CN106279438B - Novel chimeric antigen receptor and application thereof - Google Patents
Novel chimeric antigen receptor and application thereof Download PDFInfo
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- CN106279438B CN106279438B CN201610720266.2A CN201610720266A CN106279438B CN 106279438 B CN106279438 B CN 106279438B CN 201610720266 A CN201610720266 A CN 201610720266A CN 106279438 B CN106279438 B CN 106279438B
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses the recombination Chimeric antigen receptors with photo-induction guiding element, including antigen binding domain, transmembrane structure area, costimulatory signal conducting region and T cell signal transduction area functional domain, photo-induction guiding element is inserted between the N-terminal of recombination Chimeric antigen receptor, C-terminal or each functional domain.Photo-induction guiding element LOV2 gene cloning is entered the coexpression vector that plasmid Lenti-EF1 α-CD19CAR building has the recombination Chimeric antigen receptor of photo-induction guiding element with molecular cloning method by the present invention, and the slow virus carrier for having photoinduction CAR molecule is obtained using 293T cell packaging.After recombination Chimeric antigen receptor of the present invention is expressed in T cell by slow virus carrier, reversible control of the killing toxicity of T cell by LOV2 provides important channel for the CAR-T safety for improving clinical anticancer.
Description
Technical field
The present invention relates to biomedical conversion fields, more particularly to the recombination Chimeric antigen receptor with photo-induction guiding element and
Its encoding gene and application.
Background technique
Immune system is the defense system of human body, on the one hand plays the function of bacteria removal, virus, alien material, separately
On the one hand the cell of internal senile cell and mutation is eliminated, some mutant cells will become cancer cell.Cancer immunotherapy
It is exactly based on artificial intervention, its proliferation is killed and inhibited to cancer cell to transfer the immune system of body itself.
Adoptive cellular immunotherapy (adoptive cellular immunotherapy, ACI) refers to Activation In Vitro
Self or alloimmune cell infusion to patient, be current treatment malignant tumour to kill the tumour cell of patient's body
One of important means obtains good therapeutic effect in the clinical treatment of a variety of solid tumors and neoplastic hematologic disorder.Wherein, chimeric antigen by
Body (chimeric antigen receptor, CAR) T cell cure is to develop new thin of very fast one kind in recent years
Born of the same parents' immunotherapy techniques modify effector T cell by technique for gene engineering, overcome tumor by local immunosupress microenvironment and host
Immune tolerance state improves antitumor targeting ability, lethal and persistence.
CAR structure by extracellular antigen binding domain, transmembrane structure area and intracellular signal transduction district's groups at.Extracellular antigen binding domain
Mainly the variable region of the single-stranded heavy chain of antigentic specificity monoclonal antibody (VL) and light chain (VH) forms, and the two is connected with hinge area
It connects to form single-chain antibody (single chain fragment varible, scFv).The scFv that CAR passes through identification tumour antigen
With intracellular signal domain " immunoreceptor tyrosine activating motif (immunoreceptor tyrosine-based activation
Motifs, ITAM) " genetic recombination is carried out in vitro, the T lymphocyte of patient is modified by Gene transfer techniques, makes patient T
Expressions In Lymphocytes tumour antigen receptor, through purifying and extensive amplification modification after T lymphocyte, referred to as chimeric antigen by
The T lymphocyte (CAR-T) of body modification.
CAR technology clinically achieved in the treatment of hematological system tumor and solid tumor important achievement (Grupp SA,
Kalos M, Barrett D, et al. Chimeric antigen receptor-midified T cells for
acute lymphoid leukemia [J]. N Engl J Med, 2013,368:1509-1518.Davila ML,
Riciere I, Wang X, et al. Efficiacy and toxicity management of 19-28z CAR T
cell therapy in B cell acute lymphoblastic leukemia [J]. Sci Transl Med.
2014,6:224-225.
