CN117946284A

CN117946284A - Application of iNKT cells in cancer treatment

Info

Publication number: CN117946284A
Application number: CN202410095572.6A
Authority: CN
Inventors: 佟为民; 刘燕; 蒲乙琴
Original assignee: Zhongbang Stem Cell Technology Co ltd
Current assignee: Zhongbang Stem Cell Technology Co ltd
Priority date: 2024-01-24
Filing date: 2024-01-24
Publication date: 2024-04-30

Abstract

The invention belongs to the technical field of biological medicine, and in particular relates to an anti-CD 19 antibody, a chimeric antigen receptor and application of i NKT cells thereof in cancer treatment. The invention discloses a chimeric antigen receptor targeting CD19, an iNKT cell and application thereof, wherein the chimeric antigen receptor is formed by sequentially connecting a tEGFR signal peptide, a specific antibody targeting CD19, a CD8 alpha hinge region, a CD8 alpha transmembrane domain, a CD28 co-stimulatory signal domain and a CD3 zeta intracellular signal transduction domain. The CD19-CAR-i NKT cell prepared by the invention has stronger proliferation capability, cytokine release capability and tumor cell killing capability compared with the i NKT cell and the CD19-CAR-T cell, can effectively remove tumor cells, and has important application prospect in the field of tumor cell immunotherapy.

Description

Application of iNKT cells in cancer treatment

Technical Field

The invention belongs to the technical field of biological medicine, and in particular relates to an antibody targeting CD19, a chimeric antigen receptor and application of inKT cells thereof in cancer treatment.

Background

INKT cells (INVARIANT NATURAL KILLER T CELL, constant natural killer T cells) are a naturally occurring class of immune cells that mediate innate and acquired immunity, a unique subset of T lymphocytes. It expresses a constant tcrvα14 with partial identification of NK cells. iNKT cells are also considered classical type 1 NKT cells. iNKT cells are associated with a variety of tumors and autoimmune diseases, and in these tumor patients and patients the number of iNKT cells is significantly reduced, and activation of iNKT cells significantly ameliorates the symptoms of the tumor and various diseases. iNKT cells specifically recognize glycolipid antigens presented by APC surface other than classical mhc class i molecules CDld and have the ability to produce killer cytokines, thereby being involved in various immunomodulations, such as anti-tumor, anti-infection, anti-rejection of organ transplantation, and controlling the development of autoimmune diseases. The activated iNKT cells secrete a variety of cytokines such as IFN- γ and TNF- α or related apoptosis-inducing ligands (TRAIL) and the like, which can directly kill tumor cells or indirectly activate cytotoxic T cells and dendritic cells, thereby participating in different immune responses.

B cell malignancy is a group of malignant heterogeneous diseases of blood systems, including acute and chronic B lymphocyte leukemia and some lymphoma subtypes, and is a clinical difficult-to-cure blood system malignancy due to high recurrence rate and poor prognosis. CD19, a cell surface differentiation antigen specific to B cell lines, is not only expressed on the surface of pre-normal B cells and mature B cells, but also widely expressed on the surface of a variety of B cell malignancies, but is not expressed in hematopoietic stem cells, plasma cells and other normal tissue cells, nor has the presence of CD19 soluble protein been detected in the blood. In addition, the distribution of CD19 molecules on the membrane is relatively exposed, readily binds to monoclonal antibodies, and after binding there is no significant internalization, shedding, and antigen modulation, thus it is considered an ideal target for the treatment of B cell tumors. Most B-cell malignancies have high CD19 expression on their surface, and T-cell targeting with Chimeric Antigen Receptor (CAR) modification by multiple centers has been found to have unprecedented success in targeting CD 19-expressing B-cells for recurrent and refractory malignancies. Immunotherapy, such as CAR-T, has brought the hope of "curing cancer" to numerous patients.

Currently, in view of the success of CAR-T cell therapies, there is great interest in researchers who have taken over CAR engineering techniques for engineering of other types of immune cells, thereby deriving a series of novel cell therapies that are centered on CAR technology, such as CAR-NK, CAR-iNKT, CAR-macrophage (CAR-M), CAR-Treg, CAR- γδt, etc. Constant natural killer T cells (INVARIANT NATURAL KILLER TCELLS, INKT CELLS) are named for their expression of unique INVARIANT TCR, which primarily recognizes the glycolipid antigen presented by the CD1d molecule and functions by secreting large amounts of cytokines. Since CD1d molecules are expressed in only a few cells and the gene is haplotype, iNKT cell alloinfusion does not produce an acute graft versus host response and can be developed into a universal cell product. Another advantage of iNKT is that immune suppressive cells TAM, MDSC in tumor microenvironment highly express CD1d, so iNKT can kill it, improve tumor microenvironment, and enhance anti-tumor immune effect.

