CN107893055A - A kind of NK of specific chimeric antigen receptor genetic modification and its production and use - Google Patents
A kind of NK of specific chimeric antigen receptor genetic modification and its production and use Download PDFInfo
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Abstract
The present invention discloses a kind of NK of specific chimeric antigen receptor genetic modification and its production and use, wherein, the NK cell of the specific chimeric antigen receptor genetic modification includes the specific chimeric antigen receptor genes of NY ESO 1 and truncated-type spleen tyrosine kinase Syk genes.Its preparation method is by the slow virus containing the specific chimeric antigen receptor genes of NY ESO 1 and the slow virus containing truncated-type spleen tyrosine kinase Syk genes while infects NK.The NK of specific chimeric antigen receptor genetic modification of the present invention passes through the contained specific chimeric antigen receptor genes of NY ESO 1 and truncated-type spleen tyrosine kinase Syk gene collective effects, improve the targets neoplastic cells specific killing of the NK, add the ability of NK activation downstream signal, the escape of tumour immunity is avoided, so as to enhance the killing activity to tumour cell.
Description
Technical field
The present invention relates to biopharmaceutical technology, more particularly to a kind of specific chimeric antigen receptor genetic modification from
Natural killer cell and its production and use.
Background technology
NK (NK cells) is the core component of human body congenital immunity, is the arranged side by side with T, B cell
Three monoid lymphocytes.NK cells have the ability of stronger non-specific direct killing target cell, and this natural killer
Activity both need not be in advance by antigen sensibilization, it is not required that antibody participates in, and is limited without MHC.NK cells pass through various kinds of cell table
Face acceptor (such as NKG2D, CD16) and natural cytotoxicity acceptor (NKp44, NKp46, NKp30) are identified infected and disliked
The cell of property canceration.These receptor activations contain ITAM (ITAM) structure modulin DAP10,
DAP12, ITAM can active cell toxic granulations albumen release, and regulate and control the cell factors such as IFN-γ and TNF-α and chemotactic because
The secretion of son.Spleen tyrosine kinase (Syk) is the crucial downstream protein tyrosine kinases of modulin DAP12, when extracellular acceptor
When being interacted with its part, ITAM is lived by EGFR-TK phosphorylation, recruitment Syk, further phosphorylation downstream albumen transmission
Change signal, so as to activate the effect of NK cells plays.
In recent years, the T cell technology of Chimeric antigen receptor (CAR) modification is in the clinical practice for the treatment of hematological system tumor
Significant breakthrough is achieved, good targeting, lethal and persistence are shown in clinical test.But newest face
Find that CAR-T technologies have in bed experiment and cause the high risk factor such as cytokine storm and systemic neurotoxicity.In addition, by
Acted in the immunosupress of tumor microenvironment and immunologic escape, CAR-T cells are not yet clearly treated in terms for the treatment of of solid tumors
Effect.NK cells are due to its feature：Play lethal effect and do not need presensitization, also do not limited by MHC
System, the NK cells of xenogenic origin can be used without causing rejection in vivo in subject (patient)；Cell will not be produced
Factor storm (for example a large amount of releases of interleukin-6 will not occur).NK cells are modified by CAR to be expected to strengthen its targeting
The ability of killing tumor cell simultaneously develops the effector cell with powerful antitumor action.
Cancer-testis antigen (CT) is considered as one of most promising tumor associated antigen, is characterized in Various Tissues
Expressed in the tumour cell in source, but expression in the normal tissue is only limitted to testis and embryonic tissue.Due to reproduction cell not
HLA molecules, and the presence of blood-testis barrier are expressed, therefore typically will not normal tissue for the immunotherapy of tumors of CT antigens
Cell produces immunotoxicity.NY-ESO-1 (New York esophageal squamous cell carcinoma 1) is CT
Important a member in antigen gene family, because it has stronger immunogenicity in tumour antigen, and in kinds of tumors group
Knit expression in (chromoma, hepatocellular carcinoma, oophoroma etc.) and received much concern in therapeutic field of tumor.
Although nowadays occurring some contains transgenosis NK cells with the specific receptors of NY-ESO-1 specific bonds, greatly
The CAR that part is used in NK cells express is the generation CAR carriers of only T cell CD3 ζ domains, with NK itself compatibility
It is not high, while increase the load of NK cells after transfection so that transfection efficiency is relatively low；Further, since the presence of tumor escape mechanism,
So that transgenosis NK cell-stimulatings downstream signal is insufficient, the fragmentation effect of current transgenosis NK cells is reduced.
The content of the invention
The main object of the present invention is to propose a kind of NK of specific chimeric antigen receptor genetic modification, purport
Existing transgenosis NK is being solved due to the presence of tumor escape mechanism, and is causing the transgenosis NKT thin
Born of the same parents activate the technical problem of downstream signal deficiency.
To achieve the above object, the NK of specific chimeric antigen receptor genetic modification proposed by the present invention,
Including NY-ESO-1 specific chimeric antigen receptor genes and truncated-type spleen tyrosine kinase Syk genes.
Preferably, the NY-ESO-1 specific chimerics antigen receptor gene is located at NY-ESO-1/scFv-CD8-41BB-
In DAP12 genetic fragments.
Preferably, the NY-ESO-1 specific chimerics antigen receptor gene order is referring to table 1.
Preferably, the truncated-type spleen tyrosine kinase Syk gene orders are referring to table 2.
The present invention also proposes a kind of preparation method of the NK of specific chimeric antigen receptor genetic modification, should
Preparation method comprises the following steps：
First slow virus and the second slow virus are infected into NK cells simultaneously, wherein, first slow virus contains NY-ESO-
1 specific chimeric antigen receptor gene, second slow virus contain truncated-type spleen tyrosine kinase Syk genes.
Preferably, first slow virus obtains as follows：
NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments are mixed with entry vector, so that the NY-ESO-
1/scFv-CD8-41BB-DAP12 genetic fragments are embedded in the entry vector, and obtain and carry NY-ESO-1/scFv-CD8-
The entry vector of 41BB-DAP12 genetic fragments；
By the entry vector with NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments and slow virus plasmid
Mixed reorganization, to establish Cancer-testis antigen NY-ESO-1 specific NK cells receptor expression plasmids；
By the Cancer-testis antigen NY-ESO-1 specific NK cells receptor expression plasmid and viral packaging plasmid cotransfection
293FT cells, obtain the first slow virus.