Kochenderfer JN, Dudley ME, Rosenberg SA, et al. Chemotherapy-
Refractory Diffuse Large B-Cell Lymphoma and Indolent B-cell Malignancies Can
Be Effectively Treated With Autologous T Cell Expressing an Ati-CD19 Chimeric
Antigen Receptor[J]. J Clin Oncol, 2015,33:540-549.Louis CU, Savolo B, Doti
G, et al. Antitumor activity and long-ter fate of chimeric antigen reeptor-
positive T cell in patients with neuroblastoma[J]. Blood, 2011,118:6050-
6056.Tang X, Zhou Y, Li W, et al. T cell expressing a LMP1-specific chimeric
antigen receptor mediate antitumor effects against LMP1-positive
nasopharyngeal carcinoma cells in vitro and in vivo[J]. J Biomed Res, 2014,
28:468-475.Gargeet T, Fraser CK, Dotti G, et al. BRAF and MEK inhibition
variably affect GD2-specific chimeric antigen receptor (CAR) T-cell function
in vitro[J]. Immunother, 2015,38:12-23.).The third generation is had evolved at present.First generation CAR is by single-stranded
Antibody is connected by transmembrane domain with intracellular signal transduction area (ITAM), and ITAM is usually CD3 ζ or Fc ε RI γ；The second generation
The intracellular signal transduction area of CAR introduces costimulatory molecules (costimulatory molecule, CM), and predominantly CD28 points
Son；Third generation CAR introduces double costimulatory molecules (CM1 and CM2), and predominantly CD28 molecule is plus CD134 or CD137 etc..The
The research of generation CAR-T lymphocyte is more, but most of tests are in cell amplification, survival time in vitro, cytokine secretion
Etc. there is also deficiencies, do not reach expected clinical effectiveness.Studies have shown that the complete activation of T lymphocyte depend on it is double
The effect of signal and cell factor.Wherein the first signal is specific signals, by the antigen on TCR identification antigen presenting cell surface
Peptide-MHC compound is started；Second signal is costimulatory signal, by the important costimulatory molecules such as CD28/B7, is promoted
IL-2 synthesis, and activate T lymphocyte sufficiently and from apoptosis.Even if T lymphocyte and antigen contact, if do not cooperateed with
Stimulus signal, cell are difficult to function of bringing into normal play.Correspondingly, the Chimeric antigen receptor only containing CD3 ζ sequence, if do not assisted
With stimulus signal 2, it is also difficult to efficient activation CAR-T lymphocyte.Therefore, the dual signal theory activated according to T lymphocyte, the
Two generations and third generation CAR add such as CD28, CD137 costimulatory molecules in Chimeric antigen receptor, to improve T lymphocyte
Cytotoxicity, proliferation activity maintain T lymphocyte response, extend T lymphocyte time-to-live etc..Research confirms the second generation
CAR-T lymphocyte is superior to the first generation in tumor killing activity and internal time-to-live.Currently, third generation CAR-T lymph is thin
Born of the same parents' clinical application is also fewer, therefore whether its safety and validity are just centainly better than second generation CAR-T lymphocyte, Yi Jixuan
What kind of costimulatory molecules combination selected, it is also necessary to further look at.
Although CAR-T targeting therapy on tumor cell is one of current most promising modality of cancer treatment, it has
Target non-tumour " undershooting-effect " (Morgan RA, Yang JC, Kitano M, et al. Case report of a
serious adverse event following the administration of T cells transduced with
A chimeric antigen receptor recognizing ERBB2 [ J ] .Mol Ther, 2010,18 (4): 843-
And " cytokine storm " (Wrzesinski C, Paulos CM, Kaiser A, et al.Increased 851)
intensity lymphodepletion enhances tumor treatment efficacy of adoptively
Transferred tumor- specific T cells [ J ] J Immunother, 2010,33 (1): 1- 7) poison
Property effect.Activity " switch " control for increasing CAR-T cell is the important content for solving CAR-T toxicity problem, is being faced for CAR-T
The popularization and application of bed are of great significance.Therefore, the reversible light-operated clinical conversion to CAR-T of CAR-T killing activity has product
The progradation of pole.
