CN105924528B - Chimeric antigen receptor and its gene and recombinant expression vector, CARMSLN-NKT cell and its preparation method and application - Google Patents

Abstract

The invention discloses a chimeric antigen receptor and its gene and recombinant expression vector, CARMSLN-NKT cells and a preparation method and application thereof. The chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3ζ, comprising a series of large Murine growth hormone signal peptide, MSLNScFv, hinge and transmembrane regions of CD8, intracellular signaling domain of CD137, and intracellular signaling domain of CD3ζ. When the CARMSLN-NKT cells of the present invention are co-cultured with MSLN-positive mesothelioma cells, they have good specific killing activity on mesothelioma cells.

Description

Chimeric antigen receptor and its gene and recombinant expression vector, CARMSLN-NKT cells and Preparation method and application thereof

technical field

The invention belongs to the field of tumor biological products, and in particular, relates to a chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ in adoptive immunotherapy and its gene and recombinant expression vector, engineered MSLN-targeted NKT cells (CARMSLN -NKT cells) and preparation methods and applications thereof.

Background technique

Natural killer cells (NKT) are a special type of T lymphocyte subset with dual properties of T cells and NK cells. NKT cells can express two receptors, TCR of T cells and NKR-P1 of NK cells. Under the mediation of TCR and NKR, NKT cells can produce a large amount of IL-4 and INFγ, which can kill tumor cells. NKT cells bind to the Fc segment of specific antibodies through CD16 on their own surface, and exert antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity). However, in the process of antibody-dependent cell-mediated killing, because the antibody can specifically bind to the corresponding epitope on the target cell, NKT cells can kill any target cell that has been bound to the antibody, so the antibody and the target cell on the target cell can be killed. Antigen binding is specific, but killing of target cells by NKT cells is nonspecific. In addition, under normal circumstances, the half-life of infused NKT cells in patients is about 2 weeks, the validity period is short, and repeated infusions are required. Moreover, NKT cells themselves lack specific antibodies, which are insufficient to enrich around tumors or in tumor nests, which restricts the targeted therapy of NKT cells for malignant tumors. Furthermore, studies have shown that NKT cells do not have a killing effect on all tumors, and the killing effect on some tumors is relatively weak, and the specific killing activity needs to be improved.

Mesothelin (MSLN) is a 40kDa surface glycoprotein, which is expressed on the surface of various tumor cells, such as malignant mesothelioma, pancreatic tumor, ovarian cancer, etc. Among them, the expression rate of MSLN in malignant pleural mesothelioma reaches more than 90%. In normal tissues, it showed low expression. Studies have found that MSLN is highly correlated with tumor progression, metastasis and poor patient survival. Due to the above properties, the MSLN antigen is an ideal target for antibody-based therapy. At present, a large number of studies have used MSLN monoclonal antibodies or MSLN vaccines to target and kill MSLN-positive tumor cells. However, these programs have certain defects on the basis of producing certain effects, such as: MSLN monoclonal antibody targeting Transient effects and insufficient distribution of MSLN monoclonal antibodies at tumor sites.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the relatively weak killing effect of NKT cells on tumors in the prior art, the specific killing activity needs to be improved, and the above-mentioned defects of MSLN monoclonal antibody and vaccine targeted therapy in existing clinical studies, provide a kind of Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ, gene and recombinant expression vector thereof, engineered MSLN-targeted NKT cells (CARMSLN-NKT cells), and preparation methods and applications thereof.

The inventors of the present invention unexpectedly found in the research that when the NKT cells modified with the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ of the present invention are co-cultured with MSLN-positive mesothelioma cells, they have a very strong effect on mesothelioma cells. Good specific killing activity.

Therefore, in order to achieve the above object, in the first aspect, the present invention provides a chimeric antigen receptor, the chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3ζ, including tandem rat growth hormone signal peptide, MSLNScFv , the hinge region (hinge region) and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ.

In a second aspect, the present invention provides a gene encoding the above-mentioned chimeric antigen receptor.

In a third aspect, the present invention provides a recombinant expression vector containing the above-mentioned gene.

In a fourth aspect, the present invention provides an engineered MSLN-targeted NKT cell, wherein the NKT cell is the above-mentioned chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ modified NKT cell.

In a fifth aspect, the present invention provides a method for preparing engineered MSLN-targeted NKT cells, the method comprising: packaging a lentivirus carrying pWPXL-MSLNScFv-CD8-CD137-CD3ζ to obtain a virus concentrate; using the obtained The virus concentrate infects NKT cells, so that NKT cells express the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ.

In a sixth aspect, the present invention provides the engineered MSLN-targeted NKT cells prepared by the above method.

In a seventh aspect, the present invention provides the application of the above engineered MSLN-targeted NKT cells in the preparation of a preparation for treating tumors.

