CN107893055B - Natural killer cell modified by specific chimeric antigen receptor gene and preparation method and application thereof - Google Patents
Natural killer cell modified by specific chimeric antigen receptor gene and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a natural killer cell modified by a specific chimeric antigen receptor gene, a preparation method and application thereof, wherein the natural killer cell modified by the specific chimeric antigen receptor gene comprises an NY-ESO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene. The preparation method comprises the step of infecting natural killer cells with lentiviruses containing NY-ESO-1 specific chimeric antigen receptor genes and lentiviruses containing truncated spleen tyrosine kinase Syk genes. The natural killer cell modified by the specific chimeric antigen receptor gene improves the specific killing performance of the target tumor cells of the natural killer cell, increases the capability of the natural killer cell for activating downstream signals and avoids the escape of tumor immunity through the combined action of the NY-ESO-1 specific chimeric antigen receptor gene and the truncated spleen tyrosine kinase Syk gene, thereby enhancing the killing activity to the tumor cells.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a natural killer cell modified by a specific chimeric antigen receptor gene and a preparation method and application thereof.
Background
NK cells recognize infected and malignant cancerous cells through a variety of cell surface receptors (e.g., NKG2D, CD16) and natural cytotoxic receptors (NKp44, NKp46, NKp30) that activate regulatory proteins 5631 DAP 7, DAP12 containing an Immunoreceptor Tyrosine Activation Motif (ITAM) structure, which initiate release of cytotoxic granule proteins and regulate secretion of cytokines and chemokines such as IFN-. gamma.and TNF- α.
In recent years, the T cell technology modified by Chimeric Antigen Receptor (CAR) makes a significant breakthrough in the clinical application of treating the tumors in the blood system, and shows good targeting property, killing property and durability in clinical tests. However, in recent clinical trials CAR-T technology was found to have high risk factors causing cytokine storm and systemic neurotoxicity. In addition, CAR-T cells have not achieved clear efficacy in the treatment of solid tumors due to immunosuppressive and immune escape effects of the tumor microenvironment. NK cells are characterized by: the killing effect can be achieved without pre-sensitization and limitation of histocompatibility complex, and the NK cells of xenogenic origin can be used without causing rejection reaction in the body of the subject (patient); no cytokine storm is produced (e.g., no significant release of interleukin-6 occurs). The NK cell modified by the CAR is expected to enhance the capability of the NK cell in targeted killing of tumor cells and develop effector cells with strong anti-tumor effect.
Because germ cells do not express the H L A molecule and the blood testis barrier exists, the tumor immunotherapy aiming at the CT antigen generally does not generate immunotoxicity to normal tissue cells, NY-ESO-1(New York immunogenic gene cell carcinoma 1) is an important member of the CT antigen gene family, and is concerned in the field of tumor therapy because of strong immunogenicity in tumor antigens and expression in various tumor tissues (malignant melanoma, hepatocellular carcinoma, ovarian cancer and the like).
Although some transgenic NK cells containing specific receptors which are specifically combined with NY-ESO-1 appear nowadays, most of the CAR used for expression in NK cells is a generation CAR vector only having a T cell CD3 zeta domain, and has low compatibility with NK, and simultaneously, the load of the transfected NK cells is increased, so that the transfection efficiency is low; in addition, due to the existence of a tumor escape mechanism, the downstream signals of the transgenic NK cells are not sufficiently activated, and the killing effect of the current transgenic NK cells is reduced.
Disclosure of Invention
The invention mainly aims to provide a natural killer cell modified by a specific chimeric antigen receptor gene, and aims to solve the technical problem that the downstream signal of the transgenic natural killer cell is insufficient due to the existence of a tumor escape mechanism in the conventional transgenic natural killer cell.
In order to achieve the purpose, the natural killer cell modified by the specific chimeric antigen receptor gene provided by the invention comprises an NY-ESO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene.
Preferably, the NY-ESO-1 specific chimeric antigen receptor gene is arranged in a NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment.
Preferably, said NY-ESO-1 specific chimeric antigen receptor gene sequences are as described in Table 1.
Preferably, the truncated spleen tyrosine kinase Syk gene sequence is as shown in table 2.
The invention also provides a preparation method of the natural killer cell modified by the specific chimeric antigen receptor gene, which comprises the following steps:
simultaneously infecting NK cells with a first lentivirus and a second lentivirus, wherein the first lentivirus contains an NY-ESO-1 specific chimeric antigen receptor gene, and the second lentivirus contains a truncated spleen tyrosine kinase Syk gene.
Preferably, the first lentivirus is obtained as follows:
mixing the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment with an entry vector to embed the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment into the entry vector to obtain an entry vector with the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment;
mixing and recombining the entry vector with the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment and a lentiviral plasmid to establish a cancer-testis antigen NY-ESO-1 specific NK cell receptor expression plasmid;
co-transfecting 293FT cells with the cancer-testis antigen NY-ESO-1 specific NK cell receptor expression plasmid and virus packaging plasmids to obtain a first lentivirus.
Preferably, the method for obtaining the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment comprises the following steps:
obtaining NY-ESO-1 antigen immune total cDNA;
carrying out PCR on the NY-ESO-1 antigen immune total cDNA, an NY-ESO-1 antibody heavy chain variable region VH primer and a light chain variable region V L primer to obtain NY-ESO-1VH and NY-ESO-1V L gene fragments;
connecting the NY-ESO-1VH and NY-ESO-1V L gene fragments by adopting connecting peptide to obtain NY-ESO-1 antibody scFv gene fragment;
connecting the scFv gene segment of the NY-ESO-1 antibody with a pcDNA vector to obtain a pcDNA-NY-ESO-1/scFv plasmid;
designing and synthesizing a CD8-41BB-DAP12 expression frame of a chimeric antigen receptor according to CD8 α, 41BB and DAP12-ITAM sequence gene sequences, carrying out enzyme digestion by Hind III/XhoI, cloning into a pcDNA3.1(+) -NY-ESO-1/scFv plasmid to obtain a pcDNA3.1(+) -NY-ESO-1/scFv-CD8-41BB-DAP12 plasmid, and carrying out enzyme digestion by EcoR I/XhoI to obtain an NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment.
Preferably, the second lentivirus is obtained as follows:
obtaining a truncated spleen tyrosine kinase Syk gene sequence;
mixing a truncated spleen tyrosine kinase Syk gene sequence with an entry vector to embed the truncated spleen tyrosine kinase Syk gene sequence into the entry vector to obtain an entry vector with the truncated spleen tyrosine kinase Syk gene sequence;
mixing and recombining the entry vector with the truncated spleen tyrosine kinase Syk gene segment and a lentivirus plasmid to obtain a lentivirus expression plasmid p L enti 7.3-Syk;
the lentivirus expression plasmid p L enti7.3-Syk was co-transfected with viral packaging plasmid 293FT cells to obtain a second lentivirus.
Preferably, the truncated spleen tyrosine kinase Syk gene sequence is as shown in table 2.
