CN105384822A - Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered CD138 targeting NKT cell and application thereof - Google Patents

Abstract

The invention discloses a chimeric antigen receptor and its gene and recombinant expression vector, engineered CD138-targeted NKT cells and applications thereof. The chimeric antigen receptor is CD138ScFv-2-CD8-CD137-CD3ζ, It consists of CD138ScFv-2, the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ in series. The nucleotide sequence of the gene encoding the CD138ScFv-2 is shown as SEQ? ID? Shown in NO.7. When the NKT cell modified by the chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ of the present invention is used to treat CD138-positive multiple myeloma, it has certain specific killing activity on myeloma cells.

Description

Chimeric antigen receptors and their genes and recombinant expression vectors, engineered CD138-targeted NKT cells and their applications

technical field

The invention belongs to the field of tumor biological products, and in particular relates to a chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ in adoptive immunotherapy, its gene and recombinant expression vector, and engineered CD138-targeted NKT cells (CAR138-2-NKT cells) and applications thereof.

Background technique

Natural killer cells (NKT) are a special type of T lymphocyte subsets with dual properties of T cells and NK cells. NKT cells can express 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 combine CD16 on their surface with the Fc region of specific antibodies to exert the ADCC (antibody-dependent cell-mediated cytotoxicity) effect. However, in the process of antibody-dependent cell-mediated killing, since 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 Antigen binding is specific, but the killing effect of NKT cells on target cells is non-specific. In addition, under normal circumstances, the half-life of infused NKT cells in the patient's body is about 2 weeks, and the validity period is short, requiring repeated infusions. In addition, NKT cells themselves lack specific antibodies, which are not enough to enrich around tumors or in tumor nests, which restricts the targeted therapy of NKT cells on malignant tumors. Moreover, 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.

Multiple myeloma (Multiple myeloma, MM) is a malignant tumor of B cells with abnormal proliferation of plasma cells, which is characterized by the accumulation of a large number of malignant plasma cells in the bone marrow and the secretion of monoclonal immunoglobulin. Myeloma cells express a series of adhesion molecules such as CD138 on the surface. CD138, a member of the transmembrane mucin glycan family, binds to growth factors and matrix components in the bone marrow microenvironment to mediate adhesion and homing of multiple myeloma cells. With the gradual deepening of the research on CD138, people found that CD138 played a key role in the damage repair of the body and the growth and migration of tumors. At present, CD138 is considered to be the most specific surface marker of plasma cells. Studies have shown that the expression of CD138 molecules in B-cell malignancies is very complex. It is expressed in plasma cells in bone marrow and peripheral blood of MM patients, while hematopoietic precursors in bone marrow Cells or lymphocytes do not express. The increase of CD138 molecular level can be used as an effective indicator of poor prognosis in myeloma, and when MM cells show apoptosis, the expression of CD138 molecules on the membrane surface is down-regulated or lost, which can reflect the burden of tumor cells in MM patients.

Contents of the invention

The purpose of the present invention is to provide a chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ and its gene and Recombinant expression vectors, engineered CD138-targeted NKT cells (CAR138-2-NKT cells) and their application, chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ modified NKT cells in the treatment of CD138-positive multiple In the case of myeloma, it has certain specific killing activity on myeloma cells.

The inventors of the present invention unexpectedly found in the research that the chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ of the present invention has a high infection efficiency for NKT cells, and the CAR138-2-NKT cells obtained after infection are in vitro It has efficient tumor killing activity, and when CAR138-2-NKT cells are used to treat CD138-positive multiple myeloma, it has certain specific killing activity on myeloma cells.

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 CD138ScFv-2-CD8-CD137-CD3ζ, which is composed of CD138 single-chain antibody CD138ScFv-2, The hinge region (hinge region) and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ are formed in series, and the nucleotide sequence of the gene encoding the CD138ScFv-2 is shown in SEQ ID NO.7 .

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 genes.

In a fourth aspect, the present invention provides an engineered CD138-targeted NKT cell, the NKT cell is the NKT cell modified by the chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ.