When co-cultured with MSLN-positive mesothelioma cells, the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ modified NKT cells of the present invention, that is, engineered MSLN-targeted NKT cells, can specifically bind to MSLN antigens, Enhance the ability of immune cells to target and recognize MSLN antigens on the surface of cancer cells, and enhance the specific killing activity of MSLN-positive mesothelioma cells. The engineered MSLN-targeted NKT cells of the present invention provide a new option for treating MSLN-positive tumors, and have good industrial application prospects.

Other features and advantages of the present invention will be described in detail in the detailed description that follows.

Description of drawings

Figure 1 shows the results of phenotype analysis of isolated and cultured NKT cells by flow cytometry.

Figure 2 is the electrophoresis identification diagram of the double restriction endonuclease MluI/NdeI fragment of the lentiviral expression vector pWPXL-CD8-CD137-CD3ζ of the present invention.

Figure 3 is the electrophoresis identification diagram of the double restriction endonuclease BamHI/NdeI fragment of the lentiviral expression vector pWPXL-MSLNScFv-CD8-CD137-CD3ζ of the present invention.

4 is a schematic structural diagram of the lentiviral expression vector pWPXL-MSLNScFv-CD8-CD137-CD3ζ of the present invention, wherein the counterclockwise sequence is the forward gene segment, and the clockwise sequence is the reverse gene segment.

Figure 5 shows the infection efficiency of NKT cells by the virus concentrate containing the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ detected by flow cytometry.

Figure 6 shows the results of phenotype identification of chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ modified NKT cells (CARMSLN-NKT cells) detected by flow cytometry.

Fig. 7 is a graph showing the cytotoxicity analysis of the killing effect of CARMSLN-NKT cells of the present invention on MSLN-positive mesothelioma cells.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

The present invention provides a chimeric antigen receptor, the chimeric antigen receptor is MSLNScFv-CD8-CD137-CD3ζ, including tandem rat growth hormone signal peptide, MSLN single-chain antibody MSLNScFv, hinge region of CD8 and cross Membrane region, intracellular signaling domain of CD137 and intracellular signaling domain of CD3ζ.

Preferably, the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ is composed of rat growth hormone signal peptide, MSLNScFv, the hinge and transmembrane regions of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ Constructed in series. Further preferably, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO. 1, and even more preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 1.

The present invention provides a gene encoding the above-mentioned chimeric antigen receptor. Preferably, the gene has the nucleotide sequence shown in SEQ ID NO. 2, and more preferably, the nucleotide sequence of the gene encoding the above-mentioned chimeric antigen receptor is shown in SEQ ID NO. 2.

The present invention provides a recombinant expression vector containing the above-mentioned gene. Preferably, the recombinant expression vector is a lentiviral expression vector. The lentiviral expression vector is not particularly limited, as long as it can co-transfect packaging cells such as 293T packaging cells with the helper vector to obtain virus concentrate and chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ modified NKT cells, preferably In this case, the lentiviral expression vector is pWPXL-MSLNScFv-CD8-CD137-CD3ζ.

There is no particular limitation on the preparation method of the lentiviral expression vector pWPXL-MSLNScFv-CD8-CD137-CD3ζ, and various methods can be imagined by those skilled in the art. Preferably, the lentiviral expression vector pWPXL-MSLNScFv-CD8-CD137 -The preparation method of CD3ζ comprises the following steps:

(1) Amplify the hinge region and transmembrane region of CD8, the intracellular signal domain of CD137 and the intracellular signal domain of CD3ζ from NKT cell cDNA, respectively, and clone them into the vector pWPXL-GFP to construct pWPXL-CD8 -CD137-CD3ζ;

(2) The nucleotide sequences encoding rat growth hormone signal peptide and MSLNScFv were synthesized and cloned into pWPXL-CD8-CD137-CD3ζ. After sequencing and verification, the correct sequence of pWPXL-MSLNScFv-CD8-CD137-CD3ζ was obtained.

In step (1), the method for respectively amplifying the hinge region and transmembrane region of CD8, the intracellular signal domain of CD137 and the intracellular signal domain of CD3ζ from NKT cell cDNA is not particularly limited, and can be in the art. Various commonly used methods, for example, RT-PCR method can be used. Among them, NKT cells can be obtained by isolating mononuclear cells from human venous blood and then culturing them.

Specifically, the method for obtaining pWPXL-CD8-CD137-CD3ζ may include: extracting total RNA of NKT cells, reverse transcription to obtain NKT cell cDNA, using the obtained NKT cell cDNA as a template, using primers P1 (SEQ ID NO. 11) and P2 (SEQ ID NO. 12) PCR amplification was performed to obtain the hinge region and transmembrane region of the CD8 gene (SEQ ID NO. 3); primers P3 (SEQ ID NO. 13) and P4 (SEQ ID NO. 14) were used for PCR amplification to obtain the CD137 gene. Intracellular signaling domain (SEQID NO.4); using primers P5 (SEQID NO.15) and P6 (SEQID NO.16) to carry out PCR amplification to obtain the intracellular signaling domain (SEQID NO.5) of CD3ζ gene. The obtained PCR products were subjected to double digestion respectively, and then ligated with the lentiviral expression vector pWPXL-GFP after double digestion with MluI/NdeI.