The invention also provides a tumor pharmaceutical preparation, which comprises the natural killer cell modified by the specific chimeric antigen receptor gene or the natural killer cell modified by the specific chimeric antigen receptor gene prepared by the preparation method.
The natural killer cell modified by the specific chimeric antigen receptor gene contains an NY-ESO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene. The natural killer cell modified by the specific chimeric antigen receptor gene has stronger specific killing performance of target tumor cells because of containing the NY-ESO-1 specific chimeric antigen receptor gene, and meanwhile, the natural killer cell modified by the specific chimeric antigen receptor gene can increase the capability of activating downstream signals and avoid the immune escape of tumors because the natural killer cell can over-express the truncated spleen tyrosine kinase Syk gene. Therefore, the natural killer cell modified by the specific chimeric antigen receptor gene has stronger specific killing property of the targeted tumor cell, can effectively avoid escape of tumor immunity and enhances the killing activity to the tumor cell.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a schematic structural diagram of an NY-ESO-1/scFv-CD8-41BB-DAP12 gene artificially constructed in the embodiment of the present invention;
FIG. 2 is a schematic diagram of a p L enti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmid artificially constructed in the present embodiment;
FIG. 3 is an electrophoretogram of the product of the plasmid digestion of the inventive example, Entomor pENTR-NY-ESO-1/scFv-CD8-41BB-DAP 12;
FIG. 4 is a band diagram of the electrophoresis of the recombinant lentiviral expression plasmid according to the embodiment of the invention;
FIG. 5 is a diagram showing the result of electrophoresis of the restriction enzyme product of plasmid pENTR-Syk according to the embodiment of the present invention;
FIG. 6 is a diagram showing the electrophoresis results of PCR products of the colonies transformed with the recombinant lentivirus expression plasmid p L enti7.3-Syk according to the embodiment of the present invention;
FIG. 7 is a flow detection chart of NK cell killing HepG-2 cell in vitro of liver cancer according to the embodiment of the invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
If there is a description of "first", "second", etc. in an embodiment of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
The invention provides a natural killer cell modified by a specific chimeric antigen receptor gene.
In the embodiment of the invention, the specific chimeric antigen receptor gene modified natural killer cell is a chimeric antigen receptor gene modified NK cell which targets NY-ESO-1 and contains NK cell regulatory protein DAP12 immune receptor tyrosine activation motif (ITAM). Here, the specific chimeric antigen receptor gene-modified natural killer cell is represented as NY-ESO-1/scFv-CD8-41BB-DAP 12. Specifically, the natural killer cell modified by the specific chimeric antigen receptor gene comprises an NY-ESO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene. The natural killer cell modified by the specific chimeric antigen receptor gene of the invention contains the targeted NY-ESO-1 specific chimeric antigen receptor gene and the truncated spleen tyrosine kinase Syk gene, so the natural killer cell modified by the specific chimeric antigen receptor gene has higher specific killing performance of targeted tumor cells, and the truncated spleen tyrosine kinase Syk gene can increase the capability of the natural killer cell for activating downstream signals, thereby effectively avoiding the escape of tumor immunity and enhancing the killing activity to the tumor cells.
In one embodiment, the NY-ESO-1 specific chimeric antigen receptor gene is arranged in a NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment. Specifically, the NY-ESO-1 specific chimeric antigen receptor gene is obtained by infecting NK into cells by a virus containing NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragments, and the detailed introduction of the method for obtaining the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragments is described in detail below.
Wherein, the NY-ESO-1 specific chimeric antigen receptor gene sequence is shown in the following table 1:
TABLE 1
The sequence of the truncated spleen tyrosine kinase Syk gene is shown in the following table 2:
TABLE 2
The amino acid sequence of the truncated spleen tyrosine kinase Syk is shown in the following table 3:
TABLE 3
The natural killer cell modified by the specific chimeric antigen receptor gene contains an NY-ESO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene. The natural killer cell modified by the specific chimeric antigen receptor gene has stronger specific killing performance of target tumor cells because of containing the NY-ESO-1 specific chimeric antigen receptor gene, and meanwhile, the natural killer cell modified by the specific chimeric antigen receptor gene can increase the capability of activating downstream signals and avoid the immune escape of tumors because the natural killer cell can over-express the truncated spleen tyrosine kinase Syk gene. Therefore, the natural killer cell modified by the specific chimeric antigen receptor gene has stronger specific killing property of the targeted tumor cell, can effectively avoid escape of tumor immunity and enhances the killing activity to the tumor cell.
The invention also provides a preparation method of the natural killer cell modified by the specific chimeric antigen receptor gene, which is used for preparing the natural killer cell modified by the specific chimeric antigen receptor gene. Wherein, the preparation method comprises the following steps:
simultaneously infecting NK cells with a first lentivirus and a second lentivirus, wherein the first lentivirus contains an NY-ESO-1 specific chimeric antigen receptor gene, and the second lentivirus contains a truncated spleen tyrosine kinase Syk gene.
In one embodiment, the first lentivirus is obtained as follows:
s11: mixing the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment with an entry vector to embed the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment into the entry vector to obtain the entry vector with the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment.
S12: mixing and recombining the entry vector with the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment and a lentiviral plasmid to establish a cancer-testis antigen NY-ESO-1 specific NK cell receptor expression plasmid.
S13: and co-transfecting 293T cells with the cancer-testis antigen NY-ESO-1 specific NK cell receptor expression plasmid and a virus packaging plasmid to obtain a first lentivirus.
In step S11, the entry vector may be pENTR11, but is not limited thereto. Specifically, in step S11, the method for obtaining the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment comprises the following steps:
s111: obtaining NY-ESO-1 antigen immune total cDNA;
s112, carrying out PCR on the NY-ESO-1 antigen immune total cDNA, an NY-ESO-1 antibody heavy chain variable region VH primer and a light chain variable region V L primer to obtain NY-ESO-1VH and NY-ESO-1V L gene segments;
s113, connecting the NY-ESO-1VH and NY-ESO-1V L gene fragments by adopting connecting peptide to obtain an NY-ESO-1 antibody scFv gene fragment;
s114: connecting the scFv gene segment of the NY-ESO-1 antibody with a pcDNA vector to obtain a pcDNA-NY-ESO-1/scFv plasmid;
s115, designing and synthesizing a CD8-41BB-DAP12 expression frame of the chimeric antigen receptor according to the gene sequences of CD8 α, 41BB and DAP12-ITAM, carrying out enzyme digestion by Hind III/XhoI, cloning into a pcDNA3.1(+) -NY-ESO-1/scFv plasmid to obtain the pcDNA3.1(+) -NY-ESO-1/scFv-CD8-41BB-DAP12 plasmid, and carrying out enzyme digestion by EcoR I/XhoI to obtain an NY-ESO-1/scFv-CD8-41 BB-12 gene fragment.
