular antigen-binding domain encoding sequence, a transmembrane domain encoding sequence, and an intracellular signal transduction domain encoding sequence. The extracellular domain of a PD1 molecule serves as a first recognition target, and the extracellular domain of an NKG2D molecule serves as a second recognition target. When the extracellular signal domain of PD1 recognizes a PD-L1 molecule on a tumor cell, the activation of the DAP10 activating receptor pathway is triggered by downstream transduction of a negative regulatory signal, thereby converting the negative regulatory signal into a positive activating signal. Meanwhile, when the extracellular domain of the NKG2D molecule recognizes an NKG2DLs molecule on the tumor cell, the NK cell can also be activated, and under the synergistic action of co-stimulatory molecules 41BB and CD3 zeta, the antitumor treatment effect of the NK cell is greatly enhanced.

Description

A dual-targeting chimeric antigen receptor and its encoding gene, recombinant expression vector, natural killer cells, and applications Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, specifically a dual-targeting chimeric antigen receptor and its encoding gene, recombinant expression vector, natural killer cells, and their applications. Background Technology

[0002] Tumor immunotherapy can effectively overcome the mechanisms by which tumors escape from the immune system, thereby reawakening immune cells to eliminate tumor cells. Due to its fewer side effects and significant therapeutic effects, it is gradually becoming the future direction of cancer treatment and is considered the fourth major cancer treatment technology after surgery, radiotherapy, and chemotherapy.

[0003] Chimeric antigen receptors (CARs) are a crucial technology in cancer treatment. With continuous advancements in CAR technology, tumor immunotherapy is developing at an unprecedented pace, offering new hope for cancer patients. The design and application of CAR structures have provided entirely new approaches to cancer treatment. T cells were the first immune cells modified with CARs to specifically target tumor cells, and clinical results have shown that CAR-T cells have significant advantages in treating hematologic malignancies. However, as CAR-T technology has developed, its efficacy has become increasingly limited by the heterogeneity of tumor antigens, often leading to tumor recurrence. Therefore, addressing postoperative recurrence has been a key focus, leading to a recent shift towards CAR-NK cell therapy.

[0004] In recent years, with increasing attention paid to the unique characteristics and specific cytotoxicity of natural killer (NK) cells, there has been growing interest in developing CAR-NK cells for cancer therapy. Compared to CAR-T cells, CAR-NK cells offer several significant advantages, including: (1) better safety, such as less cytokine release syndrome and neurotoxicity, and less graft-versus-host disease in allogeneic settings; (2) activation of cytotoxic activity through multiple mechanisms; and (3) high feasibility of “off-the-shelf” manufacturing. CAR-NK cells can be engineered to target multiple antigens, enhance in vivo proliferation and persistence, increase infiltration into solid tumors, overcome drug-resistant tumor microenvironments, and ultimately achieve effective antitumor responses. NK cells appear to be an attractive vector for CAR expression because they are available from multiple sources and can be safely infused regardless of donor-patient matching, which significantly reduces the cost of treatment. CAR-NK cells are known to be effective against hematologic malignancies, but a growing body of preclinical findings suggests they remain active against non-hematologic malignancies.

[0005] Although the advantages of CAR-NK cell therapy over CAR-T cell therapy are obvious, there are still cases where patients are prone to developing drug resistance or tumor recurrence due to antigen escape. Therefore, researching chimeric antigen receptors that target two antigens to prevent antigen escape has become an urgent problem to be solved. Summary of the Invention

[0006] Immune checkpoints are a class of immunosuppressive molecules that regulate immune cell activity through a series of co-inhibitory or co-stimulatory signaling pathways, preventing excessive immune activation and ensuring self-tolerance, thereby avoiding damage and destruction of normal tissues. PD1 is one of the major inhibitory molecules. The PD1 / PD-L1-mediated negative immune regulation mechanism can inhibit the activation of the MHC-TCR signaling pathway, controlling the proliferation of T cells and NK cells, cytokine secretion, or responses to homologous antigens. PD1 is mainly expressed on the surface of activated immune cells. Its ligand PD-L1 has limited expression in normal tissues, but its expression is significantly increased in most solid tumor cells and immunosuppressive cells. Tumors utilize the PD1 / PD-L1 negative regulation mechanism to escape the anti-tumor immune response. Therefore, blocking this immunosuppressive signaling pathway has become an important approach in the immunotherapy of solid tumors. NKG2DLs are diverse and exist on various tumor cells. It has been reported that high expression of MICA / B is often detected in liver cancer, ovarian cancer, pancreatic cancer, colon cancer, and breast cancer, while NKG2DLs are rarely expressed in healthy human cells. Therefore, in the process of cell carcinogenesis, NK cells, which perform immune surveillance in the immune system, can effectively recognize and bind to these ligands through their own NKG2D receptors, thereby triggering the activation effect of NK cells and effectively eliminating various tumor cells.

