CN115820697B - Immune cell and preparation method and application thereof - Google Patents

Abstract

The present invention relates to a nucleic acid construct, an immune cell, and a preparation method and application thereof, wherein the nucleic acid construct comprises a CAR molecule coding sequence composed of an extracellular antigen binding region coding sequence, a transmembrane region coding sequence, and an intracellular signal transduction region coding sequence, and sGP130 or its fusion protein coding sequence; sGP130 or its fusion protein coding sequence and the CAR molecule coding sequence are connected by a shear peptide, and the shear peptide comprises: F2A, T2A, E2A or P2A. The present invention uses sGP130 in combination with CAR-T cells at the same time, and experiments show that the sGP130-HER2-CAR-NK92 cells obtained by the present invention highly express HER2 antibodies and can be used for the treatment of HER2-related tumors. Moreover, immune cells overexpress sGP130, block the binding of IL-6 to its receptor, and improve their safety in vivo. In vitro cytotoxicity tests show that sGP130-HER2-CAR-NK92 cells enhance the killing effect of CAR-T cells on JIMT-1 cells by about 2 times.

Description

Immune cell and preparation method and application thereof

Technical Field

The present invention belongs to the technical field of cell immunotherapy, and specifically relates to an immune cell and a preparation method and application thereof.

Background technique

The role of immune cells in tumor immunotherapy has received increasing attention. Natural killer cells (NK) are the core innate immune cells of the body, and T lymphocytes are important adaptive immune response cells of the body. Both NK and T cells play a major role in tumor immune surveillance. In recent years, tumor immune cell therapy has attracted much attention in tumor treatment due to its significant efficacy, especially T/NK cells (CAR-T/NK) based on chimeric antigen receptor modification. CAR-T cell therapy has shown good targeting, lethality and persistence in the clinical treatment of hematological malignancies, becoming a typical representative of tumor adoptive immunotherapy. However, there are still many problems with CAR-T cell therapy. For example, the results of clinical treatment studies of CAR-T cells show that the cytokine release syndrome (CRS) caused by CAR-T cells itself threatens the patient's life. CRS uses interleukin-6 (IL-6) as a detection indicator, and IL6 antagonists have also become the main means to control severe CRS, thereby reducing the risk of CAR-T cell therapy failure caused by severe CRS.

IL-6 is also one of the most distinctive cytokines that promotes cancer cell factors. Among the various inflammatory factors released by immune cells, IL-6 is considered to be the most core inflammatory factor that connects inflammation and tumors, and is also the key inflammatory factor that triggers cytokine storms. The combination of IL-6 and IL-6R includes two types: membrane-bound (mIL-6R) and soluble IL-6R (sIL-6R), which are called classical or non-classical signal transduction, respectively. The biological differences produced by the two signal transduction pathways are quite large. The classical signaling pathway can regenerate and has a protective effect, while the non-classical signaling pathway promotes the occurrence of inflammation. In the non-classical pathway, IL-6 forms a protein complex by binding to the non-signaling sIL-6Rα, and then dimerizes with the subunit glycoprotein 130 (gp130), and initiates the signal transduction cascade through transcription factors, Janus kinases (JAKs) and signal transducers, and transcription activators (STATs), which can not only promote the continued cell proliferation of tumors and inhibit the immune level of the tumor microenvironment, but also cause high fever, nerve damage and even death in patients.

Although immune cells have attractive prospects in tumor immunotherapy, the release of IL-6 creates an inflammatory microenvironment, triggers a cytokine storm in patients, and promotes tumor growth or immune escape. Therefore, further research is still needed in this field to further improve the efficacy of immune effector cells for tumor immunotherapy and reduce toxic side effects.

Summary of the invention

In order to solve the problems existing in the prior art, the present invention provides a nucleic acid construct, a vector, an immune cell, and a preparation method and application thereof. The ability of the immune cell to kill tumors is significantly enhanced. When used in combination with CAR-T cells, the killing effect on solid tumors is also improved, and its safety in the body is also improved.

The object of the present invention is to provide a nucleic acid construct.

The object of the present invention is to provide a vector containing the above nucleic acid construct.

Another object of the present invention is to provide immune cells containing the above nucleic acid construct and a method for preparing the same.

Another object of the present invention is to provide the application of the above immune cells.

According to a specific embodiment of the present invention, the nucleic acid construct encodes a fusion polypeptide for modifying immune cells, and the nucleic acid construct includes sequentially connected: an extracellular antigen binding region coding sequence, a transmembrane region coding sequence, an intracellular signal transduction region coding sequence, and sGP130 or its fusion protein coding sequence.

The soluble GP130 fusion protein includes but is not limited to: a fusion peptide of soluble sGP130 and Fc, or a fusion peptide of soluble sGP130 and CH3 domain.

In the present invention, sGP130 is processed/overexpressed in nucleic acid constructs or immune cells, thereby reducing the side effects of CAR-T during treatment while retaining the anti-tumor effect.

Furthermore, the extracellular antigen binding region coding sequence, the transmembrane region, and the intracellular signal transduction region coding sequence constitute the CAR molecule coding sequence, and the sGP130 or its fusion protein coding sequence and the CAR molecule coding sequence are connected by a cleavage peptide, and the cleavage peptide includes: F2A, T2A, E2A or P2A.

Wherein, the extracellular antigen binding region is an antibody that specifically binds to an antigen highly expressed in a tumor.

Furthermore, the extracellular antigen binding region recognizes any tumor-specific or tumor-related antigen, including HER2, EphA2, GD2, EpCAM, PD-1, PD-L1, LeY, CEA, EGFR, GPC3, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4EGFR, NY-ESO-1, MAGE4, CD19, CAIX, CD123, CD33, IL13R, LMP1, PLAC1, MUC1, MUC16; the transmembrane region and the intracellular signal transduction region are selected from any one of CD8, CD28 TM+ICD, 4-1BB, DAP10, DAP12, NKG2D, DNAM1, CD3ζ, or a combination of intracellular domains of at least two molecules.

Further, the sGP130 includes amino acids 1-558aa, and the amino acid sequence is shown in SEQ ID NO: 1; preferably, the nucleotide sequence of the gene encoding the sGP130 is shown in SEQ ID NO: 2. This fragment can specifically bind to the trans signaling pathway of IL6, is the best fragment selected from multiple fragments after experimental verification by this laboratory, and is different in length from the known existing GP130 extracellular segment.

Furthermore, a FLAG, His or HA tag is added to the front end of the extracellular antigen binding region coding sequence, which is conducive to detecting the expression of the target gene.

A vector comprising the nucleic acid construct.

In the CAR recognition activation induced overexpression system, the plasmid vector includes any one of pCDH-CMV-MCS-EF1-Puro, pCDH-CMV-MCS-EF1-EGFP, pCDH-CMV-MCS-EF1-EGFP-T2A-Puro, and pCDH-CMV-MCS-P2A-EGFP-T2A-Puro.

Preferably, the vector used for overexpression includes a viral vector or a PB vector.

Preferably, the viral vector comprises any one of a lentiviral vector, an adenoviral vector or a retroviral vector, or a combination of at least two of them.