In a fifth aspect, the present invention provides an application of the above-mentioned engineered CD138-targeted NKT cells in preparing a preparation for treating tumors.

When treating CD138-positive multiple myeloma, the chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ of the present invention has a high infection efficiency for NKT cells, and the CAR138-2-NKT cells obtained after infection, namely The engineered CD138-targeted NKT cells can specifically bind to the CD138 antigen, have high-efficiency tumoricidal activity in vitro, significantly prolong the survival time of immune cells in patients, and enhance the ability of immune cells to target and recognize the CD138 antigen of myeloma cells , to enhance the specific killing activity on myeloma cells. The engineered CD138-targeted NKT cells of the present invention provide a new option for treating CD138-positive multiple myeloma, 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 is the result of flow cytometry analysis on the phenotype of isolated and cultured NKT cells.

Fig. 2 is an electrophoretic identification diagram of the restriction endonuclease MluI/SalI double-digestion fragment of the lentiviral expression vector pWPT-CD8-CD137-CD3ζ of the present invention.

Fig. 3 is an electrophoresis identification diagram of the restriction endonuclease BamHI/SalI double-digested fragment of the lentiviral expression vector pWPT-CD138ScFv-2-CD8-CD137-CD3ζ of the present invention.

Fig. 4 is a schematic structural diagram of the lentiviral expression vector pWPT-CD138ScFv-2-CD8-CD137-CD3ζ of the present invention, wherein the counterclockwise sequence is the forward gene fragment, and the clockwise sequence is the reverse gene fragment.

Fig. 5 shows the infection efficiency of virus concentrate containing chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ to NKT cells detected by flow cytometry.

Fig. 6 is the result of phenotype identification of NKT cells (CAR138-2-NKT cells) modified by chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ detected by flow cytometry.

Fig. 7 is a cytotoxic analysis diagram of the killing effect of CAR138-2-NKT cells of the present invention on human multiple myeloma cells.

detailed description

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

The present invention provides a chimeric antigen receptor, the chimeric antigen receptor is CD138ScFv-2-CD8-CD137-CD3ζ, which is composed of the CD138 single-chain antibody CD138ScFv-2, the hinge region and transmembrane region of CD8, and the CD137 The intracellular signaling domain and the intracellular signaling domain of CD3ζ are formed in series, and the nucleotide sequence of the gene encoding CD138ScFv-2 is shown in SEQ ID NO.7. Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1.

The present invention provides genes encoding the above-mentioned chimeric antigen receptors. Preferably, the nucleotide sequence of the gene encoding the chimeric antigen receptor is shown in SEQ ID NO.2.

The invention provides a recombinant expression vector containing the above gene. Preferably, the recombinant expression vector is a lentiviral expression vector. There are no special restrictions on the lentiviral expression vector, 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 CD138ScFv-2-CD8-CD137-CD3ζ modified NKT cells. , preferably, the lentiviral expression vector is pWPT-CD138ScFv-2-CD8-CD137-CD3ζ.

The method for preparing the lentiviral expression vector pWPT-CD138ScFv-2-CD8-CD137-CD3ζ is not particularly limited, and can be various methods conceivable by those skilled in the art. Preferably, the lentiviral expression vector pWPT-CD138ScFv-2 -The preparation method of CD8-CD137-CD3ζ comprises the following steps:

(1) The hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ were respectively amplified from NKT cell cDNA, and cloned into the vector pWPT-GFP to construct pWPT-CD8 - CD137-CD3ζ;

(2) Synthesize the nucleotide sequence encoding rat growth hormone signal peptide and CD138ScFv-2, and clone it into pWPT-CD8-CD137-CD3ζ, and obtain pWPT-CD138ScFv-2-CD8-CD137 with the correct sequence after sequencing verification -CD3ζ.