[0007] In this invention, we synthesized a PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fusion receptor using whole-genome synthesis technology. This fusion receptor retains only the extracellular domain of the PD1 molecule as the primary recognition target, replacing the intracellular inhibitory signaling domain of the PD1 receptor with the transmembrane and intracellular segments of DAP10 and the intracellular segment of the co-stimulatory molecule 41BB. This chimeric molecular module acts as a signal switching "switch," thereby reversing the negative regulatory mechanism of PD1 / PD-L1 immunity. Simultaneously, the extracellular domain of the NKG2D molecule was selected as the secondary recognition target, and the transmembrane and intracellular segments of CD28, the intracellular segment of 41BB, and CD3ζ were used as co-stimulatory molecules. When the extracellular signaling domain of PD1 recognizes PD-L1 molecules on tumor cells, the negative regulatory signal is transmitted downstream, triggering the activation of the DAP10 activating receptor pathway, transforming it into a positive activation signal. At the same time, when the extracellular domain of NKG2D molecules recognizes NKG2DLs molecules on tumor cells, it can also activate NK cells, and under the synergistic effect of co-stimulatory molecules 41BB and CD3ζ, it can greatly enhance the anti-tumor effect of NK cells.

[0008] A dual-targeting chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO.3. This dual-targeting chimeric antigen receptor is divided into two segments: a first receptor comprising an extracellular PD1 segment, an extracellular transmembrane and intracellular DAP10 segment, and the co-stimulatory molecule CD137; and a second receptor comprising an extracellular NKG2D segment, IgG4 as a hinge region, a transmembrane and intracellular CD28 segment, CD137, and CD3ζ. Both segments can rapidly bind intracellularly to form a dual-targeting receptor. Utilizing this chimeric molecular module, on the one hand, the NKG2D binds to MICA / MICB on the tumor surface, and PD1 binds to PD-L1 on tumor cells, fully leveraging its function of targeting tumor cells; on the other hand, the combined action of NKG2D and DAP10 enhances the activation effect of NK92 cells.

[0009] Preferably, the coding gene for the dual-target chimeric antigen receptor is initiated by a single CMV promoter, with the first and second receptors linked by a P2A peptide, thus avoiding the uneven expression of target proteins that can occur with dual promoters. This nucleotide sequence conforms to codon bias in the human body, enabling better expression in vivo.

[0010] Preferably, in the target PD1 and NKG2D of the two receptors, the signal peptide region (or extracellular signal peptide structure) has a signal peptide sequence related to protein secretion, which can be selected from type I transmembrane protein signal peptides such as CD8α, PD1, DAP10, DNAM-1, and CD137 (or 4-1BB); more preferably, it is the CD8α signal peptide. The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.1, and the nucleotide sequence encoding it is shown in SEQ ID NO.14.

[0011] Preferably, in the chimeric receptor targeting the NKG2D ligand, the hinge region is preferably the monomeric IgG4 hinge region (i.e., the IgG4 molecule). The amino acid sequence of the IgG4 molecule is shown in SEQ ID NO.8, and the nucleotide sequence encoding it is shown in SEQ ID NO.21.

[0012] Preferably, in the chimeric receptor targeting the NKG2D ligand, a Flag tag sequence is further connected between the signal peptide region and the NKG2D ligand-binding domain. The amino acid sequence of the Flag tag is shown in SEQ ID NO. 6, and the nucleotide sequence encoding it is shown in SEQ ID NO. 19. In the chimeric receptor targeting the PD-L1 ligand, a MYC tag is further connected between the signal peptide region and the PD-L1 ligand-binding domain. The amino acid sequence of the MYC tag is shown in SEQ ID NO. 2, and the nucleotide sequence encoding it is shown in SEQ ID NO. 15.