Preferably, it is a lentiviral vector, and more preferably, a lentiviral vector pCDH-CMV-MCS-P2A-EGFP-T2A-Puro, which uses CMV as the promoter of the target gene to start the expression of the target gene used, including the screening gene and the reporter gene GFP, and can achieve the effect of synchronous expression of all target genes without uneven expression. The use of lentiviral packaging and lentiviral infection methods has the advantages of high transfection efficiency, large exogenous target gene fragments, and can achieve stable long-term expression of the target gene.

An immune cell, comprising the nucleic acid construct; the immune cell is an immune effector cell modified with a chimeric antigen receptor and can overexpress sGP130.

Furthermore, the immune cells are modified with one or more CAR molecules.

Furthermore, the immune cells are selected from any one or a combination of at least two of NK cells, T cells, B cells, and macrophages.

Preferably, the NK cells include NK cells removed from the human body or NK cells induced in vitro by stem cells, memory NK cells and NK cell lines; the T cells include T cells removed from the human body or T cells induced in vitro by stem cells.

Preferably, the T cells are CD4+T cells, CD8+T cells, NKT cells and memory T cells.

A method for preparing the immune cells, the method comprising: transferring the nucleic acid construct into immune effector cells.

A use of the nucleic acid construct, the vector, and the immune cell, wherein the nucleic acid construct, the vector, and the immune cell are used to prepare a drug for inhibiting tumor cells or used in tumor cell immunotherapy to alleviate CRS caused by the CAR-T cell therapy process.

Preferably, the extracellular antigen binding region in the CAR molecule recognizes and binds to the extracellular variable region of the single-chain antibody of the HER2 protein to form a HER2-CAR fragment.

Preferably, the amino acid sequence of the extracellular signal peptide CD8a Leader of the CAR molecule is shown as SEQ ID NO:5; the nucleotide sequence of its encoding gene is shown as SEQ ID NO:6.

Preferably, the amino acid sequence of the CD8αLinker in the extracellular and intracellular Linker regions of the CAR molecule is as shown in SEQ ID NO:7; the nucleotide sequence of the gene encoding it is as shown in SEQ ID NO:8.

The intracellular co-stimulatory signal transduction domain of the CAR molecule includes any one of CD8, CD28 TM+ICD, 4-1BB, DAP10, DAP12, NKG2D, DNAM1, and CD3ζ, or a combination of intracellular domains of at least two molecules.

The intracellular signal transduction region is composed of a CD28 TM+ICD intracellular region and a CD3ζ intracellular region in series.

Preferably, the amino acid sequence of the CD28 TM+ICD transmembrane and intracellular co-stimulatory region is as shown in SEQ ID NO:9; and the nucleotide sequence of the gene encoding the same is as shown in SEQ ID NO:10.

Preferably, the amino acid sequence of CD3ζ of the CAR molecule is shown in SEQ ID NO: 11. The nucleic acid sequence is shown in SEQ ID NO: 12.

Preferably, the amino acid sequence of the F2A peptide fragment is shown as SEQ ID NO:13; the nucleotide sequence of the gene encoding it is shown as SEQ ID NO:14.

Preferably, the amino acid sequence of the FLAG fragment is shown as SEQ ID NO:15; the nucleotide sequence of the gene encoding it is shown as SEQ ID NO:16.

The nucleic acid construct, the vector and the immune cell of the present invention can inhibit various tumors, including blood tumors and/or solid tumors.

It has a good inhibitory effect on solid tumors including lung cancer, liver cancer, breast cancer, Wilms' tumor, glioma, neuroblastoma, melanoma, nasopharyngeal carcinoma, mesocortical tumor, islet cell tumor, retinoblastoma, pancreatic cancer, uterine fibroids, cervical cancer or thyroid cancer, especially has better therapeutic effects on liver cancer and lung cancer.

sGP130 is a soluble GP130 that contains only the extracellular region of GP130. It is mainly produced by mRNA splicing rather than restricted protein hydrolysis. Studies have shown that sGP130 can capture the IL-6/sIL-6R complex and prevent the complex from binding to gp130 on the membrane. It can specifically inhibit non-classical signal transduction pathways, thereby inhibiting the biological activity of IL-6.

The present invention adds the extracellular segment of GP130 (sGP130: 1-558aa), which can strongly capture the IL-6/sIL-6R complex (compared to sGP130: 1-618aa), prevent the complex from binding to gp130 on the membrane, and can specifically inhibit the non-classical signaling pathway, thereby inhibiting the biological activity of IL-6 in promoting inflammation. During the research and development process, we found that the initial lentiviral expression vector pCDH-CMV-MCS-EF1-EGFP-T2A-Puro, because of the use of dual promoters to express the target gene and GFP respectively, often appears fluorescent expression may be more than 90%, but the target gene HER2 expression is less than 10%. Therefore, we first optimized the expression vector, that is, the second promoter EF1 was replaced with the self-cleaving peptide P2A, so as to couple the expression level between the target gene and GFP. During the process of lentiviral packaging of the target plasmid, the packaging efficiency of the virus containing the target gene can be visually observed through the reporter gene GFP. After the lentiviral transfection of NK92 cells in the later stage, after continuous screening with puromycin, a stably transfected cell line was quickly obtained.

In the present invention, we place F2A-sGP130 behind the HER2-CD28 TM+ICD-CD3z fragment, which not only ensures the normal cell membrane expression and function of the target fragment HER2, but also ensures the normal release and expression of sGP130.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention combines sGP130 with CAR-T cells to improve the killing effect on solid tumors and improve its safety in vivo.

2. The experiment showed that when the lentivirus obtained by the present invention was used to infect NK92 cells, the expression rate of GFP in sGP130-HER2-CAR cells was about 87.9%, and the expression of GFP in CAR-T cells was about 40.4%; the modified sGP130-HER2-CAR-NK92 cells highly expressed HER2 and could be used for the treatment of HER2-related tumors. ELISA detected the expression of sGP130 in the supernatant of the modified cells, and the content of sGP130 was high, about 50 ng/ml; blocking the binding of IL-6 to its receptor improved its safety in vivo; and, through in vitro cytotoxicity tests, it was shown that sGP130-HER2-CAR-NK92 cells enhanced the killing effect of CAR-T cells on JIMT-1 cells, which enhanced the killing effect of CAR-T cells on JIMT-1 cells by about 2 times.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1a is a pCDH-CMV-MCS-P2A-EGFP-T2A-Puro lentiviral vector;

Figure 1b is a schematic diagram of the structure of a nucleic acid construct;

FIG2 is a graph showing the detection results of 293T cells transfected with lentivirus 72 hours later, wherein a is a graph observed under an inverted microscope, and b is a graph observed under a fluorescence microscope;

FIG3 is a diagram showing the detection results of NK92 cells transfected with chimeric antigen receptors by viruses and screening stable expression cell lines, wherein a is a diagram observed under an inverted microscope and b is a diagram observed under a fluorescence microscope;