Based on the high expression characteristic of CD19 in tumor cells and the characteristic of the inhibition activity of the CD19 on immune cells, the invention provides a CD 19-targeted source chimeric antigen receptor and an inKT cell thereof, and has important application prospect in the field of tumor cell immunotherapy.

Disclosure of Invention

The invention aims to provide a CD19 targeting chimeric antigen receptor and an inKT cell thereof, and provides an application of the CD19 targeting inKT cell in a preparation for treating tumor.

In order to solve the problems, the invention provides the following technical scheme:

in a first aspect the invention provides a CD 19-targeting source chimeric antigen receptor.

Further, the chimeric antigen receptor comprises an antibody that specifically binds to CD 19;

Further, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain;

further, the chimeric antigen receptor further comprises a hinge region;

further, the chimeric antigen receptor further comprises a costimulatory signaling domain;

Further, the chimeric antigen receptor further comprises a signal peptide;

Further, the amino acid sequence of the antibody specifically binding to CD19 comprises an amino acid sequence shown as SEQ ID NO. 1, an amino acid sequence with at least 90% homology with the amino acid sequence shown as SEQ ID NO. 1, and an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 1;

more preferably, the amino acid sequence of the light chain variable region of the antibody specifically binding to CD19 is an amino acid sequence shown as SEQ ID NO.2, an amino acid sequence having at least 90% homology with the amino acid sequence shown as SEQ ID NO.2, an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown as SEQ ID NO. 2;

Furthermore, CDRs 1-3 of the light chain variable region of the antibody specifically binding to CD19 are respectively shown as an amino acid sequence shown as SEQ ID NO. 3-5, an amino acid sequence with at least 90% homology with the amino acid sequence shown as SEQ ID NO. 3-5, and an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 3-5;

More preferably, the amino acid sequence of the heavy chain variable region of the antibody specifically binding to CD19 is an amino acid sequence shown as SEQ ID NO. 6, an amino acid sequence having at least 90% homology with the amino acid sequence shown as SEQ ID NO. 6, an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown as SEQ ID NO. 6;

Furthermore, the CDRs 1-3 of the heavy chain variable region of the antibody specifically binding to CD19 are respectively shown as an amino acid sequence shown as SEQ ID NO. 7-9, an amino acid sequence with at least 90% homology with the amino acid sequence shown as SEQ ID NO. 7-9, and an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown as SEQ ID NO. 7-9;

Still further, the transmembrane domain and hinge region includes the transmembrane domain and hinge region of: CD 8a, CD28, igG1, igG4, 4-1bb, pd-1, CD34, OX40, CD3 epsilon;

Further, the transmembrane domain and hinge region are a CD 8a transmembrane domain and a CD 8a hinge region;

Further, the amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO. 10, has at least 90% of homology with the amino acid sequence shown as SE Q ID NO. 10, and is obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 10;

Further, the amino acid sequence of the CD8 alpha transmembrane domain is shown as SEQ ID NO. 11, has at least 90% of homology with the amino acid sequence shown as SEQ ID NO. 11, and is obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 11;

Still further, the costimulatory signaling domain comprises the costimulatory signaling domain of: CD28, ICOS, CD27, CD19, CD4, CD8 α, CD8 β, HVEM, LIGHT, CD, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD278;

Further, the costimulatory signaling domain is a CD28 costimulatory signaling domain;

Further, the amino acid sequence of the CD28 co-stimulatory signaling domain is shown as SEQ ID NO. 12, the amino acid sequence has at least 90% homology with the amino acid sequence shown as SEQ ID NO. 12, and the amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 12;

Still further, the intracellular signaling domain comprises an intracellular signaling domain of ：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d、DAP10、DAP12、FYN;

Further, the intracellular signaling domain is a cd3ζ intracellular signaling domain;