In step S112, the primer for VH of the heavy chain variable region of the NY-ESO-1 antibody and the primer for V L of the light chain variable region can be designed according to the sequence of VH of the heavy chain variable region and V L according to the principle of primer design, concretely, the primer for VH of the heavy chain variable region is VH-F and VH-R, the primer for V L of the light chain variable region is V L-F and V L-R, and the base sequences are as follows:
VH-F：5'-GAAGTT CAA TTG TTA GAG TCT GGT GGC GGT-3'
VH-R：5'-GCT TGA GAC GGT GAC CGT GGA CCC TTG GCC-3'
VL-F：5'-CAG AGC GAA TTG ACT CAG CCT CGC TCA GTG-3'
VL-R：5'-GAG GAC GGT GAC GTC GGT CCC TGT CCC GAA-3'
the connecting peptide in step S113 may be selected from the group consisting of Gly4Ser3The base sequence is as follows:
5'-GGT GGT GGT GGT TCT GGC GGC GGC GGC TCC GGT GGT GGT GGA TCT-3'。
the pcDNA vector in step S114 may be a vector commonly used in the pcDNA vector series, such as pcDNA3.1 (+).
CD8 α, 41BB and DAP12-ITAM gene sequences in step S115 can be directly obtained from a gene bank (Genbank (NCBI)), the expression cassette of Hinge-TM-41BB-DAP12 of the chimeric antigen receptor is designed and synthesized according to CD8 α, 41BB and DAP12-ITAM gene sequences, including the comparison chain region (Hinge) and the transmembrane region (TM) CD8 α (aa135-205), the intracellular functional region (aa214-255) of 41BB and DAP12-ITAM, therefore, the expression cassette of Hinge-TM-41BB-DAP12, i.e. the expression cassette of CD8-41BB-DAP12, wherein the amino acid sequence of DAP 45-ITAM R L VPRGRGAAEAATRKQRITETESPYQE L QGQRSDVYSD L NTYK. additionally, the expression cassette of Hinge-TM-41BB-DAP12 can be synthesized in a conventional synthesis manner, for example, the Hinge-TM-41 BB-12 expression cassette can be synthesized by NYK 7 NYK BioSci GYK, the Hinge-TM-7-BB-41 BB-DAP 7-T-K, the expression cassette can be obtained by XK cleavage of XK 7, the restriction enzyme sequence of XK of NYK 7, the XK-K.
In step S12, the lentiviral plasmid may be a commonly used lentiviral vector, for example, p L enti 7.3/V5-DEST.
In step S13, the viral packaging plasmid may be a commonly used viral packaging plasmid, such as ViraPowerTM packaging plasmid.
In one embodiment, the second lentivirus is obtained as follows:
s21: obtaining a truncated spleen tyrosine kinase Syk gene sequence;
s22: mixing a truncated spleen tyrosine kinase Syk gene sequence with an entry vector to embed the truncated spleen tyrosine kinase Syk gene sequence into the entry vector, thereby obtaining the entry vector with the truncated spleen tyrosine kinase Syk gene sequence.
S23, mixing and recombining the entry vector with the truncated spleen tyrosine kinase Syk gene sequence and a lentiviral plasmid to obtain a lentiviral expression plasmid p L enti 7.3-Syk.
S24 co-transfecting 293FT cells with the lentivirus expression plasmid p L enti7.3-Syk and a virus packaging plasmid to obtain a second lentivirus.
In step S21, the entry vector may be pENTR11, but is not limited thereto. In one example, restriction sites EcoR I/Hind III can be added to each end of the sequence of the truncated spleen tyrosine kinase Syk gene and cloned directly into pENTR11 vector.
In step S21, the functional domain of SYK protein is searched through NCBI, the N-terminal SH2 domain, C-terminal SH2 domain and protein tyrosine kinase domain of SYK protein are selected, and the corresponding gene sequences are linked, thereby obtaining the truncated spleen tyrosine kinase SYK gene sequence.
In step S22, the lentiviral plasmid may be a commonly used lentiviral vector, for example, p L enti 7.3/V5-DEST.
In step S23, the viral packaging plasmid may be a commonly used viral packaging plasmid, such as a ViraP power packaging plasmid.
In addition, in the preparation method of the natural killer cell modified by the specific chimeric antigen receptor gene, the first lentivirus and the second lentivirus infect the NK cell simultaneously, and can infect according to a normal lentivirus infection method, which is not described herein again.
The invention provides a method for preparing a natural killer cell modified by a specific chimeric antigen receptor gene
The method simultaneously infects natural killer cells by a first lentivirus containing a targeting cancer-testis antigen NY-ESO-1 antigen specific chimeric antigen receptor gene and a second lentivirus containing a truncated spleen tyrosine kinase Syk gene, so that the natural killer cells modified by the specific chimeric antigen receptor gene obtained by infection have stronger specific killing performance of the targeting tumor cells, and simultaneously increases the capability of the natural killer cells modified by the specific chimeric antigen receptor gene for activating downstream signals, avoids the immune escape of tumors and enhances the killing activity of the natural killer cells modified by the specific chimeric antigen receptor gene on the tumor cells.
The present invention will be described in further detail with reference to specific examples.
The test steps are as follows:
1. lentivirus expressing NY-ESO-1 antigen specific scFv-CD8-41BB-DAP12 chimeric receptor
1.1 obtaining NY-ESO-1 antigen immune mouse total cDNA
Immunizing BA L B/C mouse with NY-ESO-1 antigen, killing the mouse when the titer meets the requirement (1:100000 or more), taking spleen, and extracting total spleen RNA of the mouse by using a Trizol method.
Total cDNA was synthesized using the iScript cDNA Synthesis Kit, as shown in Table 4 below:
TABLE 4
| Reagent | Volume of |
| 5×iScript reaction mix | 4μl |
| iScript reverse transcriptase | 1μl |
| Nuclease-free water | 5μl |
| Total RNA | 10μl |
| Total volume | 20μl |
Reaction conditions are as follows: 5min at 25 deg.C → 30min at 42 deg.C → 5min at 85 deg.C → 4 deg.C hold.
1.2 NY-ESO-1 antibody heavy chain variable region VH and light chain variable region V L gene acquisition NY-ESO-1 antibody heavy chain variable region VH and light chain variable region V L primer sequences are shown in the following table 5:
TABLE 5
The PCR reaction system is shown in Table 6 below:
TABLE 6
| Reagent | Volume of |
| Taq DNA polymerase | 0.25 |
| 10×PCR Buffer | 5μl |
| dNTPs(2.5mM) | 4μl |
| NY-ESO-1 antigen immunized mouse total cDNA | 1μl |
| VH-F(20μM) | 1μl |
| VH-R(20μM) | 1μl |
| ddH2O | Make up to 50. mu.l |
The reaction conditions were as follows: pre-denaturation at 94 ℃ for 5min, at 94 ℃ for 45s, at 60 ℃ for 1min, at 72 ℃ for 1min for 35 cycles, and extension at 72 ℃ for 5 min.
After 1.2% agarose gel electrophoresis identification, the PCR products of the VH and V L genes of the heavy chain variable region and the light chain variable region of the NY-ESO-1 antibody are recovered.