In step (1), there is no particular limitation on the methods for respectively amplifying the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD137 and the intracellular signaling domain of CD3ζ from NKT cell cDNA, which can be Various commonly used methods include, for example, RT-PCR. Among them, NKT cells can be obtained by isolating mononuclear cells in human venous blood and then culturing them.

Specifically, the method for obtaining pWPT-CD8-CD137-CD3ζ may include: extracting the 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 ( SEQIDNO.12) to perform PCR amplification to obtain the hinge region and transmembrane region (SEQIDNO.3) of the CD8 gene; use primers P3 (SEQIDNO.13) and P4 (SEQIDNO.14) to perform PCR amplification to obtain the intracellular signal of the CD137 gene Structural domain (SEQ ID NO.4); Utilize primer P5 (SEQ ID NO.15) and P6 (SEQ ID NO.16) to carry out PCR amplification to obtain the intracellular signaling domain (SEQ ID NO.5) of CD3ζ gene, the PCR product that will obtain is doubled respectively Restriction digestion, and then ligation with the lentiviral expression vector pWPT-GFP after MluI/SalI double digestion.

In step (2), the method for synthesizing the nucleotide sequence encoding rat growth hormone signal peptide and CD138ScFv-2 is not particularly limited, and can be various methods commonly used in the art, for example, it can be synthesized by total gene synthesis technology.

Specifically, the method for obtaining pWPT-CD138ScFv-2-CD8-CD137-CD3ζ with the correct sequence may include: synthesizing the nucleotide sequence (SEQ ID NO. 8), cloned into the vector pGSI to obtain pGSI-CD138ScFv-2; then pGSI-CD138ScFv-2 was subjected to BamHI/MluI double digestion, and the recombinant plasmid pWPT- CD8-CD137-CD3ζ was connected and identified by sequencing to obtain pWPT-CD138ScFv-2-CD8-CD137-CD3ζ with the correct sequence. Wherein, the nucleotide sequence of rat growth hormone signal peptide is shown in SEQ ID NO.6, and the nucleotide sequence of CD138ScFv-2 is shown in SEQ ID NO.7.

The present invention also provides an engineered CD138-targeted NKT cell, wherein the NKT cell is an NKT cell modified by the chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ (ie, CAR138-NKT cell).

There is no particular limitation on the preparation method of engineered CD138-targeted NKT cells, and it can be any method conceivable by those skilled in the art. Preferably, the method includes: packaging pWPT-CD138ScFv-2-CD8-CD137- A lentivirus of the CD3ζ encoding gene; the obtained lentivirus is used to infect NKT cells, so that the NKT cells express the chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ.

The method for packaging the lentivirus carrying the pWPT-CD138ScFv-2-CD8-CD137-CD3ζ encoding gene is not particularly limited, and can be various methods commonly used by those skilled in the art. Preferably, the lentiviral expression vector pWPT-CD138ScFv -2-CD8-CD137-CD3ζ and helper plasmids (such as psPAX2, pMD2.G) co-transfect 293T packaging cells, collect the virus supernatant at 48-72 hours after transfection, centrifuge, filter, add 5×PEG6000-NaCl to the filtrate Mix evenly, discard the supernatant after centrifugation, and dissolve the precipitate with 0-4°C pre-cooled sterile PBS to obtain a concentrated virus solution.

The method for infecting NKT cells with lentivirus is not particularly limited, and can be various methods commonly used in the art. Preferably, the method includes: taking 1×10 ⁷ -5×10 ⁷ NKT cells (the NKT cells prepared in the present invention In cells, CD3 ⁺ CD56 ⁺ cells accounted for the average ratio of total CD3 ⁺ cells>20%), discard the old culture medium, add 2-4mL fresh GT-T551 culture medium, then add 200-400μL virus concentrate, 2- 4 μL of 1×10 ^-6 mg/mL protamine and IL-2 with a final concentration of 300-700U/mL, placed in a _CO2 incubator at 30-37°C and saturated humidity of 3-6% for 12-16h after infection , Discard the culture medium, transfer the cells to an uncoated culture flask, add 20-50mL of GT-T551 medium, then add IL-2 with a final concentration of 300-700U/mL, and a final concentration of 30-70ng/mL ml of CD3 monoclonal antibody and interleukin-15 with a final concentration of 30-70ng/mL were cultured in a CO ₂ incubator at 30-37°C and a saturated humidity of 3-6% for 12-18h to obtain chimeric antigen receptors. Somatic CD138ScFv-2-CD8-CD137-CD3ζ modified NKT cells.