In step (2), the method for synthesizing the nucleotide sequences encoding rat growth hormone signal peptide and MSLNScFv is not particularly limited, and can be various methods commonly used in the art, for example, can be synthesized by whole gene synthesis technology.

Specifically, the method for obtaining pWPXL-MSLNScFv-CD8-CD137-CD3ζ with the correct sequence may include: synthesizing a nucleotide sequence (SEQ ID NO. In the vector pGSI, pGSI-MSLNScFv is obtained; then pGSI-MSLNScFv is subjected to BamHI/MluI double digestion, and is connected with the recombinant plasmid pWPXL-CD8-CD137-CD3ζ obtained in step (1) after BamHI/MluI double digestion, After sequencing, the correct sequence of pWPXL-MSLNScFv-CD8-CD137-CD3ζ was obtained. The nucleotide sequence of rat growth hormone signal peptide is shown in SEQ ID NO.6, and the nucleotide sequence of MSLNScFv is shown in SEQ ID NO.7.

The present invention also provides an engineered MSLN-targeted NKT cell, wherein the NKT cell is an NKT cell modified by the above-mentioned chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ (ie, CARMSLN-NKT cell).

The present invention also provides a method for preparing engineered MSLN-targeted NKT cells, the method comprising: packaging a lentivirus carrying pWPXL-MSLNScFv-CD8-CD137-CD3ζ to obtain a virus concentrate; using the obtained virus concentrate NKT cells were infected to express the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ.

The method for packaging the lentivirus carrying pWPXL-MSLNScFv-CD8-CD137-CD3ζ is not particularly limited, and can be various methods commonly used by those skilled in the art. Preferably, the lentivirus expression vector pWPXL-MSLNScFv-CD8-CD137 -CD3ζ and helper plasmids (such as psPAX2, pMD2.G) were co-transfected into 293T packaging cells, and the virus supernatant was collected 48-72 hours after transfection, centrifuged and filtered, and 5×PEG6000-NaCl was added to the filtrate for mixing. After centrifugation The supernatant was discarded, and the pellet was dissolved in sterile PBS pre-cooled at 0-4°C to obtain a virus concentrate.

In the method of the present invention, it also includes preparing NKT cells by the following methods:

(1) In the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, mononuclear cells are cultured in the first stage;

(2) In the presence of interleukin-2, the cells cultured in the first stage are cultured in the second stage.

Preferably, the embodiment of the first stage of culture includes: culturing mononuclear cells in a first NKT cell culture medium, the first NKT cell culture medium containing NKT cell culture medium, CD3 monoclonal antibody, interleukin- 2 and interleukin-15; further preferably, in the first NKT cell culture medium, the concentration of the CD3 monoclonal antibody is 30-70ng/mL, and/or the concentration of the interleukin-2 is 300-700U/mL, And/or the concentration of interleukin-15 is 30-70 ng/mL.

Preferably, the embodiment of the second stage culture includes: culturing the cells cultured in the first stage in a second NKT cell culture medium, the second NKT cell culture medium containing NKT cell culture medium and interleukin -2; further preferably, in the second NKT cell culture medium, the concentration of the interleukin-2 is 300-700 U/mL.

The NKT cell medium is not particularly limited, and can be any medium commonly used in the art for culturing NKT cells, for example, GT-T551 medium.

When preparing NKT cells, the conditions for the first-stage culture and the second-stage culture are not particularly limited, and various conditions commonly used in the field can be used, for example, at 30-37°C, saturated humidity of 3-6% CO ₂ culture in an incubator. Those skilled in the art can adjust the culture time adaptively, which is well known to those skilled in the art and will not be repeated here.

In the NKT cells prepared by the present invention, the average ratio of CD3 ⁺ cells is >90%, the average ratio of CD3 ⁺ CD8 ⁺ cells to the total CD3 ⁺ cells is >70%; the average ratio of CD3 ⁺ CD56 ⁺ cells to the total CD3 ⁺ cells is > 15 %.

The method for infecting NKT cells is not particularly limited, and can be various methods commonly used in the art. Preferably, the method includes:

(1) In the presence of virus concentrate, protamine and interleukin-2, NKT cells were cultured in the first stage of infection;

(2) In the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, the first-stage infection cultured cells are subjected to second-stage infection culture.