1.3 acquisition of scFv fragment and plasmid of NY-ESO-1 antibody
NY-ESO-1VH and NY-ESO-1V L fragments obtained in 1.2 are connected by connecting peptide (Gly4Ser)3 (base sequence: 5'-GGT GGT GGT GGT TCTGGC GGC GGC GGC TCC GGT GGT GGT GGA TCT-3') through multi-gene fragment Overlap Extension PCR (OE-PCR) to obtain NY-ESO-1 scFv fragments, and enzyme cutting sites EcoR I/Hind III.OE-PCR reaction conditions are respectively introduced at the front end and the rear end, wherein (1) step 1 comprises pre-denaturation at 95 ℃ for 1min, at 94 ℃ for 1min, at 60 ℃ for 1min, at 72 ℃ for 1min, and Extension at 72 ℃ for 10min, and (2) step 2 comprises pre-denaturation at 95 ℃ for 5min, at 94 ℃ for 45s, at 62 ℃ for 1min, at 72 ℃ for 1min, and at 35, and Extension at 72 ℃ for 5 min.
After 1.2% agarose gel electrophoresis identification, recovering NY-ESO-1 antibody sc Fv fragment, EcoR I/Hind III double enzyme digestion recovered fragment and pcDNA3.1(+) vector, and connecting NY-ESO-1 antibody scFv fragment with pcDNA3.1(+) vector by using T4DNA ligase to obtain pcDNA3.1(+) -NY-ESO-1/scFv plasmid.
1.4 construction of scFv-CD8-41BB-DAP12 Gene fragment specific to NY-ESO-1 antigen
The expression cassette of Hinge-TM-41BB-DAP12 of the chimeric antigen receptor was designed according to the gene sequence of CD8 α, 41BB and DAP12-ITAM in genebank (NCBI), including the comparative chain region (Hinge) and transmembrane region (TM) CD8 α (aa135-205), the intracellular functional region of 41BB (aa214-255) and DAP12-ITAM (amino acid sequence shown in Table 7 below), restriction enzyme sites Hind III and XhoI were added to the 5 'end of Hinge and 3' end of DAP12, respectively, and Nanjing King Murray Biotech Limited was entrusted to synthesize the whole expression cassette.
TABLE 7
The synthesized CD8-41BB-DAP12 expression frame is cut by Hind III/XhoI enzyme and then cloned into pcDNA3.1(+) -NY-ESO-1/scFv plasmid to obtain pcDNA3.1(+) -NY-ESO-1/scFv-CD8-41BB-DAP12 plasmid, and then EcoRI/XhoI enzyme is cut to obtain NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment, wherein the gene structure diagram is shown in figure 1.
Construction of 5 NY-ESO-1/scFv-CD8-41BB-DAP12 entry clone
1.51 NY-ESO-1/scFv-CD8-41BB-DAP12 entry clone pENTR11 ligation reaction System is shown in Table 8 below:
TABLE 8
| NY-ESO-1/scFv-CD8-41BB-DAP12 gene sheetSegment of | 0.5μl |
| Salt solution | 1μl |
| ddH2O | 3.5μl |
| pENTR11 vector | 1μl |
| Total volume | 6μl |
TOPO cloning reaction conditions: the reaction system was shaken gently and then incubated at room temperature (21-23 ℃) for 5 min. After the incubation, the reaction system was placed on ice to further transform competent cells, and the plasmid pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 was amplified and extracted.
1.52 restriction enzyme identification of entry clone pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12
1.521 the cleavage system (EcoR I/Xho I double cleavage system) is shown in Table 9 below:
TABLE 9
1.522 enzyme cutting conditions: carrying out enzymolysis for 2h in a water bath at 37 ℃.
1.523 mu.l of the enzymatic product was taken and detected by electrophoresis on 1.2% agarose gel. The electrophoretic bands were analyzed to determine if the entry clones were correct. If the entry clone pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 is correct, EcoR I/Xho I double enzyme digestion will obtain two fragments of 1.3kb and 2.3kb respectively.
Construction of lentiviral expression plasmid 1.6 p L enti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12
1.61L R reaction
The L R reaction is a recombination reaction of an entry clone pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 and a target vector p L enti7.3/V5-DEST vector, and after recombination, a lentivirus expression plasmid p L enti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 (as shown in a figure 2) is obtained, and a L R reaction system is shown in the following table 10:
watch 10
Unfreezing on ice
LR ClonaseTMII Plus Enzyme Mix, 2. mu.l of the mixture was added to the L R reaction system, gently mixed, and then left at 25 ℃ for 1 hour.
Mu.l proteinase K was added to the reaction and incubated at 37 ℃ for 10 min.
1.62L R reaction product conversion
1.621A tube of E.coli competent cells (Invitrogen, Cat. No. C7373-03) was thawed on ice
1.622 add 2-3. mu.l of L R reaction product to the competent cell suspension, mix gently (do not blow with a pipette), incubate on ice for 30min, heat shock in a water bath at 42 ℃ for 30s, transfer the reaction tube to ice and incubate for 2 min.
1.623 add 225. mu.l of S.O.C. medium pre-warmed at room temperature.
1.624 the reaction tube was covered and incubated for 1h at 37 ℃ on a horizontal shaker at 225 rpm.
1.625 remove 100. mu.l of the transformation product and spread evenly onto pre-heated L B plates (containing ampicillin) and incubate overnight at 37 ℃ in an incubator.
1.63 screening and amplification of Positive clones
1.631A small amount of each of the 3 clones was dipped in a tip, and the tip was repeatedly blown in 10. mu.l of sterile water.
1.632 mu.l of the suspension was aspirated for PCR, and the reaction system and conditions are shown in Table 11 below:
TABLE 11
The PCR reaction conditions were as follows:
1.633 PCR products of three clones were subjected to agarose gel electrophoresis, and if a clear single band was obtained at 1.3kb, the identified positive clones were picked up in L B medium containing ampicillin for expansion culture.
1.634 plasmid DNA purification kit (Promega, Cat. No. A7500) is used to separate and purify plasmid DNA from the overnight cultured bacterial liquid, namely lentivirus expression plasmid p L enti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12, and meanwhile, the strain is preserved.
Preparation of 1.7 p L enti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 lentivirus
1.71 Day 15 × 106 293FT cells (Invitrogen, Cat. No. R700-07) were centrifuged and the supernatant discarded, resuspended in 10ml of 37 ℃ pre-warmed complete medium (D-MEM + 10% FBS +2mM L-glutamine +0.1mM non-essential amino acids +1mM sodium pyruvate + 1% P/S), plated in 10cm dishes and incubated overnight at 37 ℃ in a 5% CO2 incubator.
1.72 Day 2: the culture medium in the dish was discarded, and 5ml of FBS-10% was added
I broth (Invitrogen, Cat. No. 31985-062).
1.73
2000 preparation of the Complex：
1.731 serum-free 1.5ml
I the culture medium was added to a 5ml centrifuge tube and 9. mu.g ViraPower was addedTMThe plasmid mixture was packaged with 3. mu.g of lentiviral expression plasmid p L enti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12, and gently mixed.