Further, the amino acid sequence of the CD3 zeta intracellular signal transmission structural domain is shown as SEQ ID NO. 13, has at least 90% homology with the amino acid sequence shown as SEQ ID NO. 13, and is obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 13;

Still further, the signal peptide includes a signal peptide of: the alpha chain and beta chain of the T cell receptor, cd3ζ, cd3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, igG, tgfr;

further, the signal peptide is a tgfr signal peptide;

further, the amino acid sequence of the tEGFR signal peptide is shown as SEQ ID NO. 14, has at least 90% of homology with the amino acid sequence shown as SEQ ID NO. 14, and is obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 14;

further, the chimeric antigen receptor is sequentially connected by a tEGFR signal peptide, a specific antibody targeting CD19, a CD8 alpha hinge region, a CD8 alpha transmembrane domain, a CD28 co-stimulatory signaling domain and a CD3 zeta intracellular signaling domain;

Further, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 15, has at least 90% of homology with the amino acid sequence shown as SEQ ID NO. 15, and is obtained by modifying, substituting, deleting or adding one or more amino acids with the amino acid sequence shown as SEQ ID NO. 15;

in another aspect of the invention, polynucleotides encoding chimeric antigen receptors are provided;

In another aspect of the invention there is provided a vector comprising said polynucleotide;

Still further, the vectors include cloning vectors, expression vectors;

In another aspect the invention provides an engineered host cell comprising the polynucleotide or vector;

in another aspect the invention provides an immune cell transduced with said chimeric antigen receptor;

Still further, the immune cell is a T cell, NK cell or iNKT cell;

further, the immune cell is an iNKT cell;

In another aspect of the invention, a pharmaceutical composition is provided, comprising a chimeric antigen receptor, polynucleotide, vector, host cell or immune cell of the invention;

in another aspect, the invention provides the use of said chimeric antigen receptor, polynucleotide, vector, host cell or immune cell in the manufacture of a medicament for the treatment of cancer;

Still further, the cancer is a CD19 overexpressing cancer;

Still further, the cancer includes B-cell lymphoma, B-cell acute lymphoblastic leukemia, B-cell chronic lymphoblastic leukemia, small lymphoblastic lymphoma, acute myelogenous leukemia, non-hodgkin's lymphoma, ovarian cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, liver cancer, bladder cancer, osteosarcoma, glioma.

Compared with the prior art, the invention has the following advantages:

(1) The invention provides a CD 19-targeted chimeric antigen receptor and an inKT cell thereof based on the high expression characteristic of CD19 in tumor cells and the characteristic of the inhibition activity of the CD19 on immune cells, and experiments prove that the CD 19-targeted CAR-inKT cell prepared by the invention has stronger proliferation capability, cytokine release capability and tumor cell killing capability, can effectively remove tumor cells, and has important application prospect in the field of tumor cell immunotherapy.

(2) The invention combines the iNKT cell with the chimeric antigen receptor of CD19 of the targeted tumor cell, can have strong CD1d targeted tumor killing effect and immunoregulation effect of the iNKT cell, accurately targets the iNKT cell to the local focus of the tumor through the tumor specific targeted chimeric antigen receptor, and simultaneously utilizes the ADCC effect of the antibody to more effectively promote the specificity of tumor killing. The use of iNKT cells in combination with antibodies that increase immune function may also help patients repair the immune system. Compared with monotherapy, the combination can greatly reduce the dosage of immune cells and antibodies and the frequency of reinfusion, and reduce the pain of tumor patients and the treatment cost.

Drawings

In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 schematic structural diagram of a CD 19-targeting chimeric antigen receptor of origin (CD 19-CAR)

FIG. 2 graph of in vitro CD19-CAR-inKT cell growth

FIG. 3 results of the ability of CD19-CAR-inKT cells to secrete cytokines

FIG. 4 killing efficiency of CD19-CAR-inKT cells against tumor cells

Detailed Description

The following detailed description of embodiments of the invention, provided in the accompanying drawings, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.

Example 1 preparation of CD19 targeting nanobodies

(1) Preparation of CD19 antigen

RNA in the T cells is extracted by using an RNA extraction kit. Referring to SuperScriptTMII REVER SE TRANSCRIPTASE, the cDNA was obtained by reverse transcription using random primers. The extracellular region gene sequence of antigen CD19 was obtained by PCR using cDNA as a template. And connecting the CD19 extracellular region gene sequence into a protein expression vector for expression, and purifying by a Ni column to obtain the purified CD19-His protein.