Preferably, the acquisition methods of the NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments include following step
Suddenly：
Obtain the total cDNA of NY-ESO-1 antigen immunes；
By the total cDNA of NY-ESO-1 antigen immunes and NY-ESO-1 heavy chain of antibody variable region VH primers and light chain variable
Area's VL primers enter performing PCR, obtain NY-ESO-1VH and NY-ESO-1VL genetic fragments；
By NY-ESO-1VH the and NY-ESO-1VL genetic fragments using connection peptide connection, NY-ESO-1 antibody is obtained
ScFv genetic fragments；
It will be that NY-ESO-1 antibody scFvs genetic fragment is connected with pcDNA carriers, and obtain pcDNA-NY-ESO-1/scFv matter
Grain；
The CD8- of Chimeric antigen receptor will be synthesized according to CD8 α, 41BB and DAP12-ITAM sequence genes sequences Design
41BB-DAP12 expression cassettes, enter pcDNA3.1 (+)-NY-ESO-1/scFv plasmids with Hind III/XhoI digestion rear clones, obtain
PcDNA3.1 (+)-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmids are obtained, NY-ESO- is obtained after EcoR I/Xho I digestions
1/scFv-CD8-41BB-DAP12 genetic fragments.
Preferably, second slow virus obtains as follows：
Obtain truncated-type spleen tyrosine kinase Syk gene orders；
Truncated-type spleen tyrosine kinase Syk gene orders are mixed with entry vector, so that the truncated-type spleen tyrosine
Kinases Syk gene orders are embedded in the entry vector, and obtain the introduction with truncated-type spleen tyrosine kinase Syk gene orders
Carrier；
The entry vector with truncated-type spleen tyrosine kinase Syk genetic fragments is mixed to weight with slow virus plasmid
Group, to obtain slow virus expression plasmid pLenti7.3-Syk；
By the slow virus expression plasmid pLenti7.3-Syk and viral packaging plasmid cotransfection 293FT cells, the is obtained
Two slow virus.
Preferably, the truncated-type spleen tyrosine kinase Syk gene orders are referring to table 2.
The present invention also proposes a kind of tumor drug formulation, and the tumor drug formulation includes the specific chimeric antigen of the present invention
The NK of acceptor gene modification or the specific chimeric antigen receptor gene prepared by the preparation method of the present invention
The NK of modification.
The NK of specific chimeric antigen receptor genetic modification of the present invention contains NY-ESO-1 specific chimerics
Antigen receptor gene and truncated-type spleen tyrosine kinase Syk genes.Wherein, specific chimeric antigen receptor genetic modification of the present invention
NK swollen due to containing the NY-ESO-1 specific chimerics antigen receptor gene, thus with stronger targeting
Oncocyte specific killing, simultaneously because it can be overexpressed truncated-type spleen tyrosine kinase Syk genes, thus it can increase
The ability of the NK activation downstream signal of specific chimeric antigen receptor genetic modification of the present invention, avoids exempting from for tumour
Epidemic disease is escaped.Therefore, the NK of specific chimeric antigen receptor genetic modification of the present invention has stronger target tumor
Cell-specific is lethal, can effectively avoid the escape of tumour immunity, enhance the killing activity to tumour cell.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Structure according to these accompanying drawings obtains other accompanying drawings.
Fig. 1 is the artificial constructed NY-ESO-1/scFv-CD8-41BB-DAP12 gene structures signal of the embodiment of the present invention
Figure；
Fig. 2 is the artificial constructed pLenti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmids of the embodiment of the present invention
Schematic diagram；
Fig. 3 produces for entry clones pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 of embodiment of the present invention plasmid enzyme restrictions
The electrophoretogram of thing；
Fig. 4 is recombinant slow virus expression plasmid electrophoretic band figure of the embodiment of the present invention；
Fig. 5 is the electrophoresis result figure of entry clones pENTR-Syk plasmid enzyme restriction products of the embodiment of the present invention；
Fig. 6 is the electrophoresis that recombinant slow virus expression plasmid of embodiment of the present invention pLenti7.3-Syk converts bacterium colony PCR primer
Result figure；
Fig. 7 is the flow cytometer detection figure that NK of embodiment of the present invention cells in vitro kills HepG-2 cell line.
The realization, functional characteristics and advantage of the object of the invention will be described further referring to the drawings in conjunction with the embodiments.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only the part of the embodiment of the present invention, rather than whole embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art obtained under the premise of creative work is not made it is all its
His embodiment, belongs to the scope of protection of the invention.
If relating to the description of " first ", " second " etc. in the embodiment of the present invention, the description of " first ", " second " etc. is somebody's turn to do
It is only used for describing purpose, and it is not intended that indicating or implying its relative importance or imply the technical characteristic indicated by indicating
Quantity.Thus, " first " is defined, at least one this feature can be expressed or be implicitly included to the feature of " second ".Separately
Outside, the technical scheme between each embodiment can be combined with each other, can be real with those of ordinary skill in the art but must be
Based on existing, the combination of this technical scheme is will be understood that not when the combination appearance of technical scheme is conflicting or can not realize
In the presence of also not within the protection domain of application claims.
The present invention proposes a kind of NK of specific chimeric antigen receptor genetic modification.
In embodiments of the present invention, the NK of the specific chimeric antigen receptor genetic modification refers to target
NY-ESO-1 and the Chimeric antigen receptor base containing NK cell modulin DAP12 ITAMs (ITAM)
Because of the NK cells of modification.Here, the NK of the specific chimeric antigen receptor genetic modification is expressed as NY-ESO-1/
scFv-CD8-41BB-DAP12.Specifically, the NK of the specific chimeric antigen receptor genetic modification includes
NY-ESO-1 specific chimeric antigen receptor genes and truncated-type spleen tyrosine kinase Syk genes.Specific chimeric of the present invention resists
The NK of original receptor genetic modification is due to containing targeting NY-ESO-1 specific chimeric antigen receptor genes and truncation
Type spleen tyrosine kinase Syk genes, thus the NK of the specific chimeric antigen receptor genetic modification is with higher
Targets neoplastic cells specific killing, also, can to increase NKT thin for the truncated-type spleen tyrosine kinase Syk genes
Born of the same parents activate the ability of downstream signal, so as to effectively avoid the escape of tumour immunity, enhance the killing to tumour cell and live
Property.
In one embodiment, the NY-ESO-1 specific chimerics antigen receptor gene is located at NY-ESO-1/scFv-CD8-
In 41BB-DAP12 genetic fragments.Specifically, the NY-ESO-1 specific chimerics antigen receptor gene is by containing NY-ESO-
The virus infection NK of 1/scFv-CD8-41BB-DAP12 genetic fragments is into cell, hereafter to the NY-ESO-1/scFv-
The acquisition methods of CD8-41BB-DAP12 genetic fragments are described in detail.
Wherein, the NY-ESO-1 specific chimerics antigen receptor gene order is as shown in table 1 below：
Table 1
The truncated-type spleen tyrosine kinase Syk gene orders are as shown in table 2 below：
Table 2
The truncated-type spleen tyrosine kinase Syk amino acid sequences are as shown in table 3 below：
Table 3
The NK of specific chimeric antigen receptor genetic modification of the present invention contains NY-ESO-1 specific chimerics
Antigen receptor gene and truncated-type spleen tyrosine kinase Syk genes.Wherein, specific chimeric antigen receptor genetic modification of the present invention
NK swollen due to containing the NY-ESO-1 specific chimerics antigen receptor gene, thus with stronger targeting
Oncocyte specific killing, simultaneously because it can be overexpressed truncated-type spleen tyrosine kinase Syk genes, thus it can increase
The ability of the NK activation downstream signal of specific chimeric antigen receptor genetic modification of the present invention, avoids exempting from for tumour
Epidemic disease is escaped.Therefore, the NK of specific chimeric antigen receptor genetic modification of the present invention has stronger target tumor
Cell-specific is lethal, can effectively avoid the escape of tumour immunity, enhance the killing activity to tumour cell.