Further preferably, the method for infecting NKT cells with lentiviruses further comprises: inducing in vitro the NKT cells infected with lentiviruses obtained after the above culture with GT-T551 culture medium with a final concentration of IL-2 of 300-700U/mL, and then When the cell density is 80-90%, transfer the cells into the cell culture bag, add IL-2 at a final concentration of 300-700U/mL, and CD3 monoclonal antibody at a final concentration of 30-70ng/ml every 1.5-2.5 days 1. Fresh GT-T551 culture solution with a final interleukin-15 concentration of 30-70 ng/mL is used for expansion culture and the cells are expanded to a total of 1×10 ⁹ -2×10 ⁹ cells. The chimeric antigen receptor targeting CD138 antigen is infected with NKT cells by the lentivirus of the present invention, and the infection efficiency is as high as 30%-60%, and the obtained CAR138-2-NKT cells account for the total CD3 ⁺ CD56 ⁺ cells The ratio of CD3 ⁺ cells ranged from 15% to 40%.

The amino acid sequence of the mature protein of the chimeric antigen receptor expressed by the chimeric antigen receptor CD138ScFv-2-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 signal peptide, CD138ScFv-2, hinge region and transmembrane region of CD8, intracellular signal domain of CD137 and intracellular signal structure of CD3ζ The protein is composed of domains in series. After the protein is translated, it becomes a mature chimeric antigen receptor protein after the signal peptide is excised in the rough endoplasmic reticulum in the cell. After being secreted and exported, it is located on the cell membrane of NKT cells. The gene coding sequence corresponding to the amino acid sequence of the mature protein of the chimeric antigen receptor is shown in SEQ ID NO.2. The chimeric antigen receptor uses the structure of the hinge region and transmembrane region of the gene CD8 and the intracellular signaling domains of CD137 and CD3ζ in series as the signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.9, and the corresponding gene The coding sequence is shown in SEQ ID NO.10.

The present invention also provides the engineered CD138-targeted NKT cells prepared by the above method.

The present invention also provides the application of engineered CD138-targeted NKT cells in the preparation of preparations for treating tumors. Preferably, the tumor refers to CD138-positive multiple myeloma.

Example

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

The experimental methods in the following examples are conventional methods in the art unless otherwise specified. The experimental materials used in the following examples, unless otherwise specified, are 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 RNAisoReagent, high-fidelity DNA polymerase ( HSDNA Polymerase), T4 DNA ligase were purchased from TaKaRa Company.

RevertAid ^™ First Strand cDNASynthesis Kit was purchased from Fermentas.

BglII, EcoRI, MluI, BamHI, and SalI were purchased from Fermentas.

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

pWPT-GFP, psPAX2, and pMD2.G were all purchased from Addgene.

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

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

Lipofectamine ^TM 2000 Transfection 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 1.2M. Both PEG6000 and NaCl were purchased from Shanghai Suo Laibao Biotechnology Co., Ltd.

Fetal bovine serum was purchased from PAA, Germany.

Myeloma cell line A266 was purchased from ATCC, USA.

Primary cultured mouse myeloma cells were purchased from Shanghai Yuchen Biotechnology Co., Ltd.

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

Annexin V-RPE kit was purchased from BD Company, USA.