Preferably, the embodiment of the first-stage infection culture includes: culturing NKT cells in a third NKT cell culture medium, the third NKT cell culture medium containing NKT cell culture medium, virus concentrate, protamine and Interleukin-2; further preferably, in the third NKT cell culture medium, the concentration of interleukin-2 is 300-700 U/mL.

Preferably, the embodiment of the second-stage infection culture comprises: culturing the cells of the first-stage infection culture in the first NKT cell culture medium. For the specific composition of the first NKT cell culture medium, reference may be made to the foregoing corresponding content, which will not be repeated here.

When infecting NKT cells, the conditions of the first-stage infection culture and the second-stage infection culture are not particularly limited, and can be various conditions commonly used in the field, for example, the temperature can be 30-37°C and a saturated humidity of 3-6% cultured in a CO ₂ incubator. Those skilled in the art can adjust the culture time adaptively, which is well known to those skilled in the art and will not be repeated here.

Specifically, the method for infecting NKT cells includes: taking 1×10 ⁷ -5×10 ⁷ NKT cells, discarding the old culture medium, adding 2-4 mL of fresh GT-T551 culture medium, and then adding 200-400 μL of virus concentrate , 2-4 μL of 1× ^10-6 mg/mL protamine and IL-2 with a final concentration of 300-700 U/mL, placed in a _CO2 incubator at 30-37°C and a saturated humidity of 3-6% for infection After 12-16h, discard the culture medium, transfer the cells to an uncoated culture flask, add 20-50mL of GT-T551 medium, and then add IL-2 with a final concentration of 300-700U/mL, and the final concentration is 30-70ng/ml of CD3 monoclonal antibody and interleukin 15 at a final concentration of 30-70ng/mL were incubated at 30-37°C in a _CO2 incubator with a saturated humidity of 3-6% for 12-18h to obtain Chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ modified NKT cells.

Further preferably, the method for infecting NKT cells also includes:

(3) First, in the presence of interleukin-2, the cells infected and cultured in the second stage are induced in vitro, and when the density of the cells is 80-90%, the CD3 monoclonal antibody, interleukin-2 and interleukin-15 In the presence of cells, the cells are expanded and cultured.

Preferably, the embodiment of the in vitro induction includes: culturing the cells cultured in the second stage of infection in the second NKT cell culture medium, and the embodiment of the expansion culture includes: culturing the cells in the second NKT cell culture medium. in the first NKT cell culture medium. For the specific composition of the first NKT cell culture solution and the second NKT cell culture solution, reference may be made to the aforementioned corresponding contents, which will not be repeated here.

Specifically, the method for infecting NKT cells further includes: inducing the lentivirus-infected NKT cells obtained after the second stage of infection and culture with GT-T551 culture medium with a final concentration of IL-2 of 300-700 U/mL in vitro. When the cell density is 80-90%, the cells are transferred to cell culture bags, and the final concentration of IL-2 is 300-700U/mL, and the final concentration of CD3 monoclonal antibody is 30-70ng/ml every 1.5-2.5 days. , the final concentration of interleukin-15 is 30-70ng/mL fresh GT-T551 culture medium for expansion culture and the cells are expanded to a total of 1×10 ⁹ -2×10 ⁹ cells. The chimeric antigen receptor targeting MSLN antigen is infected with NKT cells by the lentivirus of the present invention, and the infection efficiency is as high as 30%-60%, while the obtained CARMSLN-NKT cells, the CD3 ⁺ CD56 ⁺ cells of the total CD3 ⁺ The ratio of cells is above 15%.

The protein amino acid sequence of the chimeric antigen receptor expressed by the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ modified NKT cells is shown in SEQ ID NO.1. Among them, those skilled in the art should understand that the chimeric antigen receptor precursor protein consists of rat growth hormone signal peptide, MSLNScFv, the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137, and the intracellular signal domain of CD3ζ. The signal domain is composed of tandem. After the protein is translated, the signal peptide is excised in the rough endoplasmic reticulum to become a mature chimeric antigen receptor protein, which is secreted and exported and localized on the cell membrane of NKT cells. The gene coding sequence corresponding to the protein amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.2. The chimeric antigen receptor is a signal transduction domain formed by the concatenation of the hinge region and transmembrane region of the gene CD8 and the intracellular signal domains of CD137 and CD3ζ, and its amino acid sequence is shown in SEQ ID NO. The gene coding sequence is shown in SEQ ID NO.10.

The present invention also provides the engineered MSLN-targeted NKT cells prepared by the above method.

The present invention also provides the application of the engineered MSLN-targeted NKT cells in the preparation of preparations for treating tumors. Preferably, the tumor is MSLN-positive mesothelioma, more preferably, the tumor is MSLN-positive malignant pleural mesothelioma.

In the application of the present invention, the composition of the preparation for treating MSLN-positive tumors is not particularly limited, as long as it contains the CARMSLN-NKT cells of the present invention or is prepared from CARMSLN-NKT cells. The specific composition of the preparation and preparation methods are well known to those skilled in the art and will not be repeated here.