Summary of the invention
The purpose of the present invention is to provide the recombination Chimeric antigen receptors and its encoding gene of the regulation of photo-induction guiding element.
In order to solve the above technical problems, specific technical solution of the present invention is as follows: the recombination inosculating antibody with photo-induction guiding element
Original receptor, including antigen binding domain, transmembrane structure area, costimulatory signal conducting region and T cell signal transduction area functional domain,
Photo-induction guiding element is inserted between the N-terminal of recombination Chimeric antigen receptor, C-terminal or each functional domain.
Recombination Chimeric antigen receptor of the present invention, photo-induction guiding element are quick selected from cryptochrome (CRY), ultraviolet and blue light
Feel one or more of flavoprotein (LOV and BLUF) and phytochrome (PHY), further preferred arabidopsis (rice, fruit
Fly, tomato, barley, fern, moss, algae, Africa xenopus, mouse, people etc.) CRY, the LOV2 of arabidopsis (oat etc.), plant
The PHY of object.More preferably derive from the LOV2(aa 404-546 of Avena sativa Phototropin 1, NPH1- of oat
1, GenBank:AAC05083.1) its amino acid sequence is as shown in SEQ ID No:1.
Under illumination condition, the recombination Chimeric antigen receptor containing photo-induction guiding element is caused to be able to table due to being illuminated by the light stimulation
It reaches, reaches the "ON" effect of light-operated property；Under the conditions of being protected from light, do not have the expression condition of recombination Chimeric antigen receptor to realize light
The "Off" effect of control property.
Recombination Chimeric antigen receptor of the present invention, the photo-induction guiding element can be located at recombination Chimeric antigen receptor
Between N-terminal, C-terminal or each functional domain, that is, it is located at the N-terminal (before antigen binding domain) of recombination Chimeric antigen receptor, antigen
Between combined area and transmembrane structure area, between transmembrane structure area and costimulatory signal conducting region, between costimulatory signal conducting region
Between T cell signal transduction area or C-terminal (after T cell signal transduction area).
Recombination Chimeric antigen receptor of the present invention, the antigen binding domain functional domain are selected from human antibody, source of people
Change antibody, antigen-binding fragment or combinations thereof, can be complete antibody, Fab, Fab', (Fab') 2, Fv segment or single-stranded
Variable region fragment scFv.
Preferably, functional domain combination tumour antigen in antigen binding domain of the present invention.The tumour be prostate cancer,
Clear-cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, leukaemia (chronic lymphocytic leukemia, acute lymphoblastic
Cell leukemia, small lymphocyte leukaemia, acute myelocytic leukemia, Huppert's disease, gland cancer, colorectal cancer,
Breast cancer, solid tumor, head-neck carcinoma, glioblastoma, neuroblastoma, sarcoma, metastatic carcinoma, pleomorphism colloid are female thin
Born of the same parents' tumor etc..
Preferably, the tumour antigen is selected from CD19, CD20, CD22, CD33, CD138, ROR1, Her2/neu, mesothelium
Element, CD33/IL3Ra, c-Met, PSMA, CAIX, CEA, PSCA, GD2, glycolipid F77, EGFRvIII, GD-2, FAP, FBP, NY-
ESO-1TCR, MAGEA3TCR or any combination thereof.
Preferably, costimulatory signal conducting region of the present invention is selected from CD27, CD28, CD137 (4-1BB), CD134
(OX40), the costimulatory signal of CD30, CD40, PD-1, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 protein molecular conducts
The ligand or any combination thereof in area and CD3 ζ specific binding.
Preferably, transmembrane structure area of the present invention is selected from the transmembrane structure area of CD3, CD4, CD8 or CD28 protein molecular.
Preferably, T cell signal transduction of the present invention area's functional domain be selected from CD28, CD137, Fc ε RI γ,
One or more of T cell signal transduction area of ZAP70 or CD3 ζ protein molecular.T cell signal transduction area includes immune
Receptor tyrosine activation motifs.