(2) Construction of nanobody libraries

Alpaca immunization was performed using the CD19-His protein obtained as described above. Immunization was performed 1 week for 5 total immunizations; collecting 100mL of peripheral blood after 7 days of final immunization, separating peripheral blood mononuclear cells by a density gradient centrifugation method, extracting RNA, and preparing cDNA by using a reverse transcription kit; obtaining a VHH fragment by SOE-PCR and ligating the VHH fragment into pMES phage display vector; electrotransformation of the connection product into electrotransformation competent cell TG1 colibacillus to obtain bacterial library as the constructed single domain heavy chain antibody phage display library of CD 19; after the completion of library construction, 25 clones were randomly selected for colony PCR using primers MP57 and GIII, and the PCR products were Sanger sequenced, in order to examine the insertion efficiency of the library.

(3) Enrichment screening of nanobodies

Amplification of phage nanobody library: transferring the TG1 E.coli nanobody library into a 2-YT liquid culture medium, culturing at 37 ℃ and 200rpm until the OD value is 0.5, and then adding a helper phage VCS M13 to infect the cells. After gentle mixing, incubation was carried out at 37℃for 30 minutes. Centrifuging the bacterial liquid to remove trace glucose, and re-suspending the precipitate in a 2-YT culture medium with ampicillin resistance and kanamycin resistance, and shaking and culturing at 37 ℃ and 200rpm for overnight to amplify phage displaying nano antibodies; the overnight cultures were transferred to 50mL centrifuge tubes, the supernatant was centrifuged and the phage was precipitated by adding 20% (wt/vol) PEG6000/2.5M NaCl solution. The supernatant was discarded by centrifugation, the pellet was resuspended in PBS, the supernatant was removed by centrifugation to a new centrifuge tube and the phage was reprecipitated by adding 20% (wt/vol) PEG6000/2.5M NaCl solution. The supernatant was centrifuged off and the pellet resuspended in 1mL PBS. Transferring the supernatant to a new centrifuge tube after centrifugation, adding glycerol to a final concentration of 20%, and storing at-80 ℃; phage nanobody library titer determination, phage were diluted in 10-fold gradient, TG1 bacteria in log phase were infected with phage of different dilution fold, incubated overnight at 37 ℃ and phage nanobody library titer was estimated from the number of plaques on the next day.

Phage enrichment screening: panning the nano antibody by ELISA method, coating recombinant CD19-His protein on an ELISA plate, and incubating overnight at 4 ℃; washing the ELISA plate three times by using 250 mu L of PBST, adding 200 mu L of sealing solution, and incubating the ELISA plate for 2 hours at room temperature; adding corresponding phage into each hole, and incubating for 2 hours at room temperature; 250 uL PBST wash plate 15 times; 100. Mu.L of trypsin with the concentration of 0.25mg/mL is added to each well, and the mixture is incubated for 0.5h at room temperature and 700 rpm; eluting phage with AEBSF; titer determination and phage infection amplification are carried out on the eluted phage; number of phage eluted positive: when the negative value is more than or equal to 100, the panning is stopped.

(4) Screening and identification of Positive monoclonal

Selecting single clone from TG1 colibacillus library obtained after 3 rounds of screening for expansion culture, and carrying out infection by using auxiliary phage VCSM13 to prepare monoclonal phage; adding the monoclonal phage into a culture plate coated with CD19-His protein, and incubating for 2 hours at room temperature; after PBST washes the board, add HA-HRP antibody, incubate for 1h at room temperature; after PBST cleaning the plate, adding 100 mu L of TMB single-component color development liquid, incubating for 30min at room temperature, and adding 100 mu L of stop solution; detecting absorbance at 450nm by using an enzyme-labeled instrument; positive clones were judged when the ratio of OD450 value of sample well to blank control was greater than 2; performing bacterial liquid PCR on positive clones, and performing Sanger sequencing; the Sanger sequenced monoclonal was used for sequence alignment using the software DNAMAN. And positive clones specific for the sequences were selected.