FIG4a is a diagram showing the detection results after lentivirus infection of NK92 cells, wherein the dotted line represents the parental NK92 cells, and the solid line represents the sGP130-HER2-CAR-NK92 cells; the abscissa represents the GFP fluorescence intensity (logarithmic value), and the ordinate represents the number of cells;

FIG4b is a graph showing the detection results after lentivirus infection of T cells, wherein the dotted line represents the parental NK92 cells, and the solid line represents the HER2-CAR-T cells; the abscissa represents the GFP fluorescence intensity (logarithmic value), and the ordinate represents the number of cells;

FIG5 is a graph showing the results of Western-blot detection of HER2-FLAG expression in HER2-CAR-NK92 and sGP130-HER2-CAR-NK92 cells and parental NK92 cells;

FIG6 is a graph showing the results of ELISA detection of sGP130 expression in the supernatant of sGP130-HER2-CAR cells and parental NK92 cells;

FIG7 is a diagram showing the detection results of HER2 expression in JIMT-1 cells, wherein the dotted line represents the parental JIMT-1 cells, and the solid line represents the JIMT-1 cells labeled with APC-labeled mouse anti-human HER2 monoclonal antibody;

FIG8 shows the effect of sGP130 on P-STAT3 in breast cancer JIMT-1 cells;

Figure 9 shows the cytotoxicity test results of sGP130-HER2-CAR-NK92 cells and parental NK92 cells combined with CAR-T cells.

Detailed ways

To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

Unless otherwise specified, the reagents or raw materials used in the present invention are commercially available.

In some more specific embodiments, the nucleic acid construct encodes a fusion polypeptide for modifying immune cells, and the nucleic acid construct includes sequentially connected: an extracellular antigen binding region coding sequence, a transmembrane region coding sequence, an intracellular signal transduction region coding sequence, and sGP130 or its fusion protein coding sequence.

The soluble GP130 fusion protein includes but is not limited to: a fusion peptide of soluble sGP130 and Fc, or a fusion peptide of soluble sGP130 and CH3 domain.

In the present invention, sGP130 is processed/overexpressed in nucleic acid constructs or immune cells, thereby reducing the side effects of CAR-T during treatment while retaining the anti-tumor effect.

Furthermore, the extracellular antigen binding region coding sequence, the transmembrane region, and the intracellular signal transduction region coding sequence constitute the CAR molecule coding sequence, and the sGP130 or its fusion protein coding sequence and the CAR molecule coding sequence are connected by a cleavage peptide, and the cleavage peptide includes: F2A, T2A, E2A or P2A.

Wherein, the extracellular antigen binding region is an antibody that specifically binds to an antigen highly expressed in a tumor.

Furthermore, the extracellular antigen binding region recognizes any tumor-specific or tumor-related antigen, including HER2, EphA2, GD2, EpCAM, PD-1, PD-L1, LeY, CEA, EGFR, GPC3, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4EGFR, NY-ESO-1, MAGE4, CD19, CAIX, CD123, CD33, IL13R, LMP1, PLAC1, MUC1, MUC16; the transmembrane region and the intracellular signal transduction region are selected from any one of CD8, CD28 TM+ICD, 4-1BB, DAP10, DAP12, NKG2D, DNAM1, CD3ζ, or a combination of intracellular domains of at least two molecules.

Furthermore, the amino acid sequence of the sGP130 is shown as SEQ ID NO: 1; preferably, the nucleotide sequence of the gene encoding the sGP130 is shown as SEQ ID NO: 2.

Furthermore, a FLAG, His or HA tag is added to the front end of the extracellular antigen binding region coding sequence, which is conducive to detecting the expression of the target gene.

A vector comprising the nucleic acid construct.

In the CAR recognition activation induced overexpression system, the plasmid vector includes any one of pCDH-CMV-MCS-EF1-Puro, pCDH-CMV-MCS-EF1-EGFP, pCDH-CMV-MCS-EF1-EGFP-T2A-Puro, and pCDH-CMV-MCS-P2A-EGFP-T2A-Puro.

Preferably, the vector used for overexpression includes a viral vector or a PB vector.

Preferably, the viral vector comprises any one of a lentiviral vector, an adenoviral vector or a retroviral vector, or a combination of at least two of them.

Preferably, it is a lentiviral vector, and more preferably, a lentiviral vector pCDH-CMV-MCS-P2A-EGFP-T2A-Puro, which uses CMV as the promoter of the target gene to start the expression of the target gene used, including the screening gene and the reporter gene GFP, and can achieve the effect of synchronous expression of all target genes without uneven expression. The use of lentiviral packaging and lentiviral infection methods has the advantages of high transfection efficiency, large exogenous target gene fragments, and can achieve stable long-term expression of the target gene.

An immune cell, comprising the nucleic acid construct; the immune cell is an immune effector cell modified with a chimeric antigen receptor and can overexpress sGP130.

Furthermore, the immune cells are modified with one or more CAR molecules.

Furthermore, the immune cells are selected from any one or a combination of at least two of NK cells, T cells, B cells, and macrophages.

Preferably, the NK cells include NK cells removed from the human body or NK cells induced in vitro by stem cells, memory NK cells and NK cell lines; the T cells include T cells removed from the human body or T cells induced in vitro by stem cells.

Preferably, the T cells are CD4+T cells, CD8+T cells, NKT cells and memory T cells.

A method for preparing the immune cells, the method comprising: transferring the nucleic acid construct into immune effector cells.

A use of the nucleic acid construct, the vector, and the immune cell, wherein the nucleic acid construct, the vector, and the immune cell are used to prepare a drug for inhibiting tumor cells or used in tumor cell immunotherapy to alleviate CRS caused by the CAR-T cell therapy process.

The technical scheme of the present invention is further described in detail below by embodiments and in conjunction with the accompanying drawings. However, the selected embodiments are only used to illustrate the present invention, and do not limit the scope of the present invention.

Example 1 Construction of chimeric antigen receptor sGP130-HER2-CAR

First, PCR was used with pACgp67B-Her2 plasmid as a template to design primers and amplify the Anti-HER2 ScFv fragment; primers were designed and the signal peptide CD8a Leader and FLAG tag in front of the Anti-HER2 ScFv fragment were obtained by multi-step overlapping extension PCR. Primers were designed and PCR was used with human cDNA as a template to amplify the CD28 TM+ICD transmembrane and intracellular regions and CD3ζ regions. Similarly, multi-step overlapping extension PCR was used to obtain the CD8αLinker in front of CD28 TM+ICD. Then, through overlamp, overlapping extension PCR was performed with CD8αlinker+CD28 TM+ICD and CD3ζ as templates to obtain CD8αlinker+CD28 TM+ICD+CD3ζ. Similarly, overlapping extension PCR was performed with CD8a leader+FLAG+Anti-HER2 ScFv and CD8α linker+CD28 TM+ICD+CD3ζ as templates to obtain the HER2-CAR fragment. Primers were designed, and human cDNA was used as a template to amplify the extracellular region sGP130 of GP130 by PCR. Primers were designed, and the F2A self-cleavage peptide at the front end of the extracellular region of GP130 was obtained by multi-step overlap extension PCR. The F2A-sGP130 fragment was obtained. By overlaplamp, the HER2-CAR fragment and the F2A-sGP130 fragment were used as templates, and overlap extension PCR was performed to obtain the sGP130-HER2-CAR fragment, the nucleotide sequence of which is shown in SEQ ID NO: 3; the total length is 3487 bp.