Recombination Chimeric antigen receptor of the present invention, there are also extracellular hinges between antigen binding domain and transmembrane structure area
Sequence, it is preferred that the extracellular hinge area is the hinge area or Immunoglobulin IgG hinge area or its group of CD8 protein molecular
It closes.Photo-induction guiding element can between antigen binding domain and extracellular hinge area, extracellular hinge area and transmembrane structure area it
Between, between transmembrane structure area and costimulatory signal conducting region, between costimulatory signal conducting region and T cell signal transduction area or
After T cell signal transduction area.
A preferred scheme of the invention, when photo-induction guiding element is located at after T cell signal transduction area, end can
To be connected with luminous effect element, preferably luminous effect element is human albumin, and amino acid sequence is as shown in SEQ ID No:2.
The antigen binding domain of recombination Chimeric antigen receptor of the present invention, extracellular hinge area, transmembrane structure area, altogether thorn
Energizing signal conducting region, each functional domain in T cell signal transduction area and photo-induction guiding element can be connected directly, that is, pass through clone
Addition restriction enzyme site or fusion DNA vaccine technology are expressed after these elements are connected when gene.Link peptide can also be passed through
Connect each functional domain.The preferably flexible peptide of link peptide or rigid peptide, preferably flexible peptide refer to that 1 ~ 20 is rich in Gly and/or Ala
And/or the link peptide of Ser amino acid residue, preferably rigid peptide refer to that keeping link peptide front and back to form α spiral stablizes secondary structure
Link peptide.
Preferred embodiment of the invention, photoinduction components upstream pass through flexible peptide, preferably link peptide 1 and recombination chimeric antigen by
Under the antigen binding domain of body, extracellular hinge area, transmembrane structure area, costimulatory signal conducting region or T cell signal transduction area
Trip is connected, and the amino acid sequence of link peptide 1 is as shown in SEQ ID No:25.Photoinduction member downstream passes through rigid peptide, preferably connects
Connect peptide 2 and recombinate the antigen binding domain of Chimeric antigen receptor, extracellular hinge area, transmembrane structure area, costimulatory signal conducting region,
T cell signal transduction area or the upstream of luminous effect element are connected, and the amino acid sequence of link peptide 2 is as shown in SEQ ID No:26.
A preferred scheme of the invention, the recombination Chimeric antigen receptor, photo-induction guiding element are LOV2, antigen knot
Conjunction area is CD19 scFv, extracellular hinge area is CD8 hinge area, transmembrane structure area is the transmembrane structure CD8 area, costimulatory signal
Conducting region is 4-1BB costimulatory signal conducting region, T cell signal transduction area is CD3 ζ signal transduction area.When photo-induction guiding element position
When after T cell signal transduction area, luminous effect element is human albumin.
A preferred scheme of the invention, the recombination Chimeric antigen receptor, CD19 scFv amino acid sequence is such as
Shown in SEQ ID No:3, CD8 hinge region amino acid sequence is as shown in SEQ ID No:4, the region amino acid sequence of the transmembrane structure CD8
As shown in SEQ ID No:5,4-1BB costimulatory signal conduction region amino acid sequence as shown in SEQ ID No:6, photo-induction guiding element
LOV2 amino acid sequence such as SEQ ID No:1, CD3 ζ signal transduction region amino acid sequence is as shown in SEQ ID No:7.Work as photo-induction
When guiding element is located at after T cell signal transduction area, luminous effect element amino acid sequence is as shown in SEQ ID No:2.
A preferred scheme of the invention, the recombination Chimeric antigen receptor amino acid sequence such as SEQ ID No:8-
Shown in 12.