[0013] The present invention comprises a recombinant expression vector encoding a dual-target chimeric antigen receptor gene, wherein the recombinant expression vector contains the dual-target chimeric antigen receptor encoding gene, and the amino acid sequence of the dual-target chimeric antigen receptor is shown in SEQ ID NO.13.

[0014] The above-mentioned method for preparing recombinant expression vectors includes the following steps:

[0015] 1) Synthesize the dual-targeting chimeric antigen receptor encoding gene;

[0016] 2) The dual-target chimeric antigen receptor encoding gene is ligated into a lentiviral expression vector to obtain the final product.

[0017] A highly cytotoxic antitumor NK cell, which is an NK cell modified with a dual-targeting chimeric antigen receptor, the amino acid sequence of which is shown in SEQ ID NO.13.

[0018] The anti-tumor NK cells of this invention have extremely strong cytotoxicity. Compared with the targeting effect of CAR-NK against a specific antigen, PN-CAR-NK92 cells recognize tumor cells through the NKG2D / NKG2DL and PD1 / PD-L1 pathways. Since most tumors express MICA, MICB, and PD-L1, the targeting effect of PN-CAR-NK92 on tumor cells is more broad-spectrum, rather than being limited to one or a few specific antigen targets.

[0019] The above-mentioned method for preparing anti-tumor NK cells includes the following steps:

[0020] 1) Synthesize the dual-targeting chimeric antigen receptor encoding gene;

[0021] 2) The dual-targeting chimeric antigen receptor encoding gene is ligated into a lentiviral expression vector to obtain a recombinant lentiviral expression vector;

[0022] 3) Package the recombinant lentiviral expression vector to express the virus carrying the target plasmid;

[0023] 4) Infect NK92 cells with the virus carrying the target plasmid to obtain the final product.

[0024] The anti-tumor NK cells of this invention can be allogeneically reinfused with minimal damage to normal cells, do not elicit an excessive immune response, and can precisely identify and specifically kill solid tumor cells, exhibiting stronger cytotoxicity and better therapeutic effects on tumors. Attached Figure Description

[0025] Figure 1 is a schematic diagram of the structure of the recombinant lentiviral vector CMV-PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ-pCDH containing the dual-targeting chimeric antigen receptor encoding gene of the present invention.

[0026] Figure 2 is a schematic diagram of the mRNA expression levels of PD1 and NKG2D in PN-CAR-NK92 cells after lentivirus transfection with NK92 in Example 3 of the present invention.

[0027] Figure 3 is a schematic diagram of flow cytometry detection of PD1 and NKG2D in PN-CAR-NK92 cells after lentivirus transfection with NK92 in Example 4 of the present invention.

[0028] Figure 4 is a schematic diagram of flow cytometry detection of PD-L1 and MICA / MICB in EMT6-PD-L1 and MGC-803 tumor cells in Example 5 of the present invention.

[0029] Figure 5 shows the killing efficiency of NK92, single-module PD1-CAR-NK92, NKG2D-CAR-NK92, and dual-module PN-CAR-NK92 cells against EMT6-PD-L1 and MGC-803 tumor cells in Example 6 of the present invention.

[0030] Figure 6 is a schematic diagram of PD-L1 and MICA / MICB flow cytometry detection in KATO-III and U2OS tumor cells in Example 5 of the present invention.

[0031] Figure 7 shows the killing efficiency of NK92 and PN-CAR-NK92 cells against KATO-III and U2OS tumor cells in Example 6 of the present invention.

[0032] Figure 8 is a schematic diagram of the rational design of PN-CAR-NK92 in this invention and its anti-tumor effect on modified NK cells. Detailed Implementation

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0034] The extracellular recognition binding region described in this invention recognizes any tumor-specific or related antigens, including PD1 ligands (PD-L1, PD-L2) and NKG2D ligands (MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, etc.).

[0035] Example 1

[0036] A dual-targeting chimeric antigen receptor is composed of PD1-DAP10-CD137 receptor and NKG2D-IgG4-CD28-CD137-CD3ζ receptor.

[0037] The PD1-DAP10-CD137 receptor consists of the CD8α signal peptide, the Myc tag, the extracellular domain of PD1, the transmembrane and intracellular domains of DAP10, and the intracellular domain of CD137.

[0038] The amino acid sequence of the CD8α signal peptide is: MALPVTALLLPLALLLHAARP, denoted as SEQ ID NO.1.