Example

The following examples will further illustrate the present invention, but do not limit the present invention accordingly.

The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores, wherein:

NKT cell culture medium GT-T551 was purchased from TaKaRa Company.

Lymphocyte separation medium was purchased from TBD Company.

CD3 monoclonal antibody and recombinant fibronectin (retronectin) were purchased from TaKaRa Company.

Recombinant human protein interferon-γ, recombinant human interleukin-2, and recombinant human interleukin-15 were purchased from protech company.

Total RNA extraction kit RNAiso Reagent, high-fidelity DNA polymerase ( HS DNA Polymerase) and T4 DNA ligase were purchased from TaKaRa Company.

RevertAid ^™ First Strand cDNA Synthesis Kit was purchased from Fermentas.

BglII, EcoRI, MluI, BamHI, NdeI, EcoRV were purchased from Fermentas Company.

Agarose gel DNA recovery kit, common DNA product purification kit, and plasmid mini-purification kit were purchased from Tiangen Biochemical Technology Co., Ltd.

pWPXL-GFP, psPAX2, pMD2.G were purchased from Addgene Company.

pGSI was purchased from Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

Trans1-T1 Phage Resistant chemically competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

Lipofectamine ^™ 2000Transfection Reagent was purchased from Invitrogen.

293T packaging cells were purchased from ATCC, USA.

The final concentration of PEG6000 in PEG6000-NaCl was 25.5% by mass, and the final concentration of NaCl was 1.2 M. Both PEG6000 and NaCl were purchased from Shanghai Soleibo Biotechnology Co., Ltd.

Fetal bovine serum was purchased from PAA, Germany.

The MSLN-positive mesothelioma cell line NCI-H2452 was purchased from ATCC Company in the United States.

5-Carboxyfluorescein succinimidyl ester was purchased from Shanghai Puzhen Biotechnology Co., Ltd.

Annexin V-RPE kit was purchased from BD Company of the United States.

All primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

Example 1 Preparation of NKT cells

(1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by separation of lymphocyte separation medium by density gradient centrifugation.

(2) After washing the PBMCs three times, the final cell concentration was adjusted to 2×10 ⁶ cells/mL with NKT cell medium GT-T551 containing 0.6 vol% human autologous serum; mL of retronectin-coated 75cm ² cell culture flask. Recombinant human interleukin-2, 50ng/ml CD3 monoclonal antibody, and 50ng/mL recombinant human interleukin-15 were then added to the culture medium at a final concentration of 500 U/mL, and the medium was incubated at 37°C in a _CO2 incubator with a saturated humidity of 5%. cultivated in.

(3) On the 4th day of culture, the cells were transferred to uncoated culture flasks, NKT cell culture medium GT-T551 was added every 2 days according to the cell growth number, and the control cell concentration was 1×10 ⁸ cells/mL, and Recombinant human interleukin-2 at a final concentration of 500 U/ml was added; NKT cells were obtained on the 12th day after culture, and the phenotype of NKT cells was analyzed by flow cytometry. The results are shown in Figure 1, wherein, CD3 ⁺ : 95.04%; CD3 ⁺ CD8 ⁺ : 90.99%; CD3 ⁺ CD56 ⁺ : 24.12%; CD8 ⁺ CD56 ⁺ : 24.63%.

Example 2 Construction of lentiviral expression vector pWPXL-MSLNScFv-CD8-CD137-CD3ζ

(1) Preparation of NKT cell cDNA

The NKT cells cultured in Example 1 were precipitated by centrifugation, the total RNA of the cells was extracted with a total RNA extraction kit RNAiso Reagent, and stored at -80°C for later use. The extracted total RNA was reverse transcribed with RevertAid ^™ First StrandcDNA Synthesis Kit to obtain NKT cell cDNA, which was stored at -20°C for future use.

(2) Preparation of lentiviral plasmid pWPXL-CD8-CD137-CD3ζ

The following primer sequences were designed and synthesized (wherein, underlined marks are protected bases, and boxes are restriction sites):