(5) Experimental results

Sequencing the targeted CD19 antibodies obtained by the screening gave the following results:

Table 1 sequencing results for targeting CD19 antibodies

Example 2 construction of CD 19-targeting chimeric antigen receptor of origin (CD 19-CAR)

A restriction enzyme site Nde I (CATA TG), an initiation codon (ATG) and a DNA sequence of a C-myc tag (GAGCAGAAGCTGATCAGCGAGGAGGACCTG) are sequentially added to the 5 'end of the DNA sequence encoding the CD19-CAR, and a termination codon (TAA) and a restriction enzyme SpeI (ACTA GT) DNA sequence are sequentially added to the 3' end. The DNA sequence described above was encoded by total gene synthesis. The synthesized DNA fragment was inserted into lentiviral vector pRRL through restriction enzyme sites Nde I and Spe I, and a pRRL-CD19-CAR expression plasmid was constructed using conventional genetic engineering means.

The structural schematic diagram of the constructed CD19-CAR is shown in figure 1, the CD19-CAR is obtained by sequentially connecting a tEGFR signal peptide, a specific antibody targeting CD19, a CD8 alpha hinge region, a CD8 alpha transmembrane domain, a CD28 co-stimulatory signal domain and a CD3 zeta intracellular signaling domain, wherein the amino acid sequence of the specific antibody targeting CD19 is shown as an amino acid sequence shown as SEQ ID NO. 1; the amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO. 10; the amino acid sequence of the CD8 alpha transmembrane domain is shown as SEQ ID NO. 11; the amino acid sequence of the CD28 co-stimulatory signaling domain is shown in SEQ ID NO. 12; the amino acid sequence of the CD3 zeta intracellular signal transduction domain is shown as SEQ ID NO. 13; the amino acid sequence of the tEGFR signal peptide is shown as SEQ ID NO. 14; the amino acid sequence of the CD19 targeting chimeric antigen receptor (CD 19-CAR) is shown in SEQ ID NO. 15.

Example 3 preparation of iNKT cells

(1) Collecting peripheral blood of a donor, diluting whole blood with an equal amount of physiological saline, adding lymphocyte separation liquid and diluted blood into a centrifuge tube according to a ratio of 1:2, centrifuging at 2000rpm/min for 20 minutes, collecting white membrane layer cells, washing twice with the physiological saline, and centrifuging at 1500rpm/min for 8 minutes to obtain peripheral blood mononuclear cells PBMCs.

(2) PBMCs were washed three times with AIM V cell culture medium, resuspended in AIM V cell culture medium containing 100ng/mL of alpha-galactoside ceramide, 500IU/mL of rhIL-2, and 500IU/mL of rhIL-7 to assist inKT cell growth, inoculated in 75cm ² cell culture flasks, transferred to a 37℃and cultured in a 5% CO ₂ saturated humidity cell culture incubator. Cell status was observed daily, half-way across the day, amplification was monitored by cell counting, and iNKT cell phenotypes were analyzed using flow cytometry.

(3) AIM V cell culture medium containing 100ng/mL of alpha-galactoside ceramide, 500IU/mL of rhIL-2 and 500IU/mL of rhIL-7 was added to the culture flask on the fourth and seventh days, respectively, and the culture was continued in a cell incubator at 37℃and 5% CO ₂ and saturated humidity. Cells were transferred to 225cm ² cell culture flasks on day 10, cultured to day 14, and iNKT cells in the culture were sorted using a flow cytometer.

(4) The flow-sorted iNKT cells were resuspended in AIM V cell culture medium containing 100ng/mL α -galactoside ceramide, 500IU/mL rhIL-2 and 500IU/mL rhIL-7, inoculated into 75cm ² cell culture flasks previously coated with 5 μg/mL CD3 monoclonal antibody and 5 μg/mL CD 28 monoclonal antibody, cultured for 10 days to collect cells, and 1×10 ⁷ cells per tube were dispensed into cell cryopreservation tubes and stored in liquid nitrogen.

Example 4 preparation of CD19-CAR-iNKT cells

(1) Inserting the CD19-CAR gene sequence of the designed example 2 between EcoRI and BamH1 cleavage sites in a lentiviral backbone plasmid pCDH-EF1-MCS, carrying out double cleavage on the cleavage sites at two ends of the designed gene sequence by using restriction enzymes on the recombinant lentiviral plasmid, carrying out SDS-PAGE electrophoresis, selecting a fragment region with the same size as the designed enzyme fragment for glue recovery, and obtaining positive cloning plasmids with correct sequences through sequencing identification; mixing positive cloning plasmid with DH5 alpha competent escherichia coli, adding an LB liquid culture medium without antibiotics for culture, preparing an original bacterial liquid, inoculating the original bacterial liquid into 100mL of an Amp resistant LB culture medium, shaking table 200rpm at 37 ℃ for overnight, harvesting bacterial liquid for plasmid extraction the next day, obtaining a CD19-CAR plasmid, detecting the purity and concentration of the plasmid, and reserving at-20 ℃.