The corresponding recombination Chimeric antigen receptor of SEQ ID No:8 are as follows: CD19 scFv- link peptide 1-LOV2- link peptide 2-
The transmembrane structure hinge area-CD8 CD8 area -4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor of SEQ ID No:9 are as follows: CD19 scFv-CD8 hinge area-link peptide 1-LOV2-
The transmembrane structure link peptide 2-CD8 area -4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor of SEQ ID No:10 are as follows: the transmembrane structure CD19 scFv-CD8 hinge area-CD8
Area-link peptide 1-LOV2- link peptide 2-4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor of SEQ ID No:11 are as follows: the transmembrane structure CD19 scFv- CD8 hinge area-CD8
Area's -4-1BB costimulatory signal conducting region-link peptide 1-LOV2- link peptide 2-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor of SEQ ID No:12 are as follows: the transmembrane structure CD19 scFv- CD8 hinge area-CD8
Area -4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area-link peptide 1-LOV2- link peptide 2- human albumin.
Another object of the present invention is to provide the bases for encoding the above-mentioned recombination Chimeric antigen receptor with photo-induction guiding element
Cause, including antigen binding domain, transmembrane structure area, costimulatory signal conducting region and T cell signal transduction area functional domain base
Because of sequence, photoinduction is inserted between the both ends or each functional domain gene order of recombination Chimeric antigen receptor gene order
Element genes.
The photo-induction guiding element is selected from cryptochrome (CRY), ultraviolet and sensitive to blue light flavoprotein (LOV and BLUF) and light
One or more of quick pigment (PHY), further preferred arabidopsis (rice, drosophila, tomato, barley, fern, moss, algae
Class, Africa xenopus, mouse, people etc.) CRY, the LOV2, the PHY of plant of arabidopsis (oat etc.).More preferably from oat
LOV2(aa 404-546 of Avena sativa Phototropin 1, NPH1-1, GenBank:AAC05083.1) its core
Nucleotide sequence is as shown in SEQ ID No:13.
Recombination Chimeric antigen receptor of the present invention, the antigen binding domain gene be selected from human antibody, humanized antibody,
Gene of antigen-binding fragment or combinations thereof can be complete antibody, Fab, Fab', (Fab') 2, Fv segment or single-stranded
Variable region fragment scFv gene.
Preferably, functional domain combination tumour antigen in antigen binding domain of the present invention.The tumour be prostate cancer,
Clear-cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, leukaemia (chronic lymphocytic leukemia, acute lymphoblastic
Cell leukemia, small lymphocyte leukaemia, acute myelocytic leukemia, Huppert's disease, gland cancer, colorectal cancer,
Breast cancer, solid tumor, head-neck carcinoma, glioblastoma, neuroblastoma, sarcoma, metastatic carcinoma, pleomorphism colloid are female thin
Born of the same parents' tumor etc..Preferably, the tumour antigen is selected from CD19, CD20, CD22, CD33, CD138, ROR1, Her2/neu, mesothelium
Element, CD33/IL3Ra, c-Met, PSMA, CAIX, CEA, PSCA, GD2, glycolipid F77, EGFRvIII, GD-2, FAP, FBP, NY-
ESO-1TCR, MAGEA3TCR or any combination thereof.
Preferably, costimulatory signal conducting region gene of the present invention is selected from CD27, CD28, CD137 (4-1BB), CD134
(OX40), the costimulatory signal of CD30, CD40, PD-1, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 protein molecular conducts
The ligand gene or any combination thereof of area's gene and CD3 ζ specific binding.
Preferably, transmembrane structure area of the present invention gene is selected from the cross-film knot of CD3, CD4, CD8 or CD28 protein molecular
Structure area gene.
Preferably, T cell signal transduction of the present invention area's functional domain be selected from CD28, CD137, Fc ε RI γ,
One or more of T cell signal transduction area gene of ZAP70 or CD3 ζ protein molecular.
A preferred scheme of the invention, the recombination Chimeric antigen receptor gene, in antigen binding domain gene and
Between transmembrane structure area gene there are also extracellular hinge area gene, the extracellular hinge area gene, preferably CD8 protein molecular
Hinge area or the gene of Immunoglobulin IgG hinge area or combinations thereof.Photoinduction element genes can be located at antigen binding domain with
Between extracellular hinge area gene, between extracellular hinge area and transmembrane structure area gene, transmembrane structure area and costimulatory signal
Between conducting region gene, between costimulatory signal conducting region and T cell signal transduction area gene or T cell signal transduction area base
Because after.