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

The preparation of embodiment 1 NKT cell

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

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

(3) On the 4th day of culture, transfer the cells to an uncoated culture flask, add NKT cell culture medium GT-T551 every 2 days according to the number of cell growth, control the cell concentration to ¹ ×108 cells/mL, and Add recombinant human interleukin 2 with a final concentration of 500U/ml; culture to the 12th day to obtain NKT cells, and analyze the phenotype of NKT cells by flow cytometry. The results are shown in Figure 1, where CD3 ⁺ : 95.04%; CD3 ⁺ CD8 ⁺ : 90.99%; CD3 ⁺ CD56 ⁺ : 24.12%; CD8 ⁺ CD56 ⁺ : 24.63%.

Example 2 Construction of lentiviral expression vector pWPT-CD138ScFv-2-CD8-CD137-CD3ζ

(1) Preparation of NKT cell cDNA

The NKT cells cultured in Example 1 were centrifuged to precipitate, and the total RNA of the cells was extracted with the total RNA extraction kit RNAisoReagent, and stored at -80°C for future use. The extracted total RNA was reverse-transcribed with RevertAid ^TM FirstStrandcDNASynthesisKit to obtain NKT cell cDNA, which was stored at -20°C for future use.

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

The following primer sequences were designed and synthesized (wherein, the underline marks are protected bases, and the square boxes are enzyme cleavage sites):

Using the cDNA of NKT cells in step (1) as a template, PCR amplification was carried out with primers P1 and P2 to obtain the hinge region and transmembrane region of CD8 with a length of 287 bp. The nucleotide sequence is shown in SEQ ID NO.3, and the two ends respectively contain MluI and BglII restriction sites and protective bases; use primers P3 and P4 for PCR amplification to obtain a 146bp CD137 intracellular signaling domain, the nucleotide sequence is shown in SEQ ID NO.4, and the two ends contain BglII and EcoRI restriction site and protective base; use primers P5 and P6 to perform PCR amplification to obtain the intracellular signaling domain of CD3ζ with a length of 359 bp, the nucleotide sequence is shown in SEQ ID NO.5, and both ends contain EcoRI and SalI respectively Restriction sites and protective bases. The PCR amplification reaction system of each step is the same, taking the amplification of CD137 intracellular signaling domain as an example, PCR amplification is carried out, and the PCR reaction conditions refer to Instructions for HSDNAPolymerase, the reaction system (50 μL) is as follows:

Double distilled water: 32.5 μL

5×reaction buffer: 10μL

dNTP mix (2.5 mM each): 4 μL

P3 (10mM): 1μL

P4 (10mM): 1μL

NKT cell cDNA (200ng/ul): 1μL

HSDNA Polymerase: 0.5 μL

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

The lentiviral expression vector pWPT-GFP was double-digested with MluI/SalI, the digested products were separated by 1% agarose gel, and the large carrier fragment was recovered with the agarose gel DNA recovery kit, and then combined with the previously recovered CD8 , CD137, and CD3ζ fragments were ligated by T4DNA ligase, and the ligated products were transformed into Trans1-T1PhageResistant chemically competent cells. After culturing at 37°C for 16 hours, single clones were picked, and after culturing at 37°C and 250rpm for 12 hours, plasmids were extracted with a plasmid mini-extraction kit. The extracted plasmid was identified by restriction endonuclease MluI and SalI double digestion, and the identification electropherogram is shown in Figure 2, wherein, 1 lane: DNA molecular weight marker D2000; 2 lanes: the restriction fragment (835bp) of plasmid pWPT-GFP; 3 Swimming lane: the restriction fragment (756bp) of plasmid pWPT-CD8-CD137-CD3ζ. 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 pWPT-CD8-CD137-CD3ζ.

(3) Preparation of lentiviral plasmid pWPT-CD138ScFv-2-CD8-CD137-CD3ζ

Whole gene synthesis encodes the nucleotide sequence of rat growth hormone signal peptide and CD138ScFv-2 fusion gene, the sequence is shown in SEQ ID NO.8, synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd., its 5' end contains BamHI, kozak sequence, and the 3' end contains a MluI restriction site, and the aforementioned fusion gene was cloned into the plasmid pGSI, named pGSI-CD138ScFv-2. The plasmid was digested with BamHI/MluI double enzymes, the digested product was separated by 1% agarose gel, and the target fragment was recovered with an agarose gel DNA recovery kit for future use.