[0039] The amino acid sequence of the Myc tag is: EQKLISEEDL, denoted as SEQ ID NO.2.

[0040] The amino acid sequence of the extracellular segment of PD1 is: FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV, denoted as SEQ ID NO.3.

[0041] The transmembrane and intracellular amino acid sequence of DAP10 is: QTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG, denoted as SEQ ID NO.4.

[0042] The amino acid sequence of the intracellular segment of CD137 is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, denoted as SEQ ID NO.5.

[0043] The NKG2D-IgG4-CD28-CD137-CD3ζ receptor consists of a CD8α signal peptide, a FLAG tag, an NKG2D ligand-binding domain, an IgG4 monomeric hinge region, a CD28 transmembrane and intracellular region, a CD137 intracellular region, and a CD3ζ intracellular domain.

[0044] The amino acid sequence of the FLAG tag is: DYKDDDDK, denoted as SEQ ID NO.6.

[0045] The amino acid sequence of the NKG2D ligand-binding domain is: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV, denoted as SEQ ID NO.7.

[0046] The amino acid sequence of the hinge region of the IgG4 monomer is: ESKYGPPAPPAP, denoted as SEQ ID NO.8.

[0047] The amino acid sequence of CD28 transmembrane and intracellular regions is: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS, denoted as SEQ ID NO.9.

[0048] The amino acid sequence of the CD3ζ intracellular domain is: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, denoted as SEQ ID NO.11.

[0049] The PD1-DAP10-CD137 receptor is prepared according to the following steps:

[0050] The first receptor was synthesized using a whole-genome synthesis method, consisting of a nucleotide fragment containing the extracellular segment (1-147aa) of PD1, the extracellular, transmembrane and intracellular segments (19-93aa) of DAP10, and the intracellular (214-255aa) nucleotide fragment of CD137 (also known as 4-1BB).

[0051] The NKG2D-IgG4-CD28-CD137-CD3ζ receptor was prepared according to the following steps:

[0052] A second receptor was synthesized using a whole-genome synthesis method. The receptor consists of the NKG2D extracellular domain sequence, which is sequentially linked to the IgG4 hinge region sequence, the CD28 transmembrane region sequence (153-220aa), the CD137 intracellular domain, and the CD3ζ intracellular domain.

[0053] The dual-target chimeric antigen receptor is prepared according to the following steps:

[0054] By synthesizing the entire genome, the nucleotide fragments of the first receptor and the second receptor are linked together in series and connected by the self-splitting peptide P2A to obtain the dual-targeting chimeric antigen receptor, namely the PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fragment.

[0055]

[0056] The amino acid sequence of the dual-target chimeric antigen receptor is: MALPVTALLLPLALLLHAARPDYKDDDDKLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVESKYGPPAPPAPFWVLVVVGGVLACYSLLV TVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, recorded as SEQ ID NO.13.

[0057] The nucleotide sequence of the CD8α-C1 signal peptide is: ATGGCGCTTCCGGTGACTGCTCTACTTCTACCGTTGGCTCTGCTGCTCCACGCCGCTCGCCCT, denoted as SEQ ID NO.14.

[0058] The nucleotide sequence of the Myc tag is: GAACAGAAGCTCATCTCGGAGGAAGACCTG, denoted as SEQ ID NO.15.

[0059] The extracellular nucleotide sequence of PD1: TTCCTGGACAGCCCCGACCGCCCGTGGAATCCACCCACCTTTTCACCGGCCCTGCTGGTGGTTACAGAGGGCGACAACGCCACGTTCACCTGCTCCTTCTCCAACACGTCGGAGAGTTTCGTCCTCAACTGGTACAGGATGTCTCCCAGCAACCAGACCGACAAACTTGCTGCGTTCCCCGAGGATCGCAGCCAGCCGGGCCAGGATTGCCGATTCAGAGTCACCCAGCTGCCCAACGGTCGGGACTTTCATATGTCCGTGGTCCGCGCCCGCCGCAACGACTCTGGCACCTACCTGTGCGGGGCCATCTCTTTGGCTCCCAAGGCCCAAATCAAGGAGAGCCTGCGCGCCGAGCTCCGCGTAACCGAGCGGCGTGCAGAGGTGCCAACCGCTCACCCCTCTCCTAGCCCTAGGCCTGCCGGACAGTTCCAGACCCTGGTG, denoted as SEQ ID NO.16.