P1 (SEQ ID NO. 11): GATC CTGAGCAACTCCATCATGTACTTC

MluI

P2 (SEQ ID NO. 12): GATC GCAGTAAAGGGTGATAACCAGTGA

BglII

P3 (SEQ ID NO. 13): GATC AAACGGGGCAGAAAGAAACTCC

BglII

P4 (SEQ ID NO. 14): GATC CAGTTCACATCCTCCTTCTTCTTTCT

EcoRI

P5 (SEQ ID NO. 15): GATC AGAGTGAAGTTCAGCAGGAGCG

EcoRI

P6 (SEQ ID NO. 16): GATC ATAATCAACCTCTGGATTAC

NdeI

Using the NKT cell cDNA in step (1) as a template, use primers P1 and P2 to carry out PCR amplification to obtain the hinge region and transmembrane region of CD8 with a length of 287bp. The nucleotide sequence is shown in SEQID NO.3, and the two ends are respectively Contains MluI and BglII restriction sites and protective bases; PCR amplification is performed with primers P3 and P4 to obtain a CD137 intracellular signal domain with a length of 146bp. The nucleotide sequence is shown in SEQID NO.4, and both ends contain BglII and EcoRI restriction sites and protective bases; PCR amplification was performed with primers P5 and P6 to obtain the intracellular signal domain of CD3ζ with a length of 359bp. The nucleotide sequence is shown in SEQID NO.5, and both ends contain EcoRI and NdeI restriction sites and protected bases. The PCR amplification reaction system of each step is the same. Taking the amplification of the intracellular signal domain of CD137 as an example, PCR amplification is performed. The PCR reaction conditions refer to Instructions for HS DNA Polymerase, the reaction system (50 μL) is as follows:

Double distilled water: 32.5 μL

5× reaction buffer: 10 μL

dNTP mix (2.5mM each): 4μL

P3 (10mM): 1μL

P4 (10mM): 1μL

NKT cell cDNA (200ng/ul): 1μL

HS DNA Polymerase: 0.5 μL

The above PCR products were separated by 1% agarose gel, and DNA fragments were recovered by agarose gel DNA recovery kit. After the fragments are obtained, double-enzyme digestion reactions are carried out respectively, and the digestion products are recovered with a common DNA product purification kit for use.

The lentiviral expression vector pWPXL-GFP was double digested with MluI/NdeI, and the digested product was separated by 1% agarose gel. The large vector fragment was recovered by the agarose gel DNA recovery kit, and then combined with the previously recovered CD8 , CD137, and CD3ζ fragments were ligated by T4 DNA ligase, and the ligation product was transformed into Trans1-T1 Phage Resistant chemically competent cells. After culturing at 37 °C for 16 h, single clones were picked, and after culturing at 37 °C for 12 h at 250 rpm, plasmids were extracted with a plasmid mini kit. The extracted plasmid was identified by restriction endonucleases MluI and NdeI double digestion, and the identification electropherogram was shown in Figure 2, where M1: DNA molecular weight marker D15000; Lane 1: the undigested fragment of plasmid pWPXL-CD8-CD137-CD3ζ; Lane 2: restriction fragment of plasmid pWPXL-CD8-CD137-CD3ζ (751bp); M2: DNA molecular weight marker D2000. Send the correctly identified plasmid to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to sequence the inserted fusion gene fragment. The recombinant plasmid with correct sequencing results was named pWPXL-CD8-CD137-CD3ζ, wherein the nucleotide sequences of the hinge region and transmembrane region of CD8 were shown in SEQ ID NO.3, and the nucleosides of the intracellular signal domain of CD137 The acid sequence is shown in SEQ ID NO. 4, and the nucleotide sequence of the intracellular signaling domain of CD3ζ is shown in SEQ ID NO. 5.

(3) Preparation of lentiviral plasmid pWPXL-MSLNScFv-CD8-CD137-CD3ζ

Whole-gene synthesis of the nucleotide sequence encoding the fusion gene of rat growth hormone signal peptide and MSLNScFv, the sequence is shown in SEQID NO. , the 3' end contains a MluI restriction site, and the aforementioned fusion gene was cloned into the plasmid pGSI, named pGSI-MSLNScFv. The plasmid was digested by BamHI/MluI double enzyme, the digested product was separated by 1% agarose gel, and the target fragment was recovered by agarose gel DNA recovery kit for future use.

The pWPXL-CD8-CD137-CD3ζ plasmid was digested with restriction enzymes BamHI/MluI, and the digested products were separated by 1% agarose gel, and the vector fragment was recovered with agarose gel DNA recovery kit for use. Then, it is ligated with the recovered DNA fragment containing rat growth hormone signal peptide and MSLN ScFv by T4 DNA ligase, and the specific method is shown in the instructions. The ligation product was transformed into Trans1-T1 Phage Resistant chemically competent cells, single clones were picked after culturing at 37°C for 16h, and after culturing at 37°C and 250rpm for 12h, plasmids were extracted with a plasmid mini-kit. The extracted plasmid was identified by restriction endonuclease BamHI/NdeI double digestion, and the identification result is shown in Figure 3, wherein, M1: DNA molecular weight marker D15000; 1 lane: plasmid pWPXL-MSLNScFv-CD8-CD137-CD3ζ Restriction fragment (11254bp); 2 lanes: restriction fragment (1579bp) of plasmid pWPXL-MSLNScFv-CD8-CD137-CD3ζ; M2: DNA molecular weight marker D2000. Send the correctly identified plasmid to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to sequence the inserted fusion gene fragment. The recombinant plasmid with the correct sequencing result was named pWPXL-MSLNScFv-CD8-CD137-CD3ζ, and its schematic diagram is shown in Figure 4, including rat growth hormone signal peptide (nucleotide sequence shown in SEQID NO.6), Anti-MSLN single-chain antibody (nucleotide sequence shown in SEQ ID NO. 7), the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ, wherein the chimeric antigen The receptor uses the hinge region and transmembrane region of the gene CD8 and the intracellular signaling domain of CD137 and CD3ζ as the signal transduction domain. Its amino acid sequence is shown in SEQID NO.9, and the corresponding gene coding sequence is shown in shown in SEQ ID NO.10.