The present invention also proposes a kind of preparation method of the NK of specific chimeric antigen receptor genetic modification, uses
In the NK for preparing above-mentioned specific chimeric antigen receptor genetic modification.Wherein, the preparation method includes following step
Suddenly：
First slow virus and the second slow virus are infected into NK cells simultaneously, wherein, first slow virus contains NY-ESO-
1 specific chimeric antigen receptor gene, the second slow virus contain truncated-type spleen tyrosine kinase Syk genes.
In one embodiment, first slow virus obtains as follows：
S11：NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments are mixed with entry vector, so that the NY-
ESO-1/scFv-CD8-41BB-DAP12 genetic fragments are embedded in the entry vector, and obtain and carry NY-ESO-1/scFv-
The entry vector of CD8-41BB-DAP12 genetic fragments.
S12：By the entry vector with NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments and slow virus
Plasmid mixed reorganization, to establish Cancer-testis antigen NY-ESO-1 specific NK cells receptor expression plasmids.
S13：By the Cancer-testis antigen NY-ESO-1 specific NK cells receptor expression plasmid and viral packaging plasmid,
Cotransfection 293T cells, obtain the first slow virus.
In step s 11, the entry vector can be pENTR 11, but be not limited only to this.Specifically, in step
In S11, the acquisition methods of the NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments comprise the following steps：
S111：Obtain the total cDNA of NY-ESO-1 antigen immunes；
S112：By the total cDNA of NY-ESO-1 antigen immunes and NY-ESO-1 heavy chain of antibody variable region VH primers and light chain
Variable region VL primers enter performing PCR, obtain NY-ESO-1VH and NY-ESO-1VL genetic fragments；
S113：By NY-ESO-1VH the and NY-ESO-1VL genetic fragments using connection peptide connection, NY-ESO-1 is obtained
Antibody scFv genetic fragment；
S114：It will be that NY-ESO-1 antibody scFvs genetic fragment is connected with pcDNA carriers, and obtain pcDNA-NY-ESO-1/
ScFv plasmids；
S115：Chimeric antigen receptor will be synthesized according to CD8 α, 41BB and DAP12-ITAM sequence genes sequences Design
CD8-41BB-DAP12 expression cassettes, enter pcDNA3.1 (+)-NY-ESO-1/scFv matter with Hind III/XhoI digestion rear clones
Grain, pcDNA3.1 (+)-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmids are obtained, NY- is obtained after EcoR I/Xho I digestions
ESO-1/scFv-CD8-41BB-DAP12 genetic fragments.
Wherein, in step S112, the NY-ESO-1 heavy chain of antibody variable region VH primers and the light chain variable district VL
Primer can be designed according to weight chain variable district VH and light chain variable district VL sequences according to design of primers principle.Specifically, it is described
Weight chain variable district VH primer is VH-F and VH-R, and the primer of the light chain variable district VL is VL-F and VL-R, and base sequence is such as
Under：
VH-F：5'-GAA GTT 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'
Connection peptide in step S113 can select connection peptide (Gly4Ser)3, its base sequence is as follows：
5'-GGT GGT GGT GGT TCT GGC GGC GGC GGC TCC GGT GGT GGT GGA TCT-3'。
PcDNA carriers in step S114 can be the carrier commonly used in pcDNA carrier families, such as pcDNA3.1 (+).
CD8 α, 41BB and DAP12-ITAM gene orders in step S115 can be directly from gene pool (Genebank
(NCBI) obtained in).According to CD8 α, the Hinge- of 41BB and DAP12-ITAM gene orders design synthesis Chimeric antigen receptor
TM-41BB-DAP12 expression cassettes, including compared with sequence (Hinge) and transmembrane region (TM) CD8 α (aa135-205), 41BB intracellular functions
Area (aa214-255) and DAP12-ITAM.Therefore, Hinge-TM-41BB-DAP12 expression cassettes that is to say CD8-41BB-DAP12
Expression cassette.Wherein, DAP12-ITAM amino acid sequence：
RLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK.In addition, synthesis Hinge-TM-41BB-DAP12
Expression cassette can be synthesized according to the synthesis mode of routine, such as synthesize the Hinge- by Nanjing Genscript Biotechnology Co., Ltd.
TM-41BB-DAP12 expression cassettes.In one embodiment, Hinge 5 ' ends and DAP12 3 ' ends, can be added restricted respectively
Restriction enzyme site Hind III and XhoI, therefore, digestion obtain the mistake of NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments
Cheng Zhong, directly it can carry out digestion with Eco R I/Xho I enzymes.
In step s 12, slow virus plasmid can be conventional slow virus carrier, for example, pLenti7.3/V5-DEST.
In step s 13, viral packaging plasmid can be conventional viral packaging plasmid, such as select ViraPowerTM bags
Fill plasmid.
In one embodiment, second slow virus obtains as follows：
S21：Obtain truncated-type spleen tyrosine kinase Syk gene orders；
S22：Truncated-type spleen tyrosine kinase Syk gene orders are mixed with entry vector, so that the truncated-type spleen junket
Histidine kinase Syk gene orders are embedded in the entry vector, and obtain with truncated-type spleen tyrosine kinase Syk gene orders
Entry vector.
S23：The entry vector with truncated-type spleen tyrosine kinase Syk gene orders is mixed with slow virus plasmid
Restructuring, to obtain slow virus expression plasmid pLenti7.3-Syk.
S24：By the slow virus expression plasmid pLenti7.3-Syk and viral packaging plasmid cotransfection 293FT cells, obtain
Obtain the second slow virus.
In the step s 21, the entry vector can be pENTR 11, but be not limited only to this.In an embodiment, truncated-type
The both ends of spleen tyrosine kinase Syk gene orders can add restriction enzyme site EcoR I/Hind III respectively, direct gram
It is grand to enter the carriers of pENTR 11.
In the step s 21, the functional domain of SYK albumen is searched by NCBI, selects the N-terminal SH2 structures of SYK albumen
Domain, C-terminal SH2 domains and protein tyrosine kinase domain, corresponding gene order is connected, described so as to obtain
Truncated-type spleen tyrosine kinase Syk gene orders.
In step S22, slow virus plasmid can be conventional slow virus carrier, for example, pLenti7.3/V5-DEST.
In step S23, viral packaging plasmid can be conventional viral packaging plasmid, such as select ViraP owerTM bags
Fill plasmid.
In addition, the first slow disease in the NK preparation method of above-mentioned specific chimeric antigen receptor genetic modification
Poison and the second slow virus infect NK cells simultaneously, can be infected according to normal slow-virus infection method, no longer superfluous herein
State.