1.732 serum-free 1.5ml
The culture solution I is added into another 5ml centrifuge tube, and 36 mu l of the culture solution is added
2000, incubation at room temperature for 5min after gentle mixing.
1.733 the solution from both steps 1.731 and 1.732 was transferred to a centrifuge tube and mixed gently.
1.734 incubation at RT for 20min to give
2000 of the complex.
1.74 will result
2000 complexes were added dropwise slowly to the petri dish and the dish was gently shaken back and forth. Incubate overnight at 37 ℃ in a 5% CO2 incubator.
1.75 Day 3: the dishes were removed, the medium was discarded, and 10ml of DMEM complete medium was added. Incubating at 37 ℃ in a 5% CO2 incubator for 48-72 h.
1.76 Day5 or Day 6: the culture solution in the petri dish was transferred to a 15ml centrifuge tube and centrifuged at 2000g for 15min at 4 ℃.
1.77 the supernatant was aspirated, and p L enti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 lentivirus was collected, dispensed into 1ml cryovials, and stored at-80 ℃ for a long period.
2. Lentivirus expressing truncated Syk
2.1 obtaining a truncated spleen tyrosine kinase Syk gene sequence.
The sequence of the truncated Syk gene was synthesized by Nanjing Kingsri Biotechnology Ltd, and the sequence of the truncated spleen tyrosine kinase Syk gene is shown in Table 2.
2.2 construction of truncated Syk entry clones
2.21 EcoRI/Hind III restriction sites are respectively added at two ends of the Syk gene sequence of the truncated spleen tyrosine kinase, and the restriction sites are directly cloned into a pENTR11 vector.
2.22 restriction enzyme identification of entry clone pENTR-Syk
2.221 cleavage system (EcoR I/Hind III double cleavage system) as shown in Table 12 below:
TABLE 12
2.222 enzyme cutting conditions: carrying out enzymolysis for 2h in a water bath at 37 ℃.
2.223 mu.l of the digestion product was collected and detected by electrophoresis on a 1.2% agarose gel. The electrophoretic bands were analyzed to determine if the entry clones were correct. If the entry clone pENTR-Syk is correct, two fragments of 1.4kb and 2.3kb are obtained by two enzyme cutting of EcoR I/Hind III.
Construction of 2.3 p L enti7.3-Syk Lentiviral expression plasmid
2.31L R reaction
The L R reaction is a recombination reaction between the entry clone pENTR-Syk and the target vector p L enti7.3/V5-DEST vector, and the lentivirus expression plasmid p L enti7.3-Syk is obtained after recombination, the reaction system of L R is shown in the following table 13:
watch 13
| pENTR-Syk (150 ng/reaction) | 1-7μl |
| pLenti7.3/V5-DEST vector(150ng/μl) | 1μl |
| TE Buffer，pH 8.0 | Added to a total volume of 8. mu.l |
Unfreezing on ice
LR ClonaseTMII Plus Enzyme Mix, 2. mu.l of the mixture was added to the L R reaction system, gently mixed, and then left at 25 ℃ for 1 hour.
Mu.l proteinase K was added to the reaction and incubated at 37 ℃ for 10 min.
2.32L R conversion of the reaction product
2.321A tube of E.coli competent cells (Invitrogen, Cat. No. C7373-03) was thawed on ice.
2.322 Add 2-3. mu.l of L R reaction product to the competent cell suspension, mix gently (do not blow with pipette) and incubate on ice for 30min, heat shock in water bath at 42 ℃ for 30s, transfer the reaction tube to ice and incubate for 2 min.
2.323 mu.l of S.O.C. medium pre-warmed at room temperature was added.
2.324 the reaction tubes were covered and incubated for 1h at 37 ℃ on a horizontal shaker at 225 rpm.
2.325 mu.l of the transformation product was removed and spread evenly onto pre-warmed L B plates (containing ampicillin) and incubated overnight in a 37 ℃ incubator.
2.33 screening and amplification of Positive clones
2.331A small amount of each of 3 clones was picked up with a tip, and the tip was placed in 10. mu.l of sterile water and blown up repeatedly.
2.332 mu.l of the suspension was aspirated for PCR, and the reaction system and conditions are shown in Table 14 below:
TABLE 14
| Bacterial liquid | 1μl |
| VH-F primer (20. mu.M) | 1μl |
| V5 reverse primer (20. mu.M) | 1μl |
| Bio-rad super mix | 6.25μl |
| ddH2O | 15.75μl |
| Total volume | 25μl |
The PCR reaction conditions were as follows:
2.333, and if a clear single band is obtained at 1.4kb, the identified positive clones were picked up in L B medium containing ampicillin for expansion culture.
2.334 plasmid DNA purification kit (Promega, Cat. No. A7500) is used to separate and purify the plasmid DNA from the overnight cultured bacterial liquid, namely, the lentivirus expression plasmid p L enti7.3-Syk, and the bacterial strain is preserved.
2.4 preparation of p L enti7.3-Syk lentivirus (cells expressing a genetic marker on one DNA molecule often also express a genetic marker carried by another DNA molecule, a phenomenon in which physically unrelated genes are integrated into the same integration sequence and expressed in the same transfected cell is called cotransfection)
2.41 Day 15 × 106 293FT cells (Invitrogen, Cat. No. R700-07) were centrifuged and the supernatant discarded, resuspended in 10ml of 37 ℃ pre-warmed complete medium (D-MEM + 10% FBS +2mM L-glutamine +0.1mM non-essential amino acids +1mM sodium pyruvate + 1% P/S), plated in 10cm dishes and incubated overnight at 37 ℃ in a 5% CO2 incubator.
2.42 Day 2: the culture medium in the dish was discarded, and 5ml of FBS-10% was added
I broth (Invitrogen, Cat. No. 31985-062).
2.43
2000 preparation of the complex:
2.431 serum-free 1.5ml
I the culture medium was added to a 5ml centrifuge tube and 9. mu.g ViraPower was addedTMThe plasmid mixture was packaged with 3. mu.g of the lentiviral expression plasmid p L enti7.3-Syk, gently mixed.
2.432 serum-free 1.5ml
The culture solution I is added into another 5ml centrifuge tube, and 36 mu l of the culture solution is added
2000, incubation at room temperature for 5min after gentle mixing.
2.433 the solutions obtained in the above 2.431 and 2.432 steps were transferred to a centrifuge tube and mixed gently.
2.434 incubation at room temperature for 20min to obtain
2000 of the complex.
2.44 will obtain
2000 complexes were added dropwise slowly to the petri dish and the dish was gently shaken back and forth. Incubate overnight at 37 ℃ in a 5% CO2 incubator.
2.45 Day 3: the dishes were removed, the medium was discarded, and 10ml of DMEM complete medium was added. Incubating at 37 ℃ in a 5% CO2 incubator for 48-72 h.
2.46 Day5 or Day 6: the culture solution in the petri dish was transferred to a 15ml centrifuge tube and centrifuged at 2000g for 15min at 4 ℃.