A preferred scheme of the invention, when photoinduction element genes are located at after T cell signal transduction area gene,
Its end can connect luminous effect element genes, the preferred human albumin of luminous effect element, nucleotide sequence such as SEQ ID
Shown in No:14.
Preferred embodiment of the invention, photoinduction element genes upstream pass through 1 gene of link peptide and recombination Chimeric antigen receptor
Antigen binding domain, extracellular hinge area, transmembrane structure area, costimulatory signal conducting region or T cell signal transduction area gene
Downstream is connected, and the nucleotide sequence of link peptide 1 is as shown in SEQ ID No:27.Photoinduction element genes downstream passes through link peptide 2
Antigen binding domain, extracellular hinge area, the transmembrane structure area, costimulatory signal conducting region, T of gene and recombination Chimeric antigen receptor
Cellular signal transduction area or the upstream of luminous effect element genes are connected, and the nucleotide of link peptide 2 is arranged as shown in SEQ ID No:28.
A preferred embodiment of the invention, the recombination Chimeric antigen receptor gene, antigen binding domain CD19
ScFv, nucleotide sequence is as shown in SEQ ID No:15, extracellular hinge area is CD8 hinge area, nucleotide sequence such as SEQ ID
No:16 is shown, transmembrane structure area is the transmembrane structure CD8 area, and nucleotide sequence is as shown in SEQ ID No:17, costimulatory signal passes
Leading area is 4-1BB costimulatory signal conducting region, and nucleotide sequence is as shown in SEQ ID No:18, T cell signal transduction area is CD3
ζ signal transduction area, nucleotide sequence is as shown in SEQ ID No:19.After photo-induction guiding element is located at T cell signal transduction area
When, luminous effect element nucleotide sequence is as shown in SEQ ID No:14.A preferred scheme of the invention, the recombination are embedding
Antigen receptor nucleotide sequence is closed as shown in SEQ ID No:20-24.
The corresponding recombination Chimeric antigen receptor gene of SEQ ID No:20 are as follows: CD19 scFv- link peptide 1-LOV2- connection
The transmembrane structure hinge area-CD8 peptide 2-CD8 area -4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor gene of SEQ ID No:21 are as follows: CD19 scFv-CD8 hinge area-link peptide 1-
The transmembrane structure link peptide 2-CD8 LOV2- area -4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor gene of SEQ ID No:22 are as follows: CD19 scFv-CD8 hinge area-CD8 cross-film
Structural area-link peptide 1-LOV2- link peptide 2-4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor gene of SEQ ID No:23 are as follows: CD19 scFv- CD8 hinge area-CD8 cross-film
Structural area -4-1BB costimulatory signal conducting region-link peptide 1-LOV2- link peptide 2-CD3 ζ signal transduction area.
The corresponding recombination Chimeric antigen receptor gene of SEQ ID No:24 are as follows: CD19 scFv- CD8 hinge area-CD8 cross-film
Structural area -4-1BB costimulatory signal conducting region-CD3 ζ signal transduction area-link peptide 1-LOV2- link peptide 2- human albumin.
Another object of the present invention is to provide express the recombination chimeric antigen of the present invention with photo-induction guiding element by
Recombinant vector, expression cassette, slow virus carrier or the cell of body contain above-mentioned recombination Chimeric antigen receptor gene.
A preferred embodiment of the invention, expression vector be Lenti-EF1 α, recombinant vector Lenti-liCAR, specifically
For that will recombinate Chimeric antigen receptor sequence, nucleotide sequence is inserted into expression vector as shown in SEQ ID any one of No:20-24
Obtained carrier between BamH I and EcoR the I restriction enzyme site of Lenti-EF1 α.