The preparation side of the NK of specific chimeric antigen receptor genetic modification provided by the invention
Method, by by first containing targeting Cancer-testis antigen NY-ESO-1 antigen-specific chimerics antigen receptor gene
Slow virus, and the second slow virus containing truncated-type spleen tyrosine kinase Syk genes infect NK simultaneously so that
Infecting the NK of obtained specific chimeric antigen receptor genetic modification has stronger targets neoplastic cells special
It is lethal, while add the energy of the NK activation downstream signal of the specific chimeric antigen receptor genetic modification
Power, the immunologic escape of tumour is avoided, enhance the NK of the specific chimeric antigen receptor genetic modification to tumour
The killing activity of cell.
The present invention is described in further details below by specific embodiment.
Test procedure：
1st, the slow virus of NY-ESO-1 antigentic specificity scFv-CD8-41BB-DAP12 Chimerical receptors is expressed
1.1 obtain the total cDNA of NY-ESO-1 mice immunized with antigen
BALB/C mice is immunized using NY-ESO-1 antigens, potency meets the requirements (1:More than 100000) when, place
Dead mouse, takes spleen, and mouse spleen total serum IgE is extracted with Trizol methods.
Total cDNA is synthesized using iScript cDNA Synthesis Kit, reaction system is as shown in table 4 below：
Table 4
| Reagent | Volume |
| 5×iScript reaction mix | 4μl |
| iScript reverse transcriptase | 1μl |
| Nuclease-free water | 5μl |
| Total serum IgE | 10μl |
| Cumulative volume | 20μl |
Reaction condition：25℃5min→42℃30min→85℃5min→4℃hold.
1.2 NY-ESO-1 heavy chain of antibody variable region VH and light chain variable district VL genes acquisition NY-ESO-1 heavy chain of antibody
Variable region VH and light chain variable district VL primer sequences are as shown in table 5 below：
Table 5
PCR reaction systems are as shown in table 6 below：
Table 6
| Reagent | Volume |
| Taq archaeal dna polymerases | 0.25μl |
| 10×PCR Buffer | 5μl |
| dNTPs(2.5mM) | 4μl |
| The total cDNA of NY-ESO-1 mice immunized with antigen | 1μl |
| VH-F(20μM) | 1μl |
| VH-R(20μM) | 1μl |
| ddH2O | It is supplemented to 50 μ l |
Reaction condition is as follows：94 DEG C of pre-degenerations 5min, 94 DEG C of 45s, 60 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations, 72 DEG C
Extend 5min.
After the identification of 1.2% agarose gel electrophoresis, NY-ESO-1 heavy chain of antibody variable region VH and light chain variable district VL are reclaimed
Gene PCR product.
The acquisition of 1.3 NY-ESO-1 antibody scFv fragments and plasmid
By polygenes fragment Overlap extension PCR (Overlap Extension PCR, OE-PCR) by what is obtained in 1.2
NY-ESO-1VH and NY-ESO-1VL fragments connection peptide (Gly4Ser) 3 (base sequence：5'-GGT GGT GGT GGT TCT
GGC GGC GGC GGC TCC GGT GGT GGT GGA TCT-3') connect, NY-ESO-1 antibody scFv fragments are obtained,
And introduce restriction enzyme site EcoR I/Hind III respectively in front and back end.OE-PCR reaction conditions are as follows：(1) step 1：95 DEG C pre-
1min, 94 DEG C of 1min, 60 DEG C of 1min, 72 DEG C of 1min are denatured, totally 7 circulations, 72 DEG C of extension 10min；(2) step 2：95 DEG C of pre- changes
Property 5min, 94 DEG C of 45s, 62 DEG C of 1min, 72 DEG C of 1min, totally 35 circulation, 72 DEG C extension 5min.
After the identification of 1.2% agarose gel electrophoresis, NY-ESO-1 antibody sc Fv fragments are reclaimed, EcoR I/Hind III are double
Digestion recovery fragment and pcDNA3.1 (+) carrier, using T4DNA ligases by NY-ESO-1 antibody scFv fragments with
PcDNA3.1 (+) carrier connects, and obtains pcDNA3.1 (+)-NY-ESO-1/scFv plasmids.
The structure of 1.4 NY-ESO-1 antigentic specificity scFv-CD8-41BB-DAP12 genetic fragments
According to CD8 α, 41BB and DAP12-ITAM sequence genes sequences Design synthesis chimeric antigen in Genebank (NCBI)
The Hinge-TM-41BB-DAP12 expression cassettes of acceptor, including compared with sequence (Hinge) and transmembrane region (TM) CD8 α (aa135-205),
41BB intracellulars functional areas (aa214-255) and DAP12-ITAM (amino acid sequence is as shown in table 7 below), Hinge 5 ' end and
Restriction enzyme site Hind III and XhoI are added in DAP12 3 ' ends respectively, entrust the limited public affairs of Nanjing Jin Sirui biotechnologies
Department synthesizes whole expression cassette.
Table 7
By the CD8-41BB-DAP12 expression cassettes of synthesis with Hind III/XhoI digestion rear clones enter pcDNA3.1 (+)-
NY-ESO-1/scFv plasmids, pcDNA3.1 (+)-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmids are obtained, then EcoR
NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments are obtained after I/Xho I digestions, gene structure figure is as shown in Figure 1.
The structure of 1.5 NY-ESO-1/scFv-CD8-41BB-DAP12 entry clones
1.51 NY-ESO-1/scFv-CD8-41BB-DAP12 entry clones pENTR11 coupled reactions systems such as table 8 below：
Table 8
| NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments | 0.5μl |
| Salting liquid | 1μl |
| ddH2O | 3.5μl |
| pENTR11 vector | 1μl |
| Cumulative volume | 6μl |
TOPO cloning reaction conditions：After above-mentioned reaction system is gently shaken up, 5min is incubated under room temperature (21-23 DEG C).
Reaction system is placed on ice with further transformed competence colibacillus cell by incubation after terminating, and is enlarged and is cultivated and extract pENTR-
NY-ESO-1/scFv-CD8-41BB-DAP12 plasmids.
1.52 entry clones pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 digestion identification
1.521 digestion systems (EcoR I/Xho I double digestions system) are as shown in table 9 below：
Table 9
1.522 digestion condition：2h is digested in 37 DEG C of water-baths.
1.523 take 2 μ l enzymolysis products, and electrophoresis detection is carried out with 1.2% Ago-Gel.Analytical electrophoresis band with judge into
Whether door clone is correct.If entry clones pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 is correct, EcoR I/Xho I
Double digestion obtains two respectively 1.3kb and 2.3kb fragments.