[0060] The transmembrane and intracellular nucleotide sequence of DAP10 is: CAGACTACTCCCGGTGAGCGCAGTTCCCTGCCTGCGTTTTATCCGGGGACCAGCGGCTCCTGTTCTGGATGTGGTTCGCTGTCCTTGCCCCTGCTGGCCGGCTTGGTGGCGGCGGACGCCGTGGCTTCCCTGCTGATTGTCGGTGCCGTGTTCCTGTGCGCCCGGCCTCGCCGTTCCCCAGCGCAGGAGGACGGCAAAGTGTACATTAATATGCCCGGCCGCGGC, denoted as SEQ ID NO.17.

[0061] The intracellular nucleotide sequence of CD137-C1 is: AAGCGCGGGCGAAAGAAGCTGCTGTACATCTTCAAGCAGCCATTTATGCGTCCTGTGCAGACGACCCAGGAGGAAGATGGCTGTTCATGCCGCTTCCCGGAGGAGGAGGAGGGCGGCTGCGAGTTG, denoted as SEQ ID NO.18.

[0062] The nucleotide sequence of the CD8α-C2 signal peptide is:

[0063] The nucleotide sequence of the FLAG tag is: GACTACAAAGACGATGACGACAAG, denoted as SEQ ID NO.19.

[0064] The nucleotide sequence of the NKG2D ligand binding domain is: TTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTT AAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTG, denoted as SEQ ID NO.20.

[0065] The nucleotide sequence of the hinge region of IgG4 monomer is: GAATCTAAATATGGGCCTCCAGCACCACCCGCGCCT, denoted as SEQ ID NO.21.

[0066] The nucleotide sequence of CD28 transmembrane and intracellular regions is: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC, denoted as SEQ ID NO.22.

[0067] The nucleotide sequence of the intracellular segment of CD137-C2 is: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG, denoted as SEQ ID NO.23.

[0068] The nucleotide sequence of the CD3ζ intracellular domain is: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCT CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGA, denoted as SEQ ID NO.24.

[0069] Example 2

[0070] The method for preparing the recombinant expression vector encoding the dual-target chimeric antigen receptor includes the following steps:

[0071] A first receptor, consisting of a nucleotide fragment containing the extracellular domain of PD1 (1-150 aa), the extracellular, transmembrane, and intracellular domains of DAP10 (19-93 aa), and the intracellular domain of CD137 (also known as 4-1BB) (214-255 aa), was synthesized using a whole-genome synthesis method. This NKG2D extracellular domain sequence was then sequentially linked to the IgG4 hinge region sequence (monomer mutant: ESKYGPPAPPAP), the CD28 transmembrane region sequence (153-179 aa), and the intracellular and CD3ζ domains of CD137, forming a second receptor. The two receptors are linked by a self-splitting peptide, P2A. The nucleotide fragments of the first and second receptors were then tandemly linked to obtain the PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fragment.

[0072] The coding gene for the dual-targeting chimeric antigen receptor was synthesized, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.12;

[0073] The process of ligating the dual-target chimeric antigen receptor encoding gene into a lentiviral expression vector includes the following steps:

[0074] The dual-targeting chimeric antigen receptor PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fragment and the lentiviral vector (pCDH-CMV-MCS-P2A-copGFP-T2A-Puro) were double-digested with XbaI and XhoI. The dual-targeting chimeric antigen receptor fragment and the lentiviral vector were then ligated using T4 DNA ligase. Finally, the complete lentiviral plasmid was sequenced, and the sequencing results matched the expected sequences of each fragment, thus obtaining the recombinant expression vector.

[0075] Example 3

[0076] The preparation of highly cytotoxic anti-tumor NK cells in this embodiment includes the following steps:

[0077] (I) Lentiviral Packaging

[0078] 1. Extraction of lentiviral packaging plasmids and target plasmids

[0079] 1.1 Plasmid Transformation

[0080] stbl3 competent cells and E. coli TOP10 competent cells were collected. 20 ng of the target plasmid was added to the stbl3 competent cells, and 1 μg of lentiviral packaging helper plasmid pSPAX2 and 1 μg of lentiviral packaging helper plasmid PMD2G were added to the TOP10 competent cells, respectively. After thorough mixing, the cells were incubated on ice for 30 min, then immediately placed in a 42℃ water bath for 90 s for heat shock, followed by 2 min on ice. Subsequently, the cells were placed in a 37℃ constant temperature shaking incubator and cultured at 220 rpm for 1 h with shaking. 100 μL of the culture was then evenly spread onto LB solid medium and incubated at 37℃ for 13 h.