Example 3 Preparation of Chimeric Antigen Receptor MSLNScFv-CD8-CD137-CD3ζ Modified NKT Cells

(1) Packaging and enrichment of lentivirus

The concentrations of the lentiviral expression plasmid pWPXL-MSLNScFv-CD8-CD137-CD3ζ and the helper plasmids psPAX2 and pMD2.G were measured with a spectrophotometer. The three plasmids were transfected with Lipofectamine ^TM 2000 Transfection Reagent at a mass ratio of 4:2:1. 293T packaging cells were co-transfected. 48h and 72h of transfection, respectively, collect the virus supernatant in a 50mL EP tube, centrifuge at 2000g for 10min at 4°C, transfer the supernatant obtained twice to a new EP tube, and filter the virus supernatant with a 4.5μm filter; the filtered virus Mix the supernatant and 5×PEG6000-NaCl at a volume ratio of 4:1, let stand at 4°C for 2 hours, then centrifuge at 10000g for 20 minutes at 4°C, discard the supernatant, and dissolve the precipitate with 1 mL of 4°C pre-cooled sterile PBS. That is, the virus concentrate of chimeric antigen receptor is obtained, which is divided into 100 μL per tube, and stored at -80°C for later use.

According to the above method, 293T packaging cells were co-transfected with the lentivirus expression plasmid pWPXL-GFP and the helper plasmids psPAX2 and pMD2.G, and the virus supernatant was collected and concentrated to obtain a lentivirus concentrate expressing GFP green fluorescent protein.

(2) Lentiviral infection of NKT cells and expansion of infected cells

Take 1×10 ⁷ NKT cells cultured in a 75cm ² culture flask of Example 1, discard the old culture medium, add 2 mL of fresh NKT cell culture medium GT-T551, 200 μL of the virus concentrate obtained in step (1), 2 μL of 1×10 ^-6 mg/mL protamine, recombinant human interleukin 2 with a final concentration of 500 U/mL was placed in a CO ₂ incubator at 37°C and a saturated humidity of 5% for 12 hours after infection, and the culture medium was discarded. Simultaneously, NKT cells were synchronously infected with lentivirus concentrate expressing GFP green fluorescent protein (the obtained NKT cells were called CART-GFP cells), and the infection efficiency of the virus was calculated. Transfer the infected cells to a 75cm ² culture flask without CD3 and retronectin coating, add 20mL of NKT cell culture medium GT-T551, and then add recombinant human interleukin 2 with a final concentration of 500U/mL, with a final concentration of 50ng /ml of CD3 monoclonal antibody and recombinant human interleukin-15 with a final concentration of 50ng/mL were cultured in a _CO2 incubator with a saturated humidity of 5% at 37°C for 18h, and the obtained NKT cells were called CARMSLN-NKT cells. CARMSLN-T cells were prepared by the same method (for the preparation method of T cells, refer to the literature: YajingZhang, et al. Autologous CIK Cell Immunotherapy in Patients with Renal Cell Carcinoma after Radical Nephrectomy. Clinical Study, 2013, Section 2.4, CIK Cell Preparation Method). The infection efficiency of the virus was detected by flow cytometry. The results are shown in Figure 5. The infection efficiency of CARMSLN-NKT cells was 49.21%.

(3) In vitro expansion of CARMSLN-NKT cell population

The cultured NKT cells were induced in vitro with NKT cell culture medium GT-T551 with a final concentration of recombinant human interleukin 2 of 500 U/mL. The final concentration of recombinant human interleukin 2 was 500 U/mL, the final concentration of CD3 monoclonal antibody was 50 ng/ml, and the final concentration of recombinant human interleukin 15 was 50 ng/mL. After the cells were expanded to a total of about 1.5×10 ⁹ cells, the infected cell population was identified by flow cytometry, and the cell phenotype generally reached the proportion of CD3 positive cells >90%; the proportion of CD3CD8 double positive cells >70%; CD3CD56 double positive cells ratio>15%, the results are shown in Figure 6, CD3 ⁺ : 99.15%; CD3 ⁺ CD4 ⁺ : 14.97%; CD3 ⁺ CD8 ⁺ : 85.45%; CD3 ⁺ CD56 ⁺ : 41.60%; CD8 ⁺ CD56 ⁺ : 39.81%.