The structure of 1.6 pLenti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 slow virus expression plasmids
1.61 LR reacts
LR reactions are entry clones pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 and purpose carrier
The recombining reaction that pLenti7.3/V5-DEST vector occur, slow virus expression plasmid pLenti7.3- is obtained after restructuring
NY-ESO-1/scFv-CD8-41BB-DAP12 (as described in Figure 2).LR reaction systems are as shown in table 10 below：
Table 10
Thaw on iceLR ClonaseTMII Plus Enzyme Mix, draw 2 μ l and add to above-mentioned LR reactions
In system, after soft mixing, 25 DEG C of placement 1h.
1 μ l Proteinase Ks are added to above-mentioned reaction system, 37 DEG C of incubation 10min.
The conversion of 1.62 LR reaction products
1.621 thaw a pipe E.coli competent cells (Invitrogen, Cat.No.C7373-03) on ice
1.622 add 2-3 μ l LR reaction products into competent cell suspension, and soft mix (is sure not to be blown with liquid-transfering gun
Beat).30min is incubated on ice, and thermal shock handles 30s in 42 DEG C of water-baths, and reaction tube is transferred to and continues to be incubated 2min on ice.
1.623 add the S.O.C. culture mediums of 225 μ l pre-temperatures at room temperature.
Next be placed in 37 DEG C of horizontal shakers under 225rpm rotating speeds of reaction lid is incubated 1h by 1.624.
1.625 take out 100 μ l converted products even spreads to the LB flat boards (containing ampicillin) of preheating, 37 DEG C of cultures
It is incubated overnight in case.
1.63 the screening and amplification of positive colony
After 1.631 dip 3 clones with pipette tips on a small quantity respectively, pipette tips are placed in 10 μ l sterilized waters, blown and beaten repeatedly.
1.632 drawing 1 μ l bacterium solutions is used for PCR, reaction system and condition are as shown in table 11 below：
Table 11
PCR reaction conditions are as follows：
The PCR primer of 1.633 3 clones enters row agarose gel electrophoresis reaction, if being obtained at 1.3kb clearly single
One band, then the positive colony identified is chosen in the LB nutrient solutions containing ampicillin and be enlarged culture.
1.634 use plasmid DNA purification kit (Promega, Cat.No.A7500) from the above-mentioned bacterium solution being incubated overnight
It is slow virus expression plasmid pLenti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 to isolate and purify DNA, simultaneously
Preserve strain.
The preparation of 1.7 pLenti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 slow virus
1.71 Day1：5 × 106 293FT cells (Invitrogen, Cat.No.R700-07) are taken, are abandoned after centrifugation
Clearly, with the complete medium (D-MEM+10%FBS+2mM Glu+0.1mM nonessential amino acids of 37 DEG C of preheatings of 10ml
+ 1mM Sodium Pyruvates+1%P/S) it is resuspended, it is inoculated in 10cm culture dishes, 37 DEG C, is incubated overnight in 5%CO2 incubators.
1.72 Day2：The nutrient solution in culture dish is discarded, adds 5ml containing 10%FBS'sI nutrient solutions
(Invitrogen,Cat.No.31985-062)。
1.73The preparation of 2000 compounds：
1.731 by 1.5ml serum-freesI nutrient solutions are added in 5ml centrifuge tubes, add 9 μ
gViraPowerTMPackaging plasmid mixture and 3 μ g slow virus expression plasmids pLenti7.3-NY-ESO-1/scFv-CD8-41BB-
DAP12, soft mixing.
1.732 by 1.5ml serum-freesI nutrient solutions are added in another 5ml centrifuge tube, add 36 μ l2000,5min is incubated at room temperature after soft mixing.
1.733, which walk 1.731 and 1.732 liang obtained solution, is transferred in a centrifuge tube, soft well mixed.
1.734 are incubated 20min at room temperature, obtain2000 compounds.
1.74 will obtain2000 complexes drop-wises are slowly added into culture dish, and
Culture dish is lightly rocked back and forth.37 DEG C, it is incubated overnight in 5%CO2 incubators.
1.75 Day3：Culture dish is taken out, discards nutrient solution, adds 10ml DMEM complete mediums.37 DEG C, 5%CO2 trainings
Support in case and be incubated 48-72h.
1.76 Day5 or Day6：Nutrient solution in culture dish is transferred in 15ml centrifuge tubes, the 2000g under the conditions of 4 DEG C
Centrifuge 15min.
1.77 Aspirate supernatants, collect pLenti7.3-NY-ESO-1/scFv-CD8-41BB-DAP12 slow virus, packing
Enter in 1ml cryopreservation tubes, be placed in -80 DEG C long-term preserve.
2nd, truncated-type Syk slow virus is expressed
2.1 obtain truncated-type spleen tyrosine kinase Syk gene orders.
Nanjing Genscript Biotechnology Co., Ltd.'s synthesis truncated-type Syk gene orders are entrusted, wherein, truncated-type spleen junket ammonia
Acid kinase Syk gene orders are as shown in table 2.
The structure of 2.2 truncated-type Syk entry clones
2.21 add EcoRI/Hind III enzymes respectively at truncated-type truncated-type spleen tyrosine kinase Syk gene orders both ends
Enzyme site, Direct Cloning enter the carriers of pENTR 11.
2.22 entry clones pENTR-Syk digestion identification
2.221 digestion systems (EcoR I/Hind III double digestions system) are as shown in table 12 below：
Table 12
2.222 digestion condition：2h is digested in 37 DEG C of water-baths.
2.223 take 2 μ l enzymolysis products, and electrophoresis detection is carried out with 1.2% Ago-Gel.Analytical electrophoresis band with judge into
Whether door clone is correct.If entry clones pENTR-Syk is correct, EcoR I/Hind III double digestions obtain two and are respectively
1.4kb and 2.3kb fragment.
The structure of 2.3 pLenti7.3-Syk slow virus expression plasmids
2.31 LR reacts
LR reactions are the restructuring that entry clones pENTR-Syk and purpose carrier pLenti7.3/V5-DEST vector occurs
Reaction, slow virus expression plasmid pLenti7.3-Syk is obtained after restructuring.LR reaction systems are as shown in table 13 below：
Table 13
| PENTR-Syk (150ng/ reactions) | 1-7μl |
| pLenti7.3/V5-DEST vector(150ng/μl) | 1μl |
| TE Buffer, pH 8.0 | Add to the μ l of cumulative volume 8 |
Thaw on iceLR ClonaseTMII Plus Enzyme Mix, it is anti-that 2 μ l of absorption add to above-mentioned LR
Answer in system, after soft mixing, 25 DEG C of placement 1h.
1 μ l Proteinase Ks are added to above-mentioned reaction system, 37 DEG C of incubation 10min.
The conversion of 2.32 LR reaction products
2.321 thaw a pipe E.coli competent cells (Invitrogen, Cat.No.C7373-03) on ice.
2.322 add 2-3 μ l LR reaction products into competent cell suspension, and soft mix (is sure not to be blown with liquid-transfering gun
Beat).30min is incubated on ice, and thermal shock handles 30s in 42 DEG C of water-baths, and reaction tube is transferred to and continues to be incubated 2min on ice.
2.323 add the S.O.C. culture mediums of 225 μ l pre-temperatures at room temperature.