[0081] The stbl3 competent cells were purchased from TransGenBiotech, catalog number L681212.

[0082] The target plasmid is: CMV-PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ-pCDH.

[0083] LB solid medium contains the antibiotic ampicillin (Amp) at a concentration of 50 μg / mL, added at a volume ratio of 1:1000.

[0084] 1.2 Plasmid Extraction

[0085] 1) Small-scale preparation: Pick single colonies from the above culture plates and inoculate them into 5 mL of fresh LB medium. Place them on a shaker at 37°C and shake at 220 rpm / min for 12-14 h. The LB medium contains Amp at a concentration of 50 μg / mL and is added at a ratio of 1:1000.

[0086] 2) Large extraction: Take 200 μL of bacterial culture treated in step 1) and inoculate it into 200 mL of LB medium. Shake at 37°C and 220 rpm / min. The OD value is measured to be about 1.8. The LB medium contains 200 μL of Amp.

[0087] 3) Plasmid extraction was performed using the OMEGA endotoxin-free plasmid extraction kit. The plasmid concentration and purity were measured using a Nanodrop micro spectrophotometer, and the plasmids were stored at -20°C for later use. The OMEGA endotoxin-free plasmid extraction kit was purchased from OmegaBio-Tek (USA), product code D6926-03, and the Nanodrop micro spectrophotometer was purchased from Thermoelectric (Shanghai) Technology Instruments Co., Ltd. (model: NanoDrop2000).

[0088] 2. Lentiviral packaging process

[0089] When 293T cells reach a density of 80%-90%, replace them with fresh DMEM high-glucose medium containing 6% fetal bovine serum and incubate for 1 hour. After 1 hour, package the virus.

[0090] The target plasmid CMV-PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ-pCDH was packaged with two helper plasmids pSPAX2 and PMD2G in a ratio of 4:3:1 to obtain a mixed plasmid. The mixed plasmid was added to the CaCl2 solution provided by the calcium phosphate kit, and mixed by pipetting. The mixture was then added dropwise to BBS at a uniform rate, and mixed by pipetting. The mixture was incubated at room temperature for 20 min to obtain a mixed solution. The mixture was added to 293T cells, gently mixed, and incubated in an incubator for 12 h. The supernatant was removed, the cells were gently washed once with PBS, and 15 mL of fresh DMEM medium containing 10% FBS was replaced. Viruses were collected at 48 h and 72 h.

[0091] Fetal bovine serum was purchased from Hyclone (catalog number SV30265.01B), DMEM high glucose medium was purchased from Hyclone (catalog number WH0021D031), and calcium phosphate kit was purchased from Beyotime (catalog number C0508).

[0092] 3. Lentiviral Concentration

[0093] The collected viral supernatant was centrifuged to remove cell debris, PEG-8000 was added, and the mixture was centrifuged at 4°C for more than 12 hours to collect viral particles. The concentrated viral particles were resuspended in pre-cooled PBS to obtain concentrated PD1-DAP10-41BB viral solution, which was aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles.

[0094] 4. Determination of lentivirus titer

[0095] The lentivirus titer was determined using an abm qPCR lentivirus titer kit (product number LV900). In this example, the lentivirus titer obtained was higher than 1×10⁻⁶. 9 .

[0096] (II) Lentiviral transfection of NK92 cells

[0097] Take 3-5×10 5 NK92 cells were seeded per well in 24-well plates, and concentrated PD1-DAP10-41BB virus solution was added to each well (MOI = 50-100). Polyglobulin was added to each well to a final concentration of 8 μg / mL, and the mixture was incubated at 37°C. After 12-15 hours, the cells were centrifuged, the viral supernatant was removed, and fresh culture medium was added for further culturing. Lentivirally infected NK92 cells were obtained and designated PN-CAR-NK92.

[0098] The polygluconate was purchased from Yisheng Biotechnology, catalog number 40804ES76.