The amino acid sequence of sGP130 is shown in SEQ ID NO: 1:

SEQ ID NO:1

PRT: 558

MLTLQTWLVQ ALFIFLTTES TG

ELLDPCGYIS PESPVVQLHS NFTAVCVLKE KCMDYFHVNA NYIVWKTNHF TIPKEQYTIINRTASSVTFT DIASLNIQLT CNILTFGQLE QNVYGITIIS GLPPEKPKNL SCIVNEGKKM RCEWDGGRETHLETNFTLKS EWATHKFADC KAKRDTPTSC TVDYSTVYFV NIEVWVEAEN ALGKVTSDHI NFDPVYKVKPNP PHNLSVIN SEELSSILKL TWTNPSIKSV IILKYNIQYR TKDASTWSQI PPEDTASTRS SFTVQDLKPFTEYVFRIRCM KEDGKGYWSD WSEEASGITY EDRPSKAPSF WYKIDPSHTQ GYRTVQLVWK TLPPFEANGKILDYEVTLTR WKSHLQNYTV NATKLTVNLT NDRYLATLTV RNLVGKSDAA VLTIPACDFQ ATHPVMDLKAFPKDNMLWVE WTTPRESVKK YILEWCVLSD KAPCITDWQQ EDGTVHRTYL RGNLAESKCY LITVTPVYADGPGSPESIKA YLKQAPPSKG PTVRTKKVGK NEAVLEWDQL PVDVQNGFIR NYTIFY

The nucleotide sequence of sGP130 is shown in SEQ ID NO: 2:

SEQ ID NO:2

DNA: 1674 bp

atgttga cgttgcagac

301 ttggctagtg caagccttgt ttattttcct caccactgaa tctacaggtg aacttctaga

361 tccatgtggt tatatcagtc ctgaatctcc agttgtacaa cttcattcta atttcactgc

421 agtttgtgtg ctaaaggaaa aatgtatgga ttatttcat gtaaatgcta attacattgt

481 ctggaaaaca aaccatttta ctattcctaa ggagcaatat actatcataa acagaacagc

541 atccagtgtc acctttacag atatagcttc attaaatatt cagctcactt gcaacattct

601 tacattcgga cagcttgaac agaatgttta tggaatcaca ataatttcag gcttgcctcc

661 agaaaaacct aaaaatttga gttgcattgt gaacgagggg aagaaaatga ggtgtgagtg

721 ggatggtgga agggaaacac acttggagac aaacttcact ttaaaatctg aatgggcaac

781 acacaagttt gctgattgca aagcaaaacg tgacaccccc acctcatgca ctgttgatta

841 ttctactgtg tattttgtca acattgaagt ctgggtagaa gcagagaatg cccttgggaa

901 ggttacatca gatcatatca attttgatcc tgtatataaa gtgaagccca atccgccaca

961 taatttatca gtgatcaact cagaggaact gtctagtatc ttaaaattga catggaccaa

1021 cccaagtatt aagagtgtta taatactaaa atataacatt caatataggaccaaagatgc

1081 ctcaacttgg agccagattc ctcctgaaga cacagcatcc acccgatcttcattcactgt

1141 ccaagacctt aaacctttta cagaatatgt gtttaggatt cgctgtatgaaggaagatgg

1201 taagggatac tggagtgact ggagtgaaga agcaagtggg atcacctatgaagatagacc

1261 atctaaagca ccaagtttct ggtataaaat agatccatcc catactcaaggctacagaac

1321 tgtacaactc gtgtggaaga cattgcctcc ttttgaagcc aatggaaaaatcttggatta

1381 tgaagtgact ctcacaagat ggaaatcaca tttacaaaat tacacagttaatgccacaaa

1441 actgacagta aatctcacaa atgatcgcta tctagcaacc ctaacagtaagaaatcttgt

1501 tggcaaatca gatgcagctg ttttaactat ccctgcctgt gactttcaagctactcaccc

1561 tgtaatggat cttaaagcat tccccaaaga taacatgctt tgggtggaatggactactcc

1621 aagggaatct gtaaagaaat atatacttga gtggtgtgtg ttatcagataaagcaccctg

1681 tatcacagac tggcaacaag aagatggtac cgtgcatcgc acctatttaagagggaactt

1741 agcagagagc aaatgctatt tgataacagt tactccagta tatgctgatggaccaggaag

1801 ccctgaatcc ataaaggcat accttaaaca agctccacct tccaaaggacctactgttcg

1861 gacaaaaaaa gtagggaaaa acgaagctgt cttagagtgg gaccaacttcctgttgatgt

1921 tcagaatgga tttatcagaa attatactat attttat

The amino acid sequence of Anti-HER2 ScFv is shown in SEQ ID NO: 3:

SEQ ID NO:3

PRT: 263

1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

16 Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

31 Ser Tyr Trp Ile Ala Trp Val Arg Gln MET Pro Gly Lys Gly Leu

46 Glu Tyr MET Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr

61 Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser

76 Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp

91 Ser Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr Cys Ser

106 Ser Ser Asn Cys Ala Lys Trp Pro Glu Tyr Phe Gln His Trp Gly

121 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

136 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln

151 Pro Pro Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser

166 Cys Ser Gly Ser Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp

181 Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp

196 His Thr Asn Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser

211 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg Ser

226 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu

241 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

256 Ala Ala Ala Gly Gly Gly Gly Ser

The nucleotide sequence of Anti-HER2 ScFv is shown in SEQ ID NO: 4:

SEQ ID NO:4

DNA: 789 bp

1 CAGGTGCAGC TGGTGCAGTC TGGGGCAGAG GTGAAAAAGC CCGGGGAGTC TCTGAAGATC

61 TCCTGTAAGG GTTCTGGATA CAGCTTTACC AGCTACTGGA TCGCCTGGGT GCGCCAGATG

121 CCCGGGAAAG GCCTGGAGTA CATGGGGCTC ATCTATCCTG GTGACTCTGA CACCAAATAC

181 AGCCCGTCCT TCCAAGGCCA GGTCACCATC TCAGTCGACA AGTCCGTCAG CACTGCCTAC

241 TTGCAATGGA GCAGTCTGAA GCCCTCGGAC AGCGCCGTGT ATTTTTGTGC GAGACATGAC

301 GTGGGATATT GCAGTAGTTC CAACTGCGCA AAGTGGCCTG AATACTTCCA GCATTGGGGC

361 CAGGGCACCC TGGTCACCGT CTCCTCAGGT GGAGGCGGTT CAGGCGGAGG TGGCTCTGGC

421 GGTGGCGGAT CGCAGTCTGT GTTGACGCAG CCGCCCTCAG TGTCTGCGGC CCCAGGACAG

481 AAGGTCACCA TCTCCTGCTC TGGAAGCAGC TCCAACATTG GGAATAATTA TGTATCCTGG

541 TACCAGCAGC TCCCAGGAAC AGCCCCCAAA CTCCTCATCT ATGATCACAC CAATCGGCCC

601 GCAGGGGTCC CTGACCGATT CTCTGGCTCC AAGTCTGGCA CCTCAGCCTC CCTGGCCATC

661 AGTGGGTTCC GGTCCGAGGA TGAGGCTGAT TATTACTGTG CCTCCTGGGA CTACACCCTC

721 TCGGGCTGGG TGTTCGGCGG AGGAACCAAG CTGACCGTCC TAGGTGCGGC CGCCGGCGGA

781 GGAGGATCT

The amino acid sequence of CD8a Leader is shown in SEQ ID NO: 5:

SEQ ID NO:5

PRT: 21

1 MET Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu

16 Leu His Ala Ala Arg Pro

The nucleotide sequence of CD8a Leader is shown in SEQ ID NO: 6:

SEQ ID NO:6

DNA: 63 bp;

1 ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG

61 CCG

The amino acid sequence of CD8α linker is shown in SEQ ID NO: 7:

SEQ ID NO:7

PRT: 45

1 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

16 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

31 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

The nucleotide sequence of CD8αlinker is shown in SEQ ID NO: 8:

SEQ ID NO:8

DNA: 135 bp;

1 ACCACGACGC CAGCGCCGCG ACCACCAACA CCGGCGCCCA CCATCGCGTC GCAGCCCCTG

61 TCCCTGCGCC CAGAGGCGTG CCGGCCAGCG GCGGGGGGCG CAGTGCACAC GAGGGGGCTG

121 GACTTCGCCT GTGAT

The amino acid sequence of CD28 TM+ICD is shown in SEQ ID NO: 9:

SEQ ID NO:9

PRT: 68

1 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

16 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys

31 Arg Ser Arg Leu Leu His Ser Asp Tyr MET Asn MET Thr Pro Arg

46 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

61 Arg Asp Phe Ala Ala Tyr Arg Ser

The nucleotide sequence of CD28 TM+ICD is shown in SEQ ID NO: 10:

SEQ ID NO:10

DNA: 204 bp;

1 TTTTGGGTGC TGGTGGTGGT TGGTGGAGTC CTGGCTTGCT ATAGCTTGCT AGTAACAGTG

61 GCCTTTATTA TTTTCTGGGT GAGGAGTAAG AGGAGCAGGC TCCTGCACAG TGACTACATG

121 AACATGACTC CCCGCCGCCC CGGGCCCACC CGCAAGCATT ACCAGCCCTA TGCCCCACCA

181 CGCGACTTCG CAGCCTATCG CTCC

The amino acid sequence of CD3ζ is shown in SEQ ID NO: 11:

SEQ ID NO:11

PRT: 113

1 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

16 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

31 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu MET

46 Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

61 Asn Glu Leu Gln Lys Asp Lys MET Ala Glu Ala Tyr Ser Glu Ile

76 Gly MET Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

91 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

106 His MET Gln Ala Leu Pro Pro Arg

The nucleotide sequence of CD3ζ is shown in SEQ ID NO: 12:

SEQ ID NO:12

DNA: 339 bp

1 AGAGTGAAGT TCAGCAGGAG CGCAGACGCC CCCGCGTACC AGCAGGGCCA GAACCAGCTC

61 TATAACGAGC TCAATCTAGG ACGAAGAGAG GAGTACGATG TTTTGGACAA GAGACGTGGC

121 CGGGACCCTG AGATGGGGGG AAAGCCGCAG AGAAGGAAGA ACCCTCAGGA AGGCCTGTAC

181 AATGAACTGC AGAAAGATAA GATGGCGGAG GCCTACAGTG AGATTGGGAT GAAAGGCGAG

241 CGCCGGAGGG GCAAGGGGCA CGATGGCCTT TACCAGGGTC TCAGTACAGC CACCAAGGAC

301 ACCTACGACG CCCTTCACAT GCAGGCCCTG CCCCCTCGC

The amino acid sequence of F2A is shown in SEQ ID NO: 13:

SEQ ID NO:13

PRT: 25

1 Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu

16 Ala Gly Asp Val Glu Ser Asn Pro Gly Pro

The nucleotide sequence of F2A is shown in SEQ ID NO: 14:

SEQ ID NO:14

DNA: 75 bp

1 GGAAGCGGGAGTGAAACAGAC TTTGAATTTT GACCTTCTCA AGTTGGCGGG AGACGTGGAG

61 TCCAACCCTG GACCT

The amino acid sequence of FLAG is shown in SEQ ID NO: 15:

SEQ ID NO:15

PRT: 8

DYKDDDDK

The nucleotide sequence of FLAG is shown in SEQ ID NO: 16:

SEQ ID NO:16

DNA: 24bp

1 GACTACAAAG ACGATGACGA CAAG

The nucleotide sequence of HER2-CAR is shown in SEQ ID NO: 17:

SEQ ID NO:17

DNA: 1575

GCTCTAGAGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACTACAAAGACGATGACGACAAGCAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCAT CTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGCCAGGTCACCATTCCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGA GACATGACGTGGGATATTGCAGTAGTTCCAACTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATTCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAG CAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTATGATCACACCAATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGAGGATGAGGC TGATTATTACTGTGCCTCCTGGGACTACACCCTCTCGGGCTGGGTGTTCGGCGGAGGAACCAAGCTGACCGTCCTAGGTGCGGCCGCCGGCGGAGGAGGATCTACCACGACGCCAGCGCCCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTTTGGGTGCTGGTGGTGG TTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAG CATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGA GGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAATTCC

The nucleotide sequence of sGP130-HER2-CAR is shown in SEQ ID NO: 18:

SEQ ID NO:18

DNA: 3324

GCTCTAGAGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACTACAAAGACGATGACGACAAGCAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCAT CTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACATGACGTGGGATATTGCAG TAGTTCCAACTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAA ACTCCTCATCTATGATCACACCAATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCCTCCTGGGACTACACCCTCTCGGGCTGGG TGTTCGGCGGAGGAACCAAGCTGACCGTCCTAGGTGCGGCCGCCGGCGGGAGGAGGATCTACCACGACGCCAGCGCCGCCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAAGTAACAG TGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCACCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTT ACCAGGGTCCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCTGGACCTATGTTGACGTTGCAGACTT GGCTAGTGCAAGCCTTGTTTATTTTCCTCACCACTGAATCTACAGGTGAACTTCTAGATCCATGTGGTTATATCAGTCCTGAATCTCCAGTTGTACAACTTCATTCTAATTTCACTGCAGTTTGTGTGCTAAAGGAAAAATGTATGGATTATTTTCATGTAAATGCTAATTACATTGTCTGGAAAACAAACCATTTTACTATTCCTAAGGAGCAATATACTATCATAAACAGAACAGCATCCAGTGTCACCTTTACAGATATAGCTTC ATTAAATATTCAGCTCACTTGCAACATTCTTACATTCGGACAGCTTGAACAGAATGTTTATGGAATCACAATAATTTCAGGCTTGCCTCCAGAAAAACCTAAAAATTTGAGTTGCATTGTGAACGAGGGGAAGAAAATGAGGTGTGA GTGGGATGGTGGAAGGGAAACACACTTGGAGACAAACTTCACTTTAAAATCTGAATGGGCAACACACAAGTTTGCTGATTGCAAAGCAAAACGTGACACCCCCACCTCATGCACTGTTGATTATTCTACTGTGTATTTTGTCAACATTGAAGTCTGGGTAGAAGCAGAGAATGCCCTTGGGAAGGTTACATCAGATCATATCAATTTTGATCCTGTATATAAAGTGAAGCCCAATCCGCCACATAATTTATCAGTGATCAACTCAGA GGAACTGTCTAGTATCTTAAAATTGACATGGACCAACCCAAGTATTAAGAGTGTTATAATACTAAAATATAACATTCAATATAGGACCAAAGATGCCTCAACTTGGAGCCAGATTCCTCCTGAAGACACAGCATCCACCCGATCTTCAT TCACTGTCCAAGACCTTAAACCTTTTACAGAATATGTGTTTAGGATTCGCTGTATGAAGGAAGATGGTAAGGGATACTGGAGTGACTGGAGTGAAGAAGCAAGTGGGATCACCTATGAAGATAGACCATCTAAAGCACCAAGTTTCTGGTATAAAATAGATCCATCCCATACTCAAGGCTACAGAACTGTACAACTCGTGTGGAAGACATTGCCTCCTTTTGAAGCCAATGGAAAAATTCTTGGATTATGAAGTGACTCTCACAAGA TGGAAATCACATTTACAAAATTACACAGTTAATGCCACAAAACTGACAGTAAATCTCACAAATGATCGCTATCTAGCAACCTAACAGTAAGAAATCTTGTTGGCAAATCAGATGCAGCTGTTTTAACTATCCCTGCCTGTGACTTTCA AGCTACTCACCCTGTAATGGATCTTAAAGCATTCCCCAAAGATAACATGCTTTGGGTGGAATGGACTACTCCAAGGGAATCTGTAAAGAAATATATACTTGAGTGGTGTGTTATCAGATAAAGCACCCTGTATCACAGACTGGCAACAAGAAGATGGTACCGTGCATCGCACCTATTTAAGAGGGAACTTAGCAGAGAGCAAATGCTATTTGATAACAGTTACTCCAGTATATGCTGATGGACCAGGAAGCCCTGAATCCAT AAAGGCATACCTTAAACAAGTCCCACCTTCCAAAGGACCTACTGTTCGGACAAAAAAAGTAGGGAAAAACGAAGCTGTCTTAGAGTGGGACCAACTTCCTGTTGATGTTCAGAATGGATTTATCAGAAATTATACTATATTTTATGAATTCC

Example 2: Preparation of chimeric antigen receptor recombinant expression vector

The HER2-CAR fragment and sGP130-HER2-CAR fragment in Example 1 were respectively connected to the pCDH-CMV-MCS-P2A-EGFP-T2A-Puro lentiviral vector by homologous recombination to obtain recombinant lentiviral vectors HER2-CAR-pCDH and sGP130-HER2-CAR-pCDH.

The structure is shown in Figure 1. The complete lentiviral plasmid was sequenced, and the sequencing results confirmed that the sequence was correct and consistent with the expected sequences of each fragment. The lentiviral vector is driven by the CMV promoter to promote all downstream target fragments, and there is no uneven gene expression. The expression of GFP, the selected puromycin resistance, and the expression of the target gene are basically consistent.

Example 3: Preparation of highly cytotoxic anti-tumor NK92 cells

In this example, highly cytotoxic anti-tumor NK92 cells were obtained by lentiviral infection, and the preparation thereof included the following steps:

1. Transformation and extraction of lentiviral packaging plasmid and target plasmid

The recombinant lentiviral vector obtained in Example 2, as well as the auxiliary vector psPAX2 and the auxiliary vector pMD2.G, were added to the competent cells DH5α, mixed thoroughly, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and then ice-bathed for 2 minutes. Then 500 μL of LB medium was added, and the culture was shaken at 37°C and 220 rpm for 40 minutes. After the culture was completed, 200 μL of bacterial solution was taken and spread on LB solid culture medium (containing 50 μg/mL ampicillin solution, added at a ratio of 1:1000), placed upright for 10 minutes, inverted, and cultured in a 37°C constant temperature incubator for 8-16 hours, during which the growth of the colonies was observed.

Pick the monoclonal colonies in the above LB solid medium, inoculate them into 5mL LB medium (containing 5μL of 50μg/mL ampicillin solution), place them in a 37℃ shaker, shake and culture at 220rpm/min for 8h, and the bacterial solution is turbid. Take 200μL of bacterial solution, inoculate them into 200mL LB medium (containing 200μL of 50μg/mL ampicillin solution), place them in a 37℃ shaker, shake and culture at 220rpm/min for 12-16h, and detect the OD value of the bacterial solution with a spectrophotometer. When the OD value of the bacterial solution is between 1.5-2, use the OMEGA endotoxin-free plasmid extraction kit (purchased from Omega Biotechnology, USA, product model D6926-03) to extract the plasmid, determine the concentration and purity of the target plasmid and packaging plasmid, and store them at -20℃.

2. Lentivirus packaging

When 293T cells were cultured to a density of 80%-90%, the Lipo3000 method was used for transfection, and the recombinant lentiviral vector, auxiliary vector psPAX2, and auxiliary vector pMD2.G were transfected into 293T cells at a mass ratio of 4:3:1. The viral supernatants were collected at 48h and 72h of transfection. The expression of GFP was observed under a fluorescence microscope as shown in Figure 2, where a is a white light image and b is a fluorescence image. It can be seen from the white light image a that the cells are in good condition, and from the fluorescence image b that the efficiency of lentiviral packaging is very high and the fluorescence expression is good. All collected viral supernatants need to be centrifuged at 4°C, 4000g, and 10min to remove cell debris first, then filtered using a 0.45μm sterile filter membrane, and sterilized PEG8000 (supernatant volume: PEG8000 volume = 4:1) was added, mixed, and kept at 4°C overnight. The next day, concentrate by centrifugation at 4°C, 4000g, 30min, discard the supernatant, add pre-cooled PBS, resuspend the virus, divide into aliquots, and store at -80°C. The entire process of making lentivirus must be completed on ice.

3. Lentivirus infection of NK92 cells

3-5×10 ⁵ NK92 cells/well were inoculated in a 24-well plate, 10 μl/well of concentrated virus solution (MOI=80) was added, and polybrene with a final concentration of 8 μg/mL was added to each well and mixed, and then cultured in a 37°C incubator. After 12-15 hours, the cells were centrifuged and fresh culture medium was replaced for further culture. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope. After the fluorescence expression was normal, puromycin (500-1500 ng/ml) was added in gradients with subsequent culture. After 2 weeks of screening, a cell line with stable expression of the target gene was obtained. The expression of GFP was observed under a fluorescence microscope.