Next be placed in 37 DEG C of horizontal shakers under 225rpm rotating speeds of reaction lid is incubated 1h by 2.324.
2.325 take out 100 μ l converted products even spreads to the LB flat boards (containing ampicillin) of preheating, 37 DEG C of cultures
It is incubated overnight in case.
The screening and amplification of 2.33 positive colonies
After 2.331 dip 3 clones with pipette tips on a small quantity respectively, pipette tips are placed in 10 μ l sterilized waters, blown and beaten repeatedly.
2.332 1 μ l bacterium solutions of absorption are used for PCR, reaction system and condition as shown in following table table 14：
Table 14
| Bacterium solution | 1μl |
| VH-F primers (20 μM) | 1μl |
| V5 reverse primers (20 μM) | 1μl |
| Bio-rad super mix | 6.25μl |
| ddH2O | 15.75μl |
| Cumulative volume | 25μl |
PCR reaction conditions are as follows：
The PCR primer of 2.333 3 clones enters row agarose gel electrophoresis reaction, if being obtained at 1.4kb clearly single
One band, then the positive colony identified is chosen in the LB nutrient solutions containing ampicillin and be enlarged culture.
2.334 use plasmid DNA purification kit (Promega, Cat.No.A7500) from the above-mentioned bacterium solution being incubated overnight
It is slow virus expression plasmid pLenti7.3-Syk to isolate and purify DNA, while preserves strain.
(cell of the genetic marker on one DNA molecular of expression is often for the preparation of 2.4 pLenti7.3-Syk slow virus
The genetic marker of another DNA molecular carrying is expressed, this physically disjunct gene is integrated into same integration sequence simultaneously
The phenomenon expressed in same transfectional cell is called cotransfection)
2.41 Day1：5 × 106 293FT cells (Invitrogen, Cat.No.R700-07) are taken, are abandoned after centrifugation
Clearly, with the complete medium (D-MEM+10%FBS+2mM Glu+0.1mM nonessential amino acids of 37 DEG C of preheatings of 10ml
+ 1mM Sodium Pyruvates+1%P/S) it is resuspended, it is inoculated in 10cm culture dishes, 37 DEG C, is incubated overnight in 5%CO2 incubators.
2.42 Day2：The nutrient solution in culture dish is discarded, adds 5ml containing 10%FBS'sI nutrient solutions
(Invitrogen,Cat.No.31985-062)。
2.43The preparation of 2000 compounds：
2.431 by 1.5ml serum-freesI nutrient solutions are added in 5ml centrifuge tubes, add 9 μ
gViraPowerTMPackaging plasmid mixture and 3 μ g slow virus expression plasmid pLenti7.3-Syk, soft mixing.
2.432 by 1.5ml serum-freesI nutrient solutions are added in another 5ml centrifuge tube, add 36 μ
l2000,5min is incubated at room temperature after soft mixing.
2.433, which walk above-mentioned 2.431 and 2.432 liang obtained solution, is transferred in a centrifuge tube, soft well mixed.
2.434 are incubated 20min at room temperature, obtain2000 compounds.
2.44 will obtain2000 complexes drop-wises are slowly added into culture dish, and
Culture dish is lightly rocked back and forth.37 DEG C, it is incubated overnight in 5%CO2 incubators.
2.45 Day3：Culture dish is taken out, discards nutrient solution, adds 10ml DMEM complete mediums.37 DEG C, 5%CO2 trainings
Support in case and be incubated 48-72h.
2.46 Day5 or Day6：Nutrient solution in culture dish is transferred in 15ml centrifuge tubes, the 2000g under the conditions of 4 DEG C
Centrifuge 15min.
2.47 Aspirate supernatants, pLenti7.3-Syk slow virus is collected, is distributed into 1ml cryopreservation tubes, is placed in -80 DEG C long
Phase preserves.
3rd, the preparation and amplification of Syk NY-ESO-1 specific C AR-NK cells are co-expressed
3.1 isolated genes parting HLA-A2+ healthy volunteer's PBMC cells
3.11 extract HLA-A2+ healthy volunteer peripheral blood 25ml, anticoagulant heparin, room temperature centrifugation (700g, 20min)；
Upper plasma is drawn, is placed in water-bath 56 DEG C, 30min；Then after 4 DEG C of standing 15min, centrifuge (900g, 30min), be derived from
4 DEG C of body blood plasma saves backup.
3.12 take above-mentioned 700g, 20min centrifugation rear lower cell components, add D-PBS to 50ml, mix, be added slowly to fill
In the 50ml centrifuge tubes for having 20ml human lymphocyte separating liquids, room temperature centrifugation (800g, 15min).
3.13 draw tunica albuginea confluent monolayer cells, are added in the 50ml centrifuge tubes equipped with 5ml RPMI 1640.
3.14 RPMI 1640 wash twice (600g, 10min are centrifuged), and it is PBMC cells to collect cell.
The preparation of 3.2 CAR-NK cells
3.21 use the monoclonal antibodies of CD16 containing 50ng/ml, 1000U/ml IL-2,100ng/ml IL-15 and 0.5% autologous plasma
Alys505 nutrient solutions adjustment PBMC cell densities be 1 × 106/ml, be transferred in six orifice plates, 2ml/ holes, be placed in saturated humidity,
37 DEG C, cultivate in 5.0%CO2 incubators.
3.22 every 3 days adjustment cell densities are 1 × 106/ml, add IL-2 containing 1000U/ml and 0.5% autologous plasma
Alys505 nutrient solutions.
3.23 Day7, NK cells being divided into 4 groups, every group of cell is transferred in 24 orifice plates by the density in 1 × 105/ hole,
100 μ l/ holes, every group sets three multiple holes.
1st group：Only transfection NY-ESO-1/scFv-CD8-41BB-DAP12 genes, are named as NY-ESO-1-CAR groups.
2nd group：Only transfection Syk genes, are named as Syk groups.
3rd group：NY-ESO-1/scFv-CD8-41BB-DAP12 genes and Syk are transfected, is named as NY-ESO-1-CAR-
Syk groups.
4th group：Blank control group, it is named as NC groups.
3.24, according to above-mentioned packet, add according to MOI value=20 (ratio of the viral number of MOI expressions herein and cell quantity)
Stoste containing lentiviral particle, carries out packet transfection, and each group takes slow virus solution to add Alys505 complete mediums to be configured to altogether
100 μ l, slow virus solution is added in cell with pipette, gently piping and druming mixes.It is complete that NC groups add 100 μ l Alys-505
Culture medium.
3.25 every groups of Polybrene for being separately added into final concentration of 6 μ g/ml, gently rock after being well mixed, be placed in back and forth
37 DEG C, 24h is incubated in 5%CO2 incubators.
3.26 the 8th days, each group cell took out centrifugation respectively, discards the supernatant containing slow virus, respectively with 200 μ l Alys-
505 nutrient solutions are resuspended, and add in 24 orifice plates.Fluid infusion is carried out according to cell growth state.