[0099] (III) Detection of PD1 and NKG2D mRNA expression levels in cells by qPCR

[0100] PN-CAR-NK92 and NK92 cells were collected 72 hours after lentiviral infection. mRNA was extracted from both cells, and the levels of PD1 and NKG2D in PN-CAR-NK92 and NK92 cell mRNA were detected. The detection primers used were:

[0101] PD1-F: GTGTCACACAACTGCCCAAC;

[0102] PD1-R: CCGCAGGCTCTCTTTGATCT;

[0103] NKG2D-F: GAGTGATTTTTCAACACGATGGC;

[0104] NKG2D-R:ACAGTAACTTTCGGTCAAGGGAA;

[0105] GAPDH-F: GAGGACCTGACCTGCCGTCT;

[0106] GAPDH-R:GGAGGAGTGGGTGTCGCTGT.

[0107] The detection results are shown in Figure 2. It can be seen from the figure that the mRNA expression level of PD1 / NKG2D in PN-CAR-NK92 cells is significantly higher than that in NK92 cells.

[0108] Example 4

[0109] Flow cytometry detection of the expression of target genes PD1 and NKG2D

[0110] PN-CAR-NK92 and NK92 cells were infected with lentivirus for 72 hours. The cells were centrifuged at 300g for 5 minutes, and the cell count was 1×10⁻⁶. 6 The cells were washed 1-2 times with PBS containing 1% FBS (hereinafter referred to as washbuffer). APC-labeled anti-human PD1 flow cytometry antibody and BV421-labeled anti-human NKG2D flow cytometry antibody were added to a 100 μL system. The cells were incubated on ice in the dark for 30 min, and then washed 2-3 times with washbuffer. Finally, the cells were resuspended in 400 μL of PBS and analyzed using flow cytometry.

[0111] The PD1 flow cytometry antibody was purchased from BD Biosciences, catalog number 320822, and the flow cytometer was BD FACSCantoII.

[0112] The PD1 detection results are shown in Figure 3. It can be seen that the expression rate of PD1 in PN-CAR-NK92 is about 94.3%, and the expression rate of NKG2D is about 98.1%, which is significantly higher than that in NK92 cells.

[0113] Example 5

[0114] Flow cytometry was used to detect the expression of PD-L1 in mouse breast cancer cells EMT6-PD-L1, human gastric cancer cells MGC-803, human gastric cancer cells KATO-III, and human osteosarcoma cells U2OS, as well as the expression of MICA and MICB in NKG2DL.

[0115] PD-L1 was expressed in mouse breast cancer cells EMT6-PD-L1, but MICA / MICB were not expressed; PD-L1 was not expressed in human gastric cancer cells MGC-803, but MICA / MICB were expressed; PD-L1 was not expressed in human gastric cancer cells KATO-III, but MICA / MICB were not expressed; and PD-L1 was expressed in human osteosarcoma cells U2OS, but MICA / MICB were expressed.

[0116] Take 300g of logarithmic growth phase mouse breast cancer cells EMT6-PD-L1, human gastric cancer cells MGC-803, human gastric cancer cells KATO-III, and human osteosarcoma cells U2OS, centrifuge for 5 min, and count the cells at 1×10⁻⁶. 6 The cells were washed 1-2 times with PBS containing 1% FBS (hereinafter referred to as washbuffer). APC-labeled anti-human PD-L1 flow cytometry antibody and PE-labeled anti-human MICA / B flow cytometry antibody were added to a 100 μL system. The cells were incubated on ice in the dark for 30 min, and then washed 2-3 times with washbuffer. Finally, the cells were resuspended in 400 μL of PBS and analyzed by flow cytometry.

[0117] The PD-L1 flow cytometry antibody was purchased from BD (catalog number 2460257), the MICA / B flow cytometry antibody was purchased from BD (catalog number 320906), and the flow cytometer was BD FACSCantoII.

[0118] The PD-L1 detection results are shown in Figure 4. It can be seen that PD-L1 expression is 98.1% in EMT6-PD-L1 cells, while MICA / MICB is basically not expressed. PD-L1 is basically not expressed in MGC-803 cells, while MICA / MICB expression is 98.6%. PD-L1 and MICA / MICB are basically not expressed in KATO-III cells. PD-L1 expression is about 84.5% in U2OS cells, and MICA / MICB expression is about 97.6%.

[0119] Example 6

[0120] PN-CAR-NK92 in vitro cytotoxicity assay

[0121] 1. Cell preparation

[0122] EMT6-PD-L1, MGC-803, KATO-III, and U2OS cells in the logarithmic growth phase after luciferase transfection were seeded into 96-well plates, ensuring 1 × 10⁶ cells per well. 4 Each cell type was divided into 3 sub-wells and incubated overnight in a 5% CO2, 37°C incubator.