(2) 293T cells are inoculated to a 6cm culture dish 24h in advance, so that the cell fusion rate can reach 80% during transfection. The following day of observation of 293T cells, when the cell state is confirmed to be good and the fusion degree is 80% -90%, fresh culture medium is replaced, and the recombinant lentiviral plasmid and packaging plasmid (psPAX and pMD2. G) are mixed and incubated together according to the instruction using Lipofectamine 3000 kit (Cat#L 3000008) produced by Thermofisher, and then added into a 293T cell culture dish drop by drop, and the dish is gently shaken and fully mixed. The culture dish is placed in a culture box with 5% CO ₂ at 37 ℃ for 6-8 hours, and then liquid exchange treatment is carried out. After continuous culture for 72 hours, collecting culture medium supernatant containing virus in a culture dish, filtering with a 0.45 μm filter membrane, transferring the filtrate into a sterile centrifuge tube, performing ultra-high speed centrifugation and concentration to obtain CD19-CAR virus liquid, subpackaging in virus tubes, and storing at-80deg.C.

(3) Adding 10 mu M HEPES and 6-8 mu g/mL polybrene into CD19-CAR virus liquid, uniformly mixing, re-suspending and activating the iNKT cells prepared in the example 3 by using the virus liquid, adding the iNKT cells into a 24-well plate pre-coated by retroNectin, centrifuging at 1500g and 30 ℃ for 2 hours, removing supernatant, supplementing an X-VIVO 15 serum-free immune cell culture medium containing 5% fetal bovine serum, 200U/mL IL-2, 10ng/mL IL-7 and 5ng/mL IL-15, and continuing to amplify and culture to obtain the CD19-CAR-iNKT cells.

Meanwhile, CD19-CAR-T cells were prepared using a similar method as described above.

Example 5 in vitro growth Capacity assay of CD19-CAR-inKT cells

INKT cell sorting was performed using Anti-human-iNKT MicroBea ds according to Miltenyi Biotec company operating manual.

(1) Preparation of OKT3 antibody-coated cell culture flasks: 2. Mu.g/mL of CD3 antibody working solution (0.9% sterile physiological saline) was prepared. Two brand new 75cm ² flasks were prepared, and 5mL of diluted antibody working solution was added to each flask. And (5) screwing the bottle mouth, and slightly shaking the bottle body to enable the CD3 antibody diluent to fully infiltrate the bottle bottom. Sealing the mouth of the culture flask with sealing film, and keeping in refrigerator at 4deg.C.

(2) Cells were harvested by centrifugation, the CD3 antibody-coated flask was removed from the refrigerator at 4℃and the liquid was discarded, and 10mL of 0.9% sterile physiological saline was added to wash the flask once.

(3) The cell pellet was thoroughly mixed using 2mL AIM V medium containing 100IU/mL IL-2, 3. Mu.g/mL CD28 antibody, 5ng/mL rhIL-7,5ng/mL rhIL-15, and the procedure was carefully performed to avoid air bubbles. Then, the remaining medium (30 mL or so, the cell concentration was not lower than 5X 10 ⁶ cells/mL) was added, and the cells were mixed.

(4) The well-mixed cell suspension is transferred into a 75cm ² cell culture flask, gently shaken and well mixed, and placed in a 37 ℃ incubator for culture. The cell state and the color of the culture medium are observed in the culture process, and a proper amount of culture medium can be supplemented according to the growth condition.

(5) After 4 days of stimulation with CD3 antibodies, cells can be transferred to new flasks and supplemented with corresponding volumes of medium. Cells were harvested on day 10 of culture for cell counting and flow antibody staining.