3.27 Day10, each group cell for harvesting maturation are used for subsequent analysis.
4th, Syk NY-ESO-1 specific C AR-NK cells in vitro antitumor activity detection is co-expressed
4.1 using above-mentioned each group cell as effector cell, the HepG-2 cell line of CFSE marks as target cell, according to
20:1 effect target gently mixes than melange effect cell and target cell, is placed in 5%CO2, is incubated in 37 DEG C of incubators.
After 4.2 24h, 1 μ g/ml PI dye liquors are added, are mixed, after room temperature lucifuge is incubated 15min, are examined using flow cytometer
Survey the percentage of CFSE+PI+ cells (dead HepG-2 cells).
5th, antitumor activity detection in Syk NY-ESO-1 specific C AR-NK multicellular animal bodies is co-expressed
Take the logarithm the human liver cancer HepG-2 cells in growth period, single cell suspension is made after pancreatin digestion, 1 × 107 will be contained
The suspension 0.1ml of cancer cell is subcutaneously injected in nude mice scapular region.Experiment packet and treatment be see the table below, and daily each group of observing is moved
The change of the diet of thing, activity etc., the next day measure nude mice body weight, observe changes of weight situation；Every 2 days measurement tumours most
Footpath (a) vertical greatly and maximum transverse diameter (b), observe the situation of tumour growth, are calculated according to formula：Knurl volume=1/12 π × a × b2.
The 13rd day tumour inhibiting rate for calculating each group nude mice after (3) group treatment end, tumour inhibiting rate=(1- treatment group tumors average external volume/the moon
Property control group tumor average volume) × 100%.Experiment packet and treatment are as shown in table 15 below：
Table 15
Wherein, * FAC chemotherapy regimens：Fluorouracil 500mg/M2；Adriamycin 50mg/M2；Endoxan 500mg/M2, the
It is administered within one day, 21 days courses for the treatment of.
6th, experimental result
The CAR structure charts and expression plasmid collection of illustrative plates of 6.1 structures
6.2 entry clones pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 digestion identification
Entry clones pENTR-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmids pass through EcoR I/Xho I double digestions,
Detected through agarose gel electrophoresis, there is clear band (Fig. 3) at 2.3kb and 1.3kb respectively, it is consistent with expected results.Prove
NY-ESO-1/scFv-CD8-41BB-DAP12 genes are properly inserted into pENTR carriers.
6.3 recombinant slow virus expression plasmid pLenti7.3/NY-ESO-1/scFv-CD8-41BB-DAP12 convert bacterium colony
PCR results
Three clones on random picking LB flat boards, enter performing PCR with the forward primer and V5 reverse primers of design and expand, production
Thing identifies occur clearly band (Fig. 4) at 1.3kb, illustrate NY-ESO-1/scFv-CD8- by agarose gel electrophoresis
41BB-DAP12 genes are successfully plugged into pLenti7.3/V5-DEST expression vectors.
6.4 entry clones pENTR-Syk digestion identification
The product that entry clones pENTR-Syk plasmids obtain after EcoR I/Hind III double digestions coagulates through agarose
Gel electrophoresis detect, and have clear band (Fig. 5) at about 2.3kb and 1.4kb respectively, consistent with expected piece result.Prove Syk just
Really it is inserted into carrier.
6.5 recombinant slow virus expression plasmid pLenti7.3-Syk convert the electrophoresis result of bacterium colony PCR primer
Three clones on random picking LB flat boards, enter performing PCR with the forward primer and V5 reverse primers of design and expand,
PCR primer identifies occur clearly band (Fig. 6) at 1.4kb, illustrate that Syk is successfully plugged into by agarose gel electrophoresis
In pLenti7.3/V5-DEST expression vectors.
6.6NK cells in vitro kills the flow cytometer detection result of HepG-2 cell line
It is thin using streaming as target cell with CFSE labelling liver cancer HepG-2 cells using each group cell as effector cell
Born of the same parents' art detects NK cells to the killing-efficiency of HepG-2 cell line, and experimental result is as shown in fig. 7, NY-ESO-1-CAR group cells
Killing rate to HepG-2 cell line is about that killing rate of 41.93%, the Syk groups cell to HepG-2 cell line is about
Killing rate of 18.35%, NY-ESO-1-CAR-Syk the group cell to HepG-2 cell line is about 67.56%, NC group cells pair
The killing rate of HepG-2 cell line is about 14.87%, and display coexpression Syk NY-ESO-1 specific C AR-NK cells are to liver
The killing-efficiency of cancer HepG-2 cells is significantly larger than the killing-efficiency of remaining each group cell.Illustrate coexpression prepared by this patent
Syk NY-ESO-1 specific C AR-NK cells have efficient lethal effect to liver cancer cells.
Suppress the experimental result of liver cancer cells in 6.7 coexpression Syk NY-ESO-1 specific C AR-NK cell bodies
The NY-ESO-1 specific C AR-NK cells for co-expressing Syk carry out internal anti-tumor experiment discovery, and each group nude mice is equal
Survival, using negative control group nude mice as reference, chemotherapy group nude mice Body weight loss, gross tumor volume reduces, but feed and activity are reduced.
It is more good to co-express Syk NY-ESO-1 specific C AR-NK cell therapy group nude mice survival conditions, body weight without being decreased obviously,
And gross tumor volume is significantly smaller, inhibitory rate to 82.8%.Body weights and tumor control rate statistical result are shown in Table 16.Experiment knot
Fruit illustrates that coexpression Syk prepared by this patent NY-ESO-1 specific C AR-NK cells can effectively suppress the life of in-vivo tumour
It is long.
The comparison of each group nude mice body weight of table 16, gross tumor volume and tumour inhibiting rate
It is of the invention to additionally provide a kind of tumor drug formulation in fact.The tumor drug formulation includes NK cells.Wherein, it is described
NK cells are the NK of above-mentioned specific chimeric antigen receptor genetic modification or the spy prepared by above-mentioned preparation method
The NK of different in nature Chimeric antigen receptor genetic modification.Because the tumor drug formulation employs above-mentioned all embodiments
Whole technical schemes, therefore at least with above-described embodiment technical scheme caused by all beneficial effects, herein no longer
Repeat one by one.
In addition, the NKT of specific chimeric antigen receptor genetic modification contained in tumor drug formulation of the present invention
The content or dosage of cell should be effective doses, and specifically, the effective dose refers to therapeutically effective amount, refers to foot
To show the amount of benefit or the compound of the invention of clinical meaning to individual.It will be understood to those of skill in the art that administration
Actual amount or dosage and administration time-histories are by depending on the property of treated disease and seriousness, the year of treated subject
Age and general status and administering mode etc..
It is to be understood that pharmaceutically acceptable carrier can also be included in above-mentioned pharmaceutical formulation.It is being embodied
In example, the pharmaceutically acceptable carrier refers to well known by persons skilled in the art be suitable for any auxiliary of specific mode of administration
Material.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the scope of the invention, it is every at this
Under the inventive concept of invention, the equivalent structure transformation made using description of the invention and accompanying drawing content, or directly/use indirectly
It is included in other related technical areas in the scope of patent protection of the present invention.