[0123] 2. Experimental Design

[0124] The experimental group consisted of two subgroups, each using different effector cells: PD1-CAR-NK92, NKG2D-CAR-NK92, PN-CAR-NK92, and NK92, with an effector-to-target ratio of 5:1. Three control groups were also included: target cell control wells and sample maximum enzyme activity control wells, all co-incubated with a total volume of 100 μL per well. The sample maximum enzyme activity control wells were untreated target cell wells used for subsequent lysis.

[0125] 3. Luciferase detection

[0126] This experiment used the ONE-Glo™ Luciferase Assay System Promega for detection. Effector cells and target cells were incubated together for 3-6 hours in a 5% CO2, 37°C incubator. One hour before the predetermined detection time, the 96-well plate was removed from the cell culture incubator, and Lysis Solution (10×) was added to the "Sample Maximum Enzyme Activity Control Well," at a volume equal to 10% of the original culture medium. After adding Lysis Solution, the plate was repeatedly pipetted and mixed thoroughly, and then returned to the incubator for further incubation. After the co-incubation time was reached, the detection solution was added, and the cells were detected using a microplate reader.

[0127] Cytotoxicity (%) = (RLU min – RLU sample) / (RLU min – RLU max) × 100.

[0128] The calculations revealed that for EMT6-PD-L1 cells, the single-module PD1-CAR-NK92 and dual-module PD1-CAR-NK92 cells showed significantly higher cytotoxicity than the control NK92 group, while NKG2D-CAR-NK92 cells showed virtually no cytotoxicity. For MGC-803 cells, the single-module NKG2D-CAR-NK92 and dual-module PD1-CAR-NK92 cells showed significantly higher cytotoxicity than the control NK92 group, while PD1-CAR-NK92 cells showed virtually no cytotoxicity. For U2OS cells, at a 5:1 ratio, PN-CAR-NK92 showed significantly stronger cytotoxicity than NK92, with an approximately 6-fold increase in cytotoxicity. For KATO-III cells that do not express PD-L1 and MICA / MICB, there was no change in cytotoxicity.

[0129] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0130] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0131] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A dual-targeting chimeric antigen receptor, characterized in that, The dual-targeting chimeric antigen receptor comprises a first receptor and a second receptor sequentially linked by: an extracellular recognition-binding region coding sequence, a transmembrane region coding sequence, and an intracellular signal transduction region coding sequence.

2. The dual-targeting chimeric antigen receptor according to claim 1, characterized in that, The extracellular recognition-binding region of the first receptor is PD1, containing amino acids 1-147aa, and the extracellular recognition-binding region of the second receptor is NKG2D, containing amino acids 81-216aa; the coding sequences of the first and second receptors are linked by a cleavage peptide, which includes F2A, T2A, E2A, or P2A.

3. The dual-targeting chimeric antigen receptor according to claim 1, characterized in that, The extracellular recognition-binding region recognizes any tumor-specific or related antigens, including PD1 ligand and NKG2D ligand; the transmembrane region and intracellular signal transduction region are selected from any one or a combination of intracellular domains of at least two molecules selected from CD8, CD28™+ICD, 4-1BB, DAP10™+ICD, DAP12, DNAM1, and CD3ζ.

4. A carrier, characterized in that, Contains the dual-targeting chimeric antigen receptor as described in any one of claims 1-3.

5. An immune cell, characterized in that, The immune cell contains the dual-targeting chimeric antigen receptor as described in any one of claims 1-3, and the immune cell is modified with one or more chimeric molecules.

6. The immune cells according to claim 5, characterized in that, The immune cells are selected from NK cells; the NK cells include NK cells taken from the human body or NK cells induced in vitro from stem cells, memory NK cells, and NK cell lines.

7. A method for preparing the immune cells according to claim 5 or claim 6, characterized in that, The method includes: transferring the dual-targeting chimeric antigen receptor into immune effector cells.

8. The use of a dual-targeting chimeric antigen receptor as described in any one of claims 1-3, the vector as described in claim 4, or the immune cell as described in claim 5 or 6, characterized in that, Used to prepare drugs that inhibit tumor cells or for use in tumor cell immunotherapy.

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