The results are shown in Figure 3, a is a white light image, and b is a fluorescence image. From the white light image a, it can be seen that NK92 cells are in a clustered state, indicating that the cells are in good condition. From the fluorescence image b, it can be seen that basically 80-90% of the cells express fluorescence, indicating that the stable cell line stably expressing the target gene was successfully constructed.

Example 4: Preparation of highly cytotoxic anti-tumor CAR-T cells

1. Preparation of T cells

First, take 5-10ml of peripheral blood, dilute the blood sample 1:1 with PBS in a 50ml centrifuge tube, take 3ml of lymphocyte separation solution (protected from light) and add it to a 15ml centrifuge tube. In this step, the centrifuge tube containing lymphocyte separation solution needs to be tilted 45 degrees, and an equal volume of diluted blood sample is slowly added along the tube wall. Do not break the liquid surface of the lymphocyte separation solution. After adding, slowly put it into a horizontal centrifuge, centrifuge at room temperature, 500g, and centrifuge for 30min. After taking it out, the liquid surface will be layered. Carefully take out the middle white film layer into a new 15ml centrifuge tube, add 12ml of PBS to resuspend, centrifuge at room temperature 500g for 10min, discard the supernatant, add 10ml of PBS to resuspend, centrifuge at room temperature 500g for 10min, discard the supernatant, add 10ml of PBS to resuspend, centrifuge at room temperature 500g for 10min, discard the supernatant, add 10ml of PBS again to resuspend, and centrifuge at room temperature 500g for 10min. Resuspend the cells in 1640+10% fetal bovine serum culture medium to obtain a mononuclear cell suspension. Count the cells, take 1 million mononuclear cells into a 24-well plate, add 1000U IL2, and add 30μl/ml Human T Activator CD3/CD28 TM+ICD magnetic beads to activate T cells. After 2-3 days of cell activation, remove the magnetic beads with a magnetic rack, add 300UIL2 for expansion culture for subsequent experiments.

2. Preparation of CAR-T cells

2.1. The transformation and extraction process of the lentiviral packaging plasmid and the target plasmid are as described in Example 3, step 1.

2.2 The lentiviral packaging process is as described in Example 3, step 2.

2.3. Lentivirus infection of T cells

5×10 ⁵ T cells/well were inoculated into a 24-well plate, and 30 μl/well of concentrated HER2-CAR virus solution (MOI=80) was added. The plate was cultured in a 37°C incubator. After 12-15 hours, the plate was centrifuged and fresh culture medium was replaced to continue culture. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope. After the fluorescence expression was normal, the obtained CAR-T cells could be directly used for subsequent experiments.

Example 5: Flow cytometry detection of GFP expression in stably transfected NK92 cells and transfected T cells

Take the lentivirus-infected NK92 cells (sGP130-HER2-CAR) and parental NK92 cells obtained in Example 3, the lentivirus-transfected T cells (CAR-T) and parental T cells, count them, the cell density is 1×10 ⁶ cells/ml, wash them 2-3 times with PBS buffer containing 2% fetal bovine serum, and then resuspend the cells with 400 μL washing buffer to obtain a cell suspension. Detect on the machine.

Flow cytometry was used to detect and analyze the sGP130-HER2-CAR cell suspension and the parental NK92 cell suspension, as well as the CAR-T cell suspension and the parental T cell suspension. The results are shown in Figures 4a and 4b. In the figure, the horizontal axis represents the GFP fluorescence intensity (logarithmic value), and the vertical axis represents the number of cells. Figure 4a is the detection result after lentivirus infection of NK92 cells, in which the dotted line represents the parental NK92 cells and the solid line represents the sGP130-HER2-CAR-NK92 cells. The detection results show that the expression rate of GFP in sGP130-HER2-CAR-NK92 cells is about 87.9%;

Figure 4b is a graph showing the detection results after lentivirus-infected T cells, where the dotted line represents parental NK92 cells and the solid line represents HER2-CAR-T cells. The detection results show that the expression of GFP in CAR-T cells is approximately 40.4%.

Example 6: Western blot detection of HER2 protein expression in stably transfected NK92 cells

1. Take 1 million NK92 and sGP130-HER2-CAR-NK92 cell lines in the logarithmic growth phase, add pre-cooled PBS to wash twice, add 200 μL loading buffer to each well and lyse on ice for 30 min, collect the samples and boil them in boiling water for 15 min.

2. Prepare 8% lower separation gel and 4% lower concentration gel.

3. Add 6-8 μL of cell protein sample to each well, use protein marker as control, perform electrophoresis detection, transfer the protein to the NC membrane, block with fast blocking solution at room temperature for 1 hour, incubate with primary antibody at 4°C overnight, wash with TBST 5 times, 5 minutes each time, incubate with secondary antibody at room temperature for 1 hour, wash with TBST 5 times, 5 minutes each time.

4. Development.

The test results are shown in Figure 5; it can be seen from the figure that compared with the parental NK92 cells and the empty vector, the sGP130-HER2-CAR-NK92 cells and HER2-CAR-NK92 cells that have been stably screened with antibiotics highly express HER2-FLAG, indicating that the modified NK92 cells overexpress HER2 successfully; among them, the sGP130-HER2-CAR-NK92 cells have a better effect of overexpressing HER2 and have a more obvious killing effect on tumor cells.

Example 7: ELISA detection of sGP130 expression in the supernatant of modified cells

NK92 and sGP130-HER2-CAR-NK92 cells in the logarithmic growth phase were inoculated in 6-well plates to ensure 1×10 ⁶ cells per well, and 2 replica wells were set for each cell type. The plates were cultured in a 5% CO ₂ , 37°C incubator for 48 hours. After the culture was completed, the cell culture supernatant was collected.

Preparation of standards: prepare 7 1.5 ml EP tubes, numbered 1-7, pipette 500 μl of stock solution (16 ng/ml standard), add to tube 1, add 500 μl of standard diluent to tubes 2-7, pipette 500 μl of standard stock solution from tube 1 and add to tube 2, and so on, the final concentration of tube 7 is 0 pg/ml. Set up 2 secondary wells for the seven concentrations of standard.

Prepare washing solution first, add 200μl to the well plate and wash it 5 times, add standard and sample to the well plate at the same time, incubate at room temperature for 3 h, discard the liquid in the well, wash the well plate 5 times with a spray gun, 30 s each time, add 200μl Human sgp130Conjugate to each well, incubate at room temperature for 2h, discard the liquid in the well plate, wash 5 times, add 200μl SubstrateSolution liquid to each well, incubate at room temperature for 30 min in a dark place, discard the liquid in the well plate, wash 5 times, add 50μl stop solution to each well, and immediately detect on the machine, and measure the absorbance at 450 nm respectively.

The results are shown in Figure 6. After culturing 1 million cells for 48 hours, the sGP130 content in the supernatant of sGP130-HER2-CAR-NK92 cells was about 50 ng/ml, which more effectively blocked the binding of IL-6 to its receptor and alleviated the cytokine storm (CRS) produced during CAR-T cell therapy; there was basically no release from parental NK92 cells or empty cells.