2.47 the supernatant was aspirated, and p L enti7.3-Syk lentivirus was collected and dispensed into 1ml cryovials for long-term storage at-80 ℃.
3. Preparation and amplification of NY-ESO-1 specific CAR-NK cells co-expressing Syk
3.1 isolation of genotyping H L A-A2+ PBMC cells from healthy volunteers
3.11 peripheral blood 25ml of healthy volunteers drawn H L A-A2+, heparin anticoagulation, centrifugation at room temperature (700g, 20min), sucking upper plasma, placing in a water bath kettle at 56 deg.C for 30min, standing at 4 deg.C for 15min, centrifugation (900g, 30min), and storing the autologous plasma at 4 deg.C for further use.
3.12 taking 700g of the above, centrifuging the lower cell component for 20min, adding D-PBS to 50ml, mixing uniformly, slowly adding into a 50ml centrifuge tube filled with 20ml of human lymphocyte separation liquid, and centrifuging at room temperature (800g, 15 min).
3.13 pipette the tunica albuginea cells into a 50ml centrifuge tube containing 5ml of RPMI 1640.
3.14 RPMI 1640 was washed twice (600g, 10min centrifugation) and the cells collected were PBMC cells.
3.2 preparation of CAR-NK cells
3.21 the PBMC cell density was adjusted to 1 × 106/ml by using Alys505 culture medium containing 50ng/ml CD16 monoclonal antibody, 1000U/ml I L-2, 100ng/ml I L-15 and 0.5% autologous plasma, transferred to a six-well plate, 2 ml/well, and cultured in a 5.0% CO2 incubator at 37 ℃ under saturated humidity.
3.22 cell density was adjusted to 1 × 106/ml every 3 days, and Alys505 culture medium containing 1000U/ml I L-2 and 0.5% autologous plasma was added.
3.23 Day7, dividing NK cells into 4 groups, transferring each group of cells to a 24-well plate at a density of 1 × 105/well, 100. mu.l/well, each group having three duplicate wells.
Group 1: only NY-ESO-1/scFv-CD8-41BB-DAP12 gene was transfected, named NY-ESO-1-CAR group.
Group 2: only the Syk gene was transfected, named Syk group.
Group 3: NY-ESO-1/scFv-CD8-41BB-DAP12 gene and Syk were transfected and named as NY-ESO-1-CAR-Syk group.
Group 4: blank control group, named NC group.
3.24 according to the above grouping, stock solutions containing lentiviral particles were added at an MOI value of 20 (where MOI represents the ratio of the number of viruses to the number of cells) for transfection in groups, and each group was prepared by adding a lentiviral solution to an Alys505 complete medium to 100. mu.l, adding the lentiviral solution to the cells by pipette, and gently pipetting and mixing the solution. NC groups were added with 100. mu.l of Alys-505 complete medium.
3.25 Polybrene was added to each group to a final concentration of 6. mu.g/ml, mixed by gentle shaking, and incubated at 37 ℃ in a 5% CO2 incubator for 24 hours.
3.26 day 8, each group of cells was centrifuged, the supernatant containing lentivirus was discarded, resuspended in 200. mu.l of Alys-505 culture medium, and added to 24-well plates. And (5) supplementing liquid according to the growth state of the cells.
3.27 Day10, and the mature groups of cells were harvested for subsequent analysis.
4. NY-ESO-1 specific CAR-NK cell in-vitro anti-tumor activity detection of co-expressed Syk
4.1 taking the cells of the groups as effector cells, taking the liver cancer HepG-2 cells marked by CFSE as target cells, mixing the effector cells and the target cells according to an effect-target ratio of 20:1, gently mixing the effector cells and the target cells, and placing the mixture in an incubator with 5% CO2 and 37 ℃ for incubation.
After 4.224 h, 1. mu.g/ml PI staining solution was added, mixed well, incubated at room temperature in the dark for 15min, and the percentage of CFSE + PI + cells (dead HepG-2 cells) was determined by flow cytometry.
5. Detection of antitumor activity in NY-ESO-1 specific CAR-NK cell animals co-expressing Syk
Taking human liver cancer HepG-2 cells in logarithmic growth phase, preparing single cell suspension after trypsinization, injecting 0.1ml of suspension containing 1 × 107 cancer cells into scapular part of nude mice subcutaneously, wherein the experimental grouping and the treatment condition are shown in the following table, observing the change of diet, activity and the like of animals in each group every day, measuring the body weight of the nude mice every other day, observing the change of the body weight, measuring the maximum longitudinal diameter (a) and the maximum transverse diameter (b) of tumors every other 2 days, observing the growth of the tumors, and calculating the tumor inhibition rate of the nude mice in each group according to the formula, wherein the tumor volume is 1/12 pi × a × b2., the tumor inhibition rate is 13 days after the treatment of the group (3) is finished, and the tumor inhibition rate is (1-average tumor volume of the treatment group/average tumor volume of the negative control group) is × 100% and the experimental grouping and the treatment condition are shown in the following table 15:
watch 15
Wherein, FAC chemotherapy regimen: fluorouracil 500 mg/M2; doxorubicin 50 mg/M2; cyclophosphamide 500mg/M2, administered on the first day for 21 days as a course of treatment.
6. Results of the experiment
6.1 construction of CAR construct and expression plasmid map
6.2 restriction enzyme identification of entry clone pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12
The entry clone pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmid was subjected to EcoR I/Xho I double digestion, and agarose gel electrophoresis detection, and clear bands were found at 2.3kb and 1.3kb, respectively (FIG. 3), consistent with the expected results. The NY-ESO-1/scFv-CD8-41BB-DAP12 gene was confirmed to be correctly inserted into pENTR vector.
6.3 recombinant lentivirus expression plasmid p L enti7.3/NY-ESO-1/scFv-CD8-41BB-DAP12 transformed colony PCR results
Three clones on L B plates were randomly picked, PCR amplification was performed with designed forward primer and V5 reverse primer, and the product was identified by agarose gel electrophoresis, and a clear band appeared at 1.3kb (FIG. 4), indicating that the NY-ESO-1/scFv-CD8-41BB-DAP12 gene was successfully inserted into the p L enti7.3/V5-DEST expression vector.
6.4 restriction enzyme identification of entry clone pENTR-Syk
The products obtained by double restriction of the plasmid Englor pENTR-Syk with EcoR I/Hind III were detected by agarose gel electrophoresis and showed clear bands at about 2.3kb and 1.4kb, respectively (FIG. 5), consistent with the expected fragment results. The correct insertion of Syk into the vector was confirmed.
6.5 electrophoresis results of PCR products from transformed colonies of the recombinant lentivirus expression plasmid p L enti7.3-Syk
Three clones on L B plates were picked at random and PCR amplified with the designed forward and V5 reverse primers, and the PCR products were identified by agarose gel electrophoresis with a clear band at 1.4kb (FIG. 6), indicating successful insertion of Syk into the p L enti7.3/V5-DEST expression vector.