The pWPT-CD8-CD137-CD3ζ plasmid was digested with restriction endonuclease BamHI/MluI, the digested product was separated by 1% agarose gel, and the vector fragment was recovered with an agarose gel DNA recovery kit for future use. Then it is ligated with the recovered DNA fragment containing rat growth hormone signal peptide and CD138ScFv-2 through T4 DNA ligase, and the specific method is shown in the instructions. The ligation product was transformed into Trans1-T1PhageResistant chemically competent cells, cultured at 37°C for 16 hours, and single clones were picked, and after cultured at 37°C, 250rpm for 12 hours, the plasmid was extracted with a plasmid mini-extraction kit. The extracted plasmid was identified by restriction endonuclease BamHI/SalI double digestion, and the identification result is shown in Figure 3, wherein, M1: DNA molecular weight marker D15000; 1 lane: the enzyme-cut fragment (853bp) of plasmid pWPT-GFP; 2 Swimming lane: enzyme-cut fragment (774bp) of plasmid pWPT-CD8-CD137-CD3ζ; lane 3: enzyme-cut fragment (1575bp) of plasmid pWPT-CD138ScFv-2-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 is named as pWPT-CD138ScFv-2-CD8-CD137-CD3ζ, and its structural schematic diagram is shown in Figure 4, including rat growth hormone signal peptide (nucleotide sequence as shown in SEQIDNO.6) , anti-CD138 single-chain antibody (nucleotide sequence as 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 takes the structure formed by the hinge region and transmembrane region of the gene CD8 and the intracellular signal domains of CD137 and CD3ζ in series as the signal transduction domain. Its amino acid sequence is shown in SEQ ID NO.9, and the corresponding gene coding sequence is shown in SEQ ID NO. .10 shown.

Example 3 Preparation of NKT Cells Modified by Chimeric Antigen Receptor CD138ScFv-2-CD8-CD137-CD3ζ

(1) Packaging and concentration of lentivirus

Measure the concentration of the lentiviral expression plasmid pWPT-CD138ScFv-2-CD8-CD137-CD3ζ and the helper plasmids psPAX2 and pMD2.G with a spectrophotometer, and transfect the three plasmids with Lipofectamine ^TM 2000TransfectionReagent at a mass ratio of 4:2:1 Reagents co-transfect 293T packaging cells. Collect the virus supernatant at 48h and 72h of transfection respectively in 50mLEP tubes, centrifuge at 2000g for 10min at 4°C, transfer the supernatant obtained twice to a new EP tube, filter the virus supernatant with a 4.5μm filter; 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 10,000 g for 20 minutes at 4°C, discard the supernatant, and dissolve the precipitate with 1 mL of 4°C pre-cooled sterile PBS, namely The chimeric antigen receptor virus concentrate was obtained, divided into 100 μL per tube, and stored at -80°C for future use.

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

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

Get the ¹ × 107 NKT cells cultivated in the 75cm culture flask of Example ¹ , discard the old culture solution, add 2 mL of fresh NKT cell culture medium GT-T551, 200 μ L of the virus concentrate that step (1) obtains, 2 μL of 1×10 ^-6 mg/mL protamine and recombinant human interleukin-2 at a final concentration of 500 U/mL were placed in a CO ₂ incubator at 37°C with a saturated humidity of 5% for 12 hours, and the culture medium was discarded. At the same time, the NKT cells were simultaneously infected with the lentivirus concentrate expressing GFP green fluorescent protein (the obtained NKT cells were called CART-GFP cells), which was used to calculate the infection efficiency of the virus. Transfer the infected cells to a 75cm ² culture flask not coated with CD3 and retronectin, add 20mL of NKT cell culture medium GT-T551, and then add recombinant human interleukin 2 with a final concentration of 500U/mL and a final concentration of 50ng /ml CD3 monoclonal antibody and recombinant human interleukin-15 with a final concentration of 50ng/mL were cultured in a _CO2 incubator at 37°C and a saturated humidity of 5% for 18h, and the NKT cells obtained were called CAR138-2-NKT cells . The same method was used to prepare CAR138-2-T cells (refer to the literature for the preparation method of T cells: Yajing Zhang, et al. Autologous CIK Cell Immunotherapy in Patients with Renal Cell Carcinoma after Radical Nephrectomy. Clinical Study, 2013, 2.4 Part 2.4 CIK cell preparation method). The infection efficiency of the virus was detected by flow cytometry, as shown in Figure 5, the infection efficiency of CAR138-2-NKT cells was 34.33%.