Example 6 in vitro cytokine secretion Capacity test of CD19-CAR-inKT cells

After the CD19-CAR-iNKT cells and CD19-CAR-T cells prepared in example 4 were cultured until day 21, 3×10 ⁵ CD19-CAR-iNKT cells, CD19-CAR-T cells, and 1×10 ⁵ CD 19-expressing KMHC cells were co-cultured in 0.5ml X-VIVO 15 serum-free immune cell culture medium (containing no IL-2 and α -GalCer) for 24 hours at an effector cell to target cell ratio of 3:1, the cells were collected, centrifuged at 1000g for 5 minutes, and the supernatant was assayed for IFN- γ and IL-2 content using an ELISA kit.

The results in FIG. 3 show that the ability of CD19-CAR-iNKT cells to secrete IFN-gamma and IL-2 is stronger than that of ordinary CAR-T cells, and has significant differences from the iNKT cells, indicating that the CD19-CAR-iNKT cells have stronger proliferation ability and killing ability to tumors.

Example 7 in vitro tumor cell killing experiments with CD19-CAR-inKT cells

After the CD19-CAR-iNKT cells and CD19-CAR-T cells prepared in example 4 were cultured until day 21, 0.33×10 ⁵ CD19-CAR-iNKT cells, 1×10 ⁵ CD19-CAR-iNKT cells, 3×10 ⁵ CD19-CAR-iNKT cells, 0.33×10 ⁵ CD19-CAR-T cells, 1×10 ⁵ CD19-CAR-T cells, 3×10 ⁵ CD19-CAR-T cells were co-cultured with 1×10 ⁵ CD 19-expressing KMCH cells (mixed with fluorescent dye CFSE for 10 minutes) for 6 hours, respectively, KMCH cells expressing CD19 alone were set as control groups, and the fluorescent intensity (the greater the fluorescence intensity released in the KMCH cells was calculated as the fluorescent intensity of CD19-CAR-iNKT cells was increased) in each group culture supernatant was measured with an enzyme-labeling instrument, and the fluorescent efficiency of each nkt cell group was calculated as the fluorescent intensity was increased.

The results in FIG. 4 show that CD19-CAR-iNKT cells and CD19-CAR-T cells can kill KMCH cells effectively, but the killing ability of CD19-CAR-iNKT cells is strongest, which is consistent with the higher ability of secreting IFN-gamma and IL-2 cytokines.

Based on the foregoing description one skilled in the art will appreciate that the present disclosure may be embodied in different specific forms without departing from its technical spirit or essential characteristics. The above embodiments are, therefore, to be understood as not being limiting in all respects, but rather illustrative. The scope of the present disclosure is defined by the appended claims, rather than by the description preceding them, and all changes and modifications that fall within the metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.

Claims

1. A chimeric antigen receptor targeting CD19, wherein said chimeric antigen receptor is sequentially linked by a tgfr signal peptide, a specific antibody targeting CD19, a CD 8a hinge region, a CD 8a transmembrane domain, a CD28 costimulatory signal domain, a CD3 zeta intracellular signaling domain;

The amino acid sequence of the specific antibody targeting CD19 is shown as the amino acid sequence of SEQ ID NO. 1;

The amino acid sequence of the CD8 alpha hinge region is shown as SEQ ID NO. 10;

The amino acid sequence of the CD8 alpha transmembrane domain is shown as SEQ ID NO. 11;

the amino acid sequence of the CD28 co-stimulatory signaling domain is shown in SEQ ID NO. 12;

The amino acid sequence of the CD3 zeta intracellular signal transduction domain is shown as SEQ ID NO. 13;

the amino acid sequence of the tEGFR signal peptide is shown as SEQ ID NO. 14.

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. 15.

3. A polynucleotide encoding the chimeric antigen receptor of claims 1-2.

4. A vector comprising the polynucleotide of claim 3, wherein the vector comprises a cloning vector and an expression vector.

5. An engineered host cell comprising the polynucleotide of claim 3 or the vector of claim 4.

6. An immune cell transduced with the chimeric antigen receptor according to claim 1.

7. The immune cell according to claim 6, wherein the immune cell is a T cell, NK cell or iNKT cell.

8. The immune cell according to claim 7, wherein the immune cell is an iNKT cell.

9. A pharmaceutical composition comprising the chimeric antigen receptor of claims 1-2, the polynucleotide of claim 3, the vector of claim 4, the host cell of claim 5, or the immune cell of claims 6-8.

10. Use of the chimeric antigen receptor according to claims 1-2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5 or the immune cell according to claims 6-8 for the preparation of a medicament for the treatment of a CD19 overexpressing cancer.