6.6 flow assay result of NK cells killing liver cancer HepG-2 cells in vitro
The cell groups are used as effector cells, the CFSE is used for marking liver cancer HepG-2 cells and used as target cells, the killing efficiency of NK cells on the liver cancer HepG-2 cells is detected by using flow cytometry, the experimental result is shown in figure 7, the killing rate of NY-ESO-1-CAR cells on the liver cancer HepG-2 cells is about 41.93%, the killing rate of Syk cells on the liver cancer HepG-2 cells is about 18.35%, the killing rate of NY-ESO-1-CAR-Syk cells on the liver cancer HepG-2 cells is about 67.56%, the killing rate of NC cells on the liver cancer HepG-2 cells is about 14.87%, and the killing efficiency of NY-ESO-1 specific NK-CAR cells co-expressing Syk on the liver cancer HepG-2 cells is far higher than that of the rest cell groups. The NY-ESO-1 specific CAR-NK cells co-expressing Syk prepared by the method have a high-efficiency killing effect on liver cancer cells.
6.7 Experimental results of in vivo inhibition of hepatoma cells by NY-ESO-1 specific CAR-NK cells co-expressing Syk
NY-ESO-1 specific CAR-NK cells co-expressing Syk are subjected to in vivo anti-tumor experiments, and the results show that all groups of nude mice survive, and the weight of the nude mice in a chemotherapy group is reduced, the tumor volume is reduced, but the feeding and activity are reduced by taking the nude mice in a negative control group as a reference. The nude mice of the NY-ESO-1 specific CAR-NK cell treatment group co-expressing Syk have good survival condition, no obvious weight reduction, obvious smaller tumor volume and 82.8 percent of tumor inhibition rate. The body weight and tumor suppression statistics are shown in Table 16. Experimental results show that the NY-ESO-1 specific CAR-NK cells co-expressing Syk prepared by the method can effectively inhibit the growth of in vivo tumors.
TABLE 16 comparison of body weight, tumor volume and tumor inhibition rate of each group of nude mice
The invention also provides a tumor pharmaceutical preparation. The tumor pharmaceutical formulation comprises NK cells. Wherein, the NK cell is the natural killer cell modified by the specific chimeric antigen receptor gene or the natural killer cell modified by the specific chimeric antigen receptor gene prepared by the preparation method. Since the tumor pharmaceutical preparation adopts all technical schemes of all the embodiments, at least all the beneficial effects brought by the technical schemes of the embodiments are achieved, and no details are repeated here.
In addition, the amount or dosage of specific chimeric antigen receptor genetically modified natural killer cells contained in the tumor pharmaceutical preparation of the present invention should be an effective dose, specifically, a therapeutically effective dose, which refers to an amount of the compound of the present invention sufficient to show a benefit or clinical significance to an individual. One skilled in the art will appreciate that the actual amount or dose administered and the time course of administration will depend on the nature and severity of the disease being treated, the age and general condition of the subject being treated, and the mode of administration, among other factors.
It will be appreciated that a pharmaceutically acceptable carrier may also be included in the above pharmaceutical formulations. In particular embodiments, the pharmaceutically acceptable carrier refers to any adjuvant known to those skilled in the art to be suitable for a particular mode of administration.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen Murashel biomedical science and technology development Limited
<120> specific chimeric antigen receptor gene modified natural killer cell, preparation method and application thereof
<130>2017.10.30
<160>7
<170>PatentIn version 3.5
<210>1
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<213> Artificial sequence
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ccggaattcc ggatgcttct cctggtgaca agccttctgc tctgtgagtt accacaccca 60
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ggtggttctt tacgtctttc ttgcgctgct tccggattca ctttctctac ttaccagatg 180
tcttgggttc gccaagctcc tggtaaaggt ttggagtggg tttctggtat cgtttcttct 240
ggtggctcta ctgcttatgc tgactccgtt aaaggtcgct tcactatctc tagagacaac 300
tctaagaata ctctctactt gcagatgaac agcttaaggg ctgaggacac tgcagtctac 360
tattgtgcgg gggagctact tccctactac ggtatggacg tctggggcca agggaccacg 420
gtcaccgtct caagcggtgg tggtggttct ggcggcggcg gctccggtgg tggtggatct 480
cagagcgaat tgactcagcc tcgctcagtg tccgggtctc ctggacagtc agtcaccatc 540
tcctgcactg ggaccagccg tgatgttggt ggttataact atgtctcctg gtaccaacaa 600
cacccaggca aagcccccaa actcataatt catgatgtca tagagcggtc gtcaggggtc 660
cctgatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 720
caggctgagg atgaggctga ttattattgc tggtcatttg caggctccta ttatgtcttc 780
gggacaggga ccgacgtcac cgtcctcccc aagcttgggg cgaagcccac cacgacgcca 840
gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 900
gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 960
gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1020
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acctacgcca tcgccggtgg caggacccat gccagccccg ccgacctctg ccactaccac 240
tcccaggagt ctgatggcct ggtctgcctc ctcaagaagc ccttcaaccg gccccaaggg 300
gtgcagccca aggaaaaaat gccttggttc catggaaaaa tctctcggga agaatctgag 360
caaattgtcc tgataggatc aaagacaaat ggaaagttcc tgatccgagc cagagacaac 420
aacggctcct acgccctgtg cctgctgcac gaagggaagg tgctgcacta tcgcatcgac 480
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caaaaaatca aagaactggg ctctggtaat tttggaactg tgaaaaaggg ctactaccaa 660
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gaacttggtc ccctcaataa gtatttgcag cagaacagac atgtcaagga taagaacatc 900
atagaactgg ttcatcaggt ttccatgggc atgaagtact tggaggagag caattttgtg 960
cacagagatc tggctgcaag aaatgtgttg ctagttaccc aacattacgc caagatcagt 1020
gatttcggac tttccaaagc actgcgtgct gatgaaaact actacaaggc ccagacccat 1080
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aaaagcgatg tctggagctt tggagtgttg atgtgggaag cattctccta tgggcagaag 1200
ccatatcgag ggatgaaagg aagtgaagtc accgctatgt tagagaaagg agagcggatg 1260
gggtgccctg cagggtgtcc aagagagatg tacgatctca tgaatctgtg ctggacatac 1320
gatgtggaaa acaggcccgg attcgcagca gtggaactgc ggctgcgcaa ttactactat 1380
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<213> Artificial sequence
<400>4
gcttgagacg gtgaccgtgg acccttggcc 30
<210>5
<211>30
<212>DNA
<213> Artificial sequence
<400>5
cagagcgaat tgactcagcc tcgctcagtg 30
<210>6
<211>30
<212>DNA
<213> Artificial sequence
<400>6
gaggacggtg acgtcggtcc ctgtcccgaa 30
<210>7
<211>45
<212>DNA
<213>(Gly4Ser)3
<400>7
ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg gatct 45
Claims (6)
1. A natural killer cell modified by a specific chimeric antigen receptor gene is characterized by comprising an NY-ESO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene, wherein the NY-ESO-1 specific chimeric antigen receptor gene sequence is shown in Table 1, and the NY-ESO-1 specific chimeric antigen receptor gene is arranged in an NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment in the natural killer cell; the truncated spleen tyrosine kinase Syk gene sequence is shown in table 2.