Example 4 Cytotoxicity analysis of the killing effect of CARMSLN-NKT cells on mesothelioma cells

The CARMSLN-NKT cells prepared in Example 3, the CARMSLN-T cells and the NKT cells cultured in Example 1 were seeded in 96-well plates, stained with 5-carboxyfluorescein succinimidyl ester (CFSE), and mixed with The MSLN-positive mesothelioma cell line NCI-H2452 was co-cultured at the ratio of effector to target (killer cells: target cells) 5:1, 10:1, 20:1, 40:1, after 24 hours of co-culture , the cells were stained with Annexin V-RPE kit, and the control group was set as the mesothelioma cell line NCI-H2452 without immune cell killing treatment, and the cells were stained with Annexin V-RPE kit. Cell apoptosis was detected by flow cytometry, and the amount of cell apoptosis was calculated according to the following formula: apoptosis rate=(control-sample)/control×100%, and the control was the number of cells that survived without immune cell killing treatment; The sample is the number of surviving cells killed by immune cells added with the corresponding effector-to-target ratio (killer cells:target cells), see Figure 7 . It can be seen from Figure 7 that NKT cells modified with chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ have specific killing activity against MSLN-positive mesothelioma cells, and the specific killing activity of CARMSLN-NKT cells is significantly better than that of CARMSLN-T cells. and NKT cells.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. In order to avoid unnecessary repetition, the present invention provides The combination method will not be specified otherwise.

In addition, the various embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the spirit of the present invention, they should also be regarded as the contents disclosed in the present invention.

Claims (6)

1. a preparation method of the NKT cell of engineered MSLN targeting, is characterized in that, described method comprises: Packaging the lentivirus carrying pWPXL-MSLNScFv-CD8-CD137-CD3ζ to obtain a virus concentrate; using the obtained virus concentrate to infect NKT cells to make NKT cells express the chimeric antigen receptor MSLNScFv-CD8-CD137-CD3ζ; The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1; The method of infecting NKT cells includes: Take 1×10 ⁷ NKT cells cultured in a 75cm ² culture flask, discard the old culture medium, add 2 mL of fresh NKT cell culture medium GT-T551, 200 μL of virus concentrate, 2 μL of 1×10 ^-6 mg/mL fish Protamine, recombinant human interleukin 2 with a final concentration of 500 U/mL, placed in a CO ₂ incubator at 37°C and a saturated humidity of 5% for 12 hours after infection, the culture medium was discarded; the infected cells were transferred to non-CD3 and recombinant fibronectin-coated ^75cm2 culture flask, add 20mL of NKT cell culture medium GT-T551, then add recombinant human interleukin 2 with a final concentration of 500U/mL, and CD3 monoclonal antibody with a final concentration of 50ng/mL. and recombinant human interleukin-15 with a final concentration of 50ng/mL, cultured in a _CO2 incubator with a saturated humidity of 5% at 37°C for 18h; The cultured NKT cells were induced in vitro with NKT cell culture medium GT-T551 with a final concentration of recombinant human interleukin 2 of 500 U/mL. The final concentration of recombinant human interleukin 2 was 500 U/mL, the final concentration of CD3 monoclonal antibody was 50 ng/ml, and the final concentration of recombinant human interleukin 15 was 50 ng/mL. . 2. The method of claim 1, wherein the method further comprises preparing NKT cells by: (1) In the presence of CD3 monoclonal antibody, interleukin-2 and interleukin-15, the mononuclear cells are cultured in the first stage; the embodiment of the first stage culture includes: culturing the mononuclear cells on the first NKT cells In the culture solution, the first NKT cell culture solution contains NKT cell culture medium, CD3 monoclonal antibody, interleukin-2 and interleukin-15; in the first NKT cell culture solution, the concentration of the CD3 monoclonal antibody is 30- 70ng/mL, and/or the concentration of the interleukin-2 is 300-700U/mL, and/or the concentration of the interleukin-15 is 30-70ng/mL; (2) In the presence of interleukin-2, the cells cultured in the first stage are cultured in a second stage; an embodiment of the second stage culture includes: culturing the cells cultured in the first stage in a second NKT In the cell culture solution, the second NKT cell culture solution contains NKT cell culture medium and interleukin-2; in the second NKT cell culture solution, the concentration of the interleukin-2 is 300-700 U/mL. 3. The engineered MSLN-targeted NKT cells prepared by the method of claim 1 or 2. 4. The application of the engineered MSLN-targeted NKT cells of claim 3 in the preparation of a preparation for treating tumors. 5. The use of claim 4, wherein the tumor is MSLN-positive mesothelioma. 6. The use according to claim 5, wherein the tumor is MSLN-positive malignant pleural mesothelioma.