Example 8: Detection of HER2 expression on the surface of breast cancer cells JIMT-1 by flow cytometry

Take JIMT-1 cell line in logarithmic growth phase, count, cell density is 1×10 ⁶ /ml, wash 2-3 times with PBS buffer containing 2% fetal bovine serum, add APC-labeled mouse anti-human HER2 monoclonal antibody in 100μL system, incubate on ice in dark for 30min, wash 2-3 times with wash buffer. Finally, add 400μL PBS solution to resuspend the cells and detect on the machine.

The results are shown in Figure 7, where the horizontal axis represents the HER2 fluorescence intensity (logarithmic value) and the vertical axis represents the number of cells. The test results show that the expression rate of HER2 on the surface of JIMT-1 cells is about 99.8%, which is basically all expressed.

Example 9: Western blot detection of the effect of sGP130 on P-STAT3 in breast cancer JIMT-1 cells

1. Take 200,000 JIMT-1 cells in the logarithmic growth phase and seed them into 6-well plates. After the cells adhere to the wall, add IL6 (10 ng/ml) and IL6/sIL6R (10 ng/ml) cytokines, and add 1 ml of sGP130 supernatant (final concentration 25 ng/ml) at the same time, and culture together for 24 hours. Add 200 μL of loading buffer to each well and lyse on ice for 30 minutes. Collect the samples and boil them in boiling water for 15 minutes.

2. Prepare 8% lower separation gel and 4% lower concentration gel.

3. Add 6-8 μL of cell protein sample to each well, use protein marker as control, perform electrophoresis detection, transfer the protein to the NC membrane, block with fast blocking solution at room temperature for 1 hour, incubate with primary antibody at 4°C overnight, wash with TBST 5 times, 5 minutes each time, incubate with secondary antibody at room temperature for 1 hour, wash with TBST 5 times, 5 minutes each time.

4. Development.

The test results are shown in Figure 8; it can be seen from the figure that compared with the NK92-empty vector supernatant, the sGP130-HER2-CAR-NK92 cell supernatant that has been stably screened by antibiotics contains sGP130, which can inhibit the transmission of the non-classical signaling pathway of IL6/sIL6R, directly leading to a decrease in the phosphorylation level of the downstream signal STAT3. This shows that the cell culture supernatant containing sGP130 can significantly antagonize the transmission of the IL6 signaling pathway.

Example 10: In vitro cytotoxicity assay

1. Cell Preparation

The breast cancer cells JIMT-1-Luc in the logarithmic growth phase after the transfer of luciferase were inoculated into a 96-well plate, ensuring that 1×10 ⁴ cells were present in each well. Three duplicate wells were set for each cell type and cultured overnight in a 5% CO ₂ , 37°C incubator.

2. Experimental Grouping

Four experimental groups were set up, and each group was first added with 20,000 CAR-T cells of the same concentration. The first group was a blank group, the second group was added with 20,000 NK92 cells, the third group was added with 20,000 HER2-CAR-NK92 cells, and the fourth group was added with 20,000 sGP130-HER2-CAR-NK92 cells. At the same time, three groups of controls were set up, including target cell control wells and sample maximum enzyme activity control wells (target cell wells for subsequent lysis without effector cell treatment), with a total volume of 100 μL per well.

3. Luciferase Assay

This experiment uses the ONE-Glo ^TM Luciferase Assay System Promega for detection. Effector cells and target cells were placed in a 5% CO ₂ , 37°C incubator and co-incubated for 6 hours. One hour before the scheduled detection point, the 96-well plate was removed from the cell incubator and Lysis Solution (10×) was added to the "sample maximum enzyme activity control well" at an amount of 10% of the original culture solution volume. After adding Lysis Solution, repeatedly pipet and mix, and then continue to culture in the incubator. After reaching the co-incubation time, add the detection solution and detect on the microplate reader. Cytotoxicity (%) = (RLU min–RLU sample)/(RLU min–RLU max)×100.

The calculated results are shown in Figure 9. After the addition of NK92 cells, HER2-CAR-NK92 cells and sGP130-HER2-CAR-NK92 cells, the killing effect of CAR-T cells on JIMT-1 cells was correspondingly enhanced. Among them, the killing effect of sGP130-HER2-CAR-NK92 cells on JIMT-1 cells was the most obvious, which enhanced the killing effect of CAR-T cells on JIMT-1 cells by about 2 times.

The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (10)

1. A nucleic acid construct, characterized in that the nucleic acid construct comprises: an extracellular antigen binding region coding sequence, a transmembrane region coding sequence, an intracellular signal transduction region coding sequence and an sGP130 coding sequence connected in sequence, The extracellular antigen binding region coding sequence, the transmembrane region coding sequence, and the intracellular signal transduction region coding sequence constitute the CAR molecule coding sequence, and the sGP130 coding sequence and the CAR molecule coding sequence are connected by a cleavage peptide, and the cleavage peptide includes: F2A, T2A, E2A or P2A; The amino acid sequence of sGP130 is shown in SEQ ID NO: 1. 2. The nucleic acid construct according to claim 1, characterized in that the extracellular antigen binding region recognizes any tumor-specific or tumor-related antigen, including HER2, EphA2, GD2, EpCAM, PD-1, PD-L1, LeY, CEA, EGFR, GPC3, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4EGFR, NY-ESO-1, MAGE4, CD19, CAIX, CD123, CD33, IL13R, LMP1, PLAC1, MUC1, MUC16; the transmembrane region is selected from any one of CD8, CD28, 4-1BB, DAP10, DAP12, NKG2D, DNAM1 or a combination of at least two molecules; The intracellular signal transduction domain contains CD3ζ. 3. The nucleic acid construct according to claim 2, characterized in that the intracellular signal transduction region further comprises any one of CD8, CD28, 4-1BB, DAP10, DAP12, NKG2D, DNAM1 or a combination of intracellular domains of at least two molecules. 4. The nucleic acid construct according to any one of claims 1 to 3, wherein the nucleotide sequence of the gene encoding sGP130 is shown in SEQ ID NO: 2. 5. The nucleic acid construct according to claim 2 or 3, characterized in that a coding sequence of a FLAG, His or HA tag is added to the front end of the coding sequence of the extracellular antigen binding region. 6. A vector, characterized in that the vector contains the nucleic acid construct according to any one of claims 1 to 5. 7. An immune cell, characterized in that the immune cell contains the nucleic acid construct described in any one of claims 1-5, and the immune cell is modified with one or more CAR molecules; the immune cell overexpresses sGP130, and the immune cell is a NK cell. 8. The immune cell according to claim 7, characterized in that the NK cells include NK cells taken from the human body or NK cells induced in vitro by stem cells, memory NK cells and NK cell strains. 9. A method for preparing the immune cell according to claim 7, characterized in that the method comprises: transferring the nucleic acid construct according to any one of claims 1 to 5 into the immune cell. 10. Use of the nucleic acid construct according to any one of claims 1 to 5, the vector according to claim 6, or the immune cell according to claim 7 or 8, characterized in that it is used for preparing a drug for inhibiting tumor cells.