(3) Induction and expansion of CAR138-2-NKT cell population in vitro

The above-mentioned 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, and when the density of the cells was 85%, the cells were transferred into cell culture bags, and the Add fresh NKT cell medium GT-T551 with a final concentration of recombinant human interleukin 2 of 500 U/mL, a final concentration of CD3 monoclonal antibody of 50 ng/ml, and a final concentration of recombinant human interleukin 15 of 50 ng/mL on the first day for expansion and culture , after the cells 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 positive cells > 70% %; the proportion of CD3CD56 double-positive cells > 5%, the results are shown in Figure 6, CD3 ⁺ : 98.51%; CD3 ⁺ CD8 ⁺ : 87.15%; CD3 ⁺ CD56 ⁺ : 25.16%; CD8 ⁺ CD56 ⁺ : 29.88%.

Example 4 Cytotoxicity analysis of the killing effect of CAR138-2-NKT cells on human myeloma cells

The CAR138-2-NKT cells prepared in Example 3, the CAR138-2-T cells and the NKT cells cultured in Example 1 were inoculated in 96-well plates, and treated with 5-carboxyfluorescein succinimidyl ester (CFSE) For staining, co-culture with myeloma cell line A266 and primary cultured mouse myeloma cells at an effect-to-target ratio (killer cells: target cells) of 5:1, 10:1, 20:1, 40:1, After 24 hours of co-cultivation, the cells were stained with the Annexin V-RPE kit, and the control groups were the myeloma cell line A266 without immune cell killing treatment and the primary cultured mouse myeloma cells. And cells were stained with Annexin V-RPE kit. Flow cytometry was used to detect cell apoptosis, 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 surviving cells without immune cell killing treatment; The sample is the number of surviving cells treated with immune cell killing treatment with corresponding effect-to-target ratio (killer cells: target cells), as shown in FIG. 7 . Chimeric antigen receptor CD138ScFv-2-CD8-CD137-CD3ζ modified NKT cells (CAR138-2-NKT) have specific killing activity against myeloma cells, and the specific killing activity of CAR138-2-NKT cells is significantly better than that of CAR138- 2-T cells.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above 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 various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (10)

1. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor is CD138ScFv-2-CD8-CD137-CD3ζ, composed of the hinge region and transmembrane region of CD138ScFv-2, CD8, and the intracellular region of CD137 The signaling domain and the intracellular signaling domain of CD3ζ are formed in series, and the nucleotide sequence of the gene encoding the CD138ScFv-2 is shown in SEQ ID NO.7. 2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1. 3. A gene encoding the chimeric antigen receptor of claim 1 or 2. 4. The gene according to claim 3, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.2. 5. A recombinant expression vector containing the gene according to claim 3 or 4. 6. The recombinant expression vector according to claim 5, wherein the recombinant expression vector is a lentiviral expression vector. 7. The recombinant expression vector according to claim 6, wherein the lentiviral expression vector is pWPT-CD138ScFv-2-CD8-CD137-CD3ζ. 8. An engineered CD138-targeted NKT cell, characterized in that the NKT cell is an NKT cell modified by the chimeric antigen receptor according to claim 1 or 2. 9. The application of the engineered CD138-targeted NKT cells according to claim 8 in the preparation of preparations for treating tumors. 10. The application according to claim 9, wherein the tumor refers to CD138-positive multiple myeloma.