2. A method for preparing a natural killer cell genetically modified by a specific chimeric antigen receptor, which comprises the following steps:
simultaneously infecting NK cells with a first lentivirus and a second lentivirus to obtain natural killer cells of claim 1; wherein the first lentivirus contains an NY-ESO-1 specific chimeric antigen receptor gene, and the second lentivirus contains a truncated spleen tyrosine kinase Syk gene.
3. The method of claim 2, wherein the first lentivirus is obtained by:
mixing the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment with an entry vector to embed the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment into the entry vector to obtain an entry vector with the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment;
mixing and recombining the entry vector with the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment and a lentiviral plasmid to establish a cancer-testis antigen NY-ESO-1 specific NK cell receptor expression plasmid;
co-transfecting 293FT cells with the cancer-testis antigen NY-ESO-1 specific NK cell receptor expression plasmid and virus packaging plasmids to obtain a first lentivirus.
4. The method of claim 3, wherein the NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment is obtained by the following steps:
obtaining NY-ESO-1 antigen immune total cDNA;
carrying out PCR on the NY-ESO-1 antigen immune total cDNA, an NY-ESO-1 antibody heavy chain variable region VH primer and a light chain variable region V L primer to obtain NY-ESO-1VH and NY-ESO-1V L gene fragments;
connecting the NY-ESO-1VH and NY-ESO-1V L gene fragments by adopting connecting peptide to obtain NY-ESO-1 antibody scFv gene fragment;
connecting the scFv gene segment of the NY-ESO-1 antibody with a pcDNA vector to obtain a pcDNA-NY-ESO-1/scFv plasmid;
designing and synthesizing a CD8-41BB-DAP12 expression frame of a chimeric antigen receptor according to CD8 α, 41BB and DAP12-ITAM sequence gene sequences, carrying out enzyme digestion by Hind III/XhoI, cloning into a pcDNA3.1(+) -NY-ESO-1/scFv plasmid to obtain a pcDNA3.1(+) -NY-ESO-1/scFv-CD8-41BB-DAP12 plasmid, and carrying out enzyme digestion by EcoR I/XhoI to obtain an NY-ESO-1/scFv-CD8-41BB-DAP12 gene fragment.
5. The method of any one of claims 2 to 4, wherein the second lentivirus is obtained by:
obtaining a truncated spleen tyrosine kinase Syk gene sequence;
mixing a truncated spleen tyrosine kinase Syk gene sequence with an entry vector to embed the truncated spleen tyrosine kinase Syk gene sequence into the entry vector to obtain an entry vector with the truncated spleen tyrosine kinase Syk gene sequence;
mixing and recombining the entry vector with the truncated spleen tyrosine kinase Syk gene segment and a lentivirus plasmid to obtain a lentivirus expression plasmid p L enti 7.3-Syk;
the lentivirus expression plasmid p L enti7.3-Syk was co-transfected with viral packaging plasmid 293FT cells to obtain a second lentivirus.
6. A pharmaceutical preparation for tumor comprising the specific chimeric antigen receptor gene-modified natural killer cell according to claim 1 or the specific chimeric antigen receptor gene-modified natural killer cell prepared by the preparation method according to any one of claims 2 to 5.
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| CN109182470A (en) * | 2018-09-20 | 2019-01-11 | 南京卡提医学科技有限公司 | It is a kind of to detect using DAP12 as the method and its kit of intracellular signal structure C AR-T cell |
| CN112063639B (en) * | 2019-06-10 | 2023-05-26 | 深圳市罗湖区人民医院 | Double-signal chimeric antigen receptor and preparation method and application thereof |
| CN112080512A (en) * | 2019-06-14 | 2020-12-15 | 深圳市罗湖区人民医院 | CAR-NK (CAR-natural killer) with safety switch and preparation method thereof |
| CN112279923B (en) * | 2019-07-22 | 2023-07-18 | 南京助天中科科技发展有限公司 | Chimeric antigen receptor and application thereof |
| CN112300288B (en) * | 2019-07-29 | 2022-08-02 | 济南赛尔生物科技股份有限公司 | Chimeric antigen receptor CAR of CIK cell and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105131126A (en) * | 2015-10-10 | 2015-12-09 | 北京康爱瑞浩生物科技股份有限公司 | Chimeric antigen receptor for treating malignant tumor and preparation method and application of chimeric antigen receptor |
| CN106456670A (en) * | 2014-04-25 | 2017-02-22 | 蓝鸟生物公司 | Improved methods for manufacturing adoptive cell therapies |
| WO2017049166A1 (en) * | 2015-09-17 | 2017-03-23 | Novartis Ag | Car t cell therapies with enhanced efficacy |
| CN106793780A (en) * | 2014-06-06 | 2017-05-31 | 蓝鸟生物公司 | The T cell composition of improvement |
| CN106755023A (en) * | 2015-10-15 | 2017-05-31 | 中国人民解放军军事医学科学院附属医院 | Chimeric antigen receptor immunocyte with safety switch and preparation method and application |
| WO2017123956A1 (en) * | 2016-01-15 | 2017-07-20 | Etubics Corporation | Methods and compositions for t-cell immunotherapy |
-
2017
- 2017-11-03 CN CN201711069205.5A patent/CN107893055B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106456670A (en) * | 2014-04-25 | 2017-02-22 | 蓝鸟生物公司 | Improved methods for manufacturing adoptive cell therapies |
| CN106793780A (en) * | 2014-06-06 | 2017-05-31 | 蓝鸟生物公司 | The T cell composition of improvement |
| WO2017049166A1 (en) * | 2015-09-17 | 2017-03-23 | Novartis Ag | Car t cell therapies with enhanced efficacy |
| CN105131126A (en) * | 2015-10-10 | 2015-12-09 | 北京康爱瑞浩生物科技股份有限公司 | Chimeric antigen receptor for treating malignant tumor and preparation method and application of chimeric antigen receptor |
| CN106755023A (en) * | 2015-10-15 | 2017-05-31 | 中国人民解放军军事医学科学院附属医院 | Chimeric antigen receptor immunocyte with safety switch and preparation method and application |
| WO2017123956A1 (en) * | 2016-01-15 | 2017-07-20 | Etubics Corporation | Methods and compositions for t-cell immunotherapy |
Non-Patent Citations (2)
| Title |
|---|
| 《Dap10 and Dap12 Form Distinct, but Functionally Cooperative, Receptor Complexes in Natural Killer Cells》;Jun Wu等;《J Exp Med.》;20001002;第192卷(第7期);参见对比文件2摘要 * |
| 《Preclinical characterization of LV305, a lentiviral vector targeting tumors expressing NY-ESO-1》;Albershardt, Tina C等;《CANCER RESEARCH》;20150801;第75卷(第15期);全文 * |
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