SEQUENCE LISTING
<110>Shenzhen Mo Saier biomedicine developments in science and technology Co., Ltd
<120>A kind of NK of specific chimeric antigen receptor genetic modification and its production and use
<130> 2017.10.30
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1314
<212> DNA
<213>Artificial sequence
<400> 1
ccggaattcc ggatgcttct cctggtgaca agccttctgc tctgtgagtt accacaccca 60
gcattcctcc tgatcccaga agttcaattg ttagagtctg gtggcggtct tgttcagcct 120
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
atcacccttt acaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080
agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140
gaaggaggat gtgaactgcg gctggtccct cgggggcgag gggctgcgga ggcagcgacc 1200
cggaaacagc gtatcactga gaccgagtcg ccttatcagg agctccaggg tcagaggtcg 1260
gatgtctaca gcgacctcaa cacacagagg ccgtattaca aaccgctcga gcgg 1314
<210> 2
<211> 1380
<212> DNA
<213>Artificial sequence
<400> 2
ctgcccttct ttttcggcaa catcacccgg gaggaggcag aagattacct ggtccagggg 60
ggcatgagtg atgggcttta tttgctgcgc cagagccgca actacctggg tggcttcgcc 120
ctgtccgtgg cccacgggag gaaggcacac cactacacca tcgagcggga gctgaatggc 180
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
aaagacaaga cagggaagct ctccatcccc gagggaaaga agttcgacac gctctggcag 540
ctagtcgagc attattctta taaagcagat ggtttgttaa gagttcttac tgtcccatgt 600
caaaaaatca aagaactggg ctctggtaat tttggaactg tgaaaaaggg ctactaccaa 660
atgaaaaaag ttgtgaaaac cgtggctgtg aaaatactga aaaacgaggc caatgacccc 720
gctcttaaag atgagttatt agcagaagca aatgtcatgc agcagctgga caacccgtac 780
atcgtgcgga tgatcgggat atgcgaggcc gagtcctgga tgctggttat ggagatggca 840
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
ggaaagtggc ctgtcaagtg gtacgctccg gaatgcatca actactacaa gttctccagc 1140
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
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence
<400> 3
gaagttcaat tgttagagtc tggtggcggt 30
<210> 4
<211> 30
<212> DNA
<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 (10)
1. a kind of NK of specific chimeric antigen receptor genetic modification, it is characterised in that special including NY-ESO-1
Different in nature Chimeric antigen receptor gene and truncated-type spleen tyrosine kinase Syk genes.
2. the NK of specific chimeric antigen receptor genetic modification as claimed in claim 1, it is characterised in that institute
NY-ESO-1 specific chimeric antigen receptors gene is stated in NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments.
3. the NK of specific chimeric antigen receptor genetic modification as claimed in claim 2, it is characterised in that institute
NY-ESO-1 specific chimeric antigen receptor gene orders are stated referring to table 1.
4. the NK of specific chimeric antigen receptor genetic modification as claimed in claim 1, it is characterised in that institute
Truncated-type spleen tyrosine kinase Syk gene orders are stated referring to table 2.
A kind of 5. preparation method of the NK of specific chimeric antigen receptor genetic modification, it is characterised in that the system
Preparation Method comprises the following steps：
First slow virus and the second slow virus are infected into NK cells simultaneously, wherein, first slow virus contains NY-ESO-1 spies
Different in nature Chimeric antigen receptor gene, second slow virus contain truncated-type spleen tyrosine kinase Syk genes.
6. preparation method as claimed in claim 5, it is characterised in that first slow virus obtains as follows：
NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments are mixed with entry vector, so that the NY-ESO-1/
ScFv-CD8-41BB-DAP12 genetic fragments are embedded in the entry vector, and obtain and carry NY-ESO-1/scFv-CD8-41BB-
The entry vector of DAP12 genetic fragments；
The entry vector with NY-ESO-1/scFv-CD8-41BB-DAP12 genetic fragments is mixed with slow virus plasmid
Restructuring, to establish Cancer-testis antigen NY-ESO-1 specific NK cells receptor expression plasmids；
By the Cancer-testis antigen NY-ESO-1 specific NK cells receptor expression plasmid and viral packaging plasmid cotransfection
293FT cells, obtain the first slow virus.
7. preparation method as claimed in claim 6, it is characterised in that the NY-ESO-1/scFv-CD8-41BB-DAP12 bases
Because the acquisition methods of fragment comprise the following steps：
Obtain the total cDNA of NY-ESO-1 antigen immunes；
By the total cDNA of NY-ESO-1 antigen immunes and NY-ESO-1 heavy chain of antibody variable region VH primers and light chain variable district VL
Primer enters performing PCR, obtains NY-ESO-1VH and NY-ESO-1VL genetic fragments；
By NY-ESO-1VH the and NY-ESO-1VL genetic fragments using connection peptide connection, NY-ESO-1 antibody scFv bases are obtained
Because of fragment；
It will be that NY-ESO-1 antibody scFvs genetic fragment is connected with pcDNA carriers, and obtain pcDNA-NY-ESO-1/scFv plasmids；
The CD8-41BB- of Chimeric antigen receptor will be synthesized according to CD8 α, 41BB and DAP12-ITAM sequence genes sequences Design
DAP12 expression cassettes, enter pcDNA3.1 (+)-NY-ESO-1/scFv plasmids with Hind III/XhoI digestion rear clones, obtain
PcDNA3.1 (+)-NY-ESO-1/scFv-CD8-41BB-DAP12 plasmids, NY-ESO-1/ is obtained after EcoR I/Xho I digestions
ScFv-CD8-41BB-DAP12 genetic fragments.
8. the preparation method as described in claim 5 to 7 any one, it is characterised in that second slow virus is according to as follows
Method obtains：
Obtain truncated-type spleen tyrosine kinase Syk gene orders；
Truncated-type spleen tyrosine kinase Syk gene orders are mixed with entry vector, so that the truncated-type spleen tyrosine kinase
Syk gene orders are embedded in the entry vector, and obtain the introduction with truncated-type spleen tyrosine kinase Syk gene orders and carry
Body；
By the entry vector with truncated-type spleen tyrosine kinase Syk genetic fragments and slow virus plasmid mixed reorganization, with
Obtain slow virus expression plasmid pLenti7.3-Syk；
By the slow virus expression plasmid pLenti7.3-Syk and viral packaging plasmid cotransfection 293FT cells, it is slow to obtain second
Virus.
9. preparation method as claimed in claim 8, it is characterised in that the truncated-type spleen tyrosine kinase Syk gene orders
Referring to table 2.
10. a kind of tumor drug formulation, it is characterised in that resist including the specific chimeric as described in any one of Claims 1-4
The NK of original receptor genetic modification or the spy prepared as the preparation method as described in any one of claim 5 to 9
The NK of different in nature Chimeric antigen receptor genetic modification.
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