A preferred embodiment of the invention, the cell are selected from self or transgenosis T cell, NK cell, cell toxicant
Property T lymphocyte or regulatory T-cell, memory t cell, bispecific T cell, CIK cell.
It is an object of the present invention to provide the recombination Chimeric antigen receptor preparation methods with photo-induction guiding element, it may include
Following steps:
1) building of coexpression vector
Photo-induction guiding element LOV2 gene cloning is entered into plasmid Lenti-EF1 α-CD19CAR with molecular cloning method
(CD3ZetaQQ) building has the coexpression vector of the recombination Chimeric antigen receptor of photo-induction guiding element；
2) packaging of slow virus carrier
The slow virus carrier for having photoinduction CAR molecule is obtained using 293T cell packaging.The slow virus carrier is will
Above-mentioned coexpression vector and slow virus pack necessary structural proteins expression plasmid corotation and efficiently assemble institute in the cell of slow virus
What is obtained has the slow virus of expression recombination.A preferred embodiment of the invention, by above-mentioned coexpression vector and slow virus knot
Structure protein expression vector pGP and pVSVG transfect HEK 293T cell culture jointly and obtain slow virus carrier；
3) there is lentivirus mediated the recombination Chimeric antigen receptor of photo-induction guiding element to transfect cell.
Method provided by the invention includes the following steps: building target gene slow virus package carrier, packs slow virus, slowly
Virus-mediated T cell transgenosis, recombinant C AR-T cell have reversible photocontrol special the specific killing activity of target cell
Property, i.e., only there is under illumination condition killing ability.
The invention has the advantages that
The present invention provides a kind of recombination Chimeric antigen receptors with photo-induction guiding element, it is demonstrated experimentally that the recombinant protein
After being expressed in T cell by slow virus carrier, reversible control of the killing toxicity of T cell by LOV2.It is of the present invention
Recombination Chimeric antigen receptor with photo-induction guiding element maintains the targeting of CAR-T and anti-personnel while to this two kinds of characteristics
Control switch is increased, provides important channel for the CAR-T safety for improving clinical anticancer.
Detailed description of the invention
Fig. 1 is the recombination Chimeric antigen receptor structural schematic diagram of the present invention with photo-induction guiding element.
Fig. 2 be transfect the Jurkat-CAR5 of the recombination Chimeric antigen receptor of the present invention with photo-induction guiding element~
Jurkat-CAR9 cell activity photoinduction controls result of study.
Fig. 3 Jurkat-CAR5 tests knot in the cellkilling capacity of illumination effect target ratio different under the conditions of being protected from light respectively
Fruit.
Fig. 4 Jurkat-CAR6 tests knot in the cellkilling capacity of illumination effect target ratio different under the conditions of being protected from light respectively
Fruit.
Fig. 5 Jurkat-CAR7 tests knot in the cellkilling capacity of illumination effect target ratio different under the conditions of being protected from light respectively
Fruit.
Fig. 6 Jurkat-CAR8 tests knot in the cellkilling capacity of illumination effect target ratio different under the conditions of being protected from light respectively
Fruit.
Fig. 7 Jurkat-CAR9 tests knot in the cellkilling capacity of illumination effect target ratio different under the conditions of being protected from light respectively
Fruit.
Specific embodiment
Illustrate specific steps of the invention by the following examples, but is not limited by the example.
Term as used in the present invention generally there are those of ordinary skill in the art usually to manage unless otherwise indicated
The meaning of solution.
The present invention is described in further detail combined with specific embodiments below and referring to data.It should be understood that the embodiment is
In order to demonstrate the invention, it rather than limits the scope of the invention in any way.
In the examples below, the various processes and method being not described in detail are conventional methods as known in the art.
The present invention is further described combined with specific embodiments below.
Material used in following examples, reagent etc. commercially obtain unless otherwise specified.
Embodiment 1, the design of recombination Chimeric antigen receptor with photo-induction guiding element
The present invention selects the preferable CD19-CAR gene of clinical effectiveness, it is successively as follows to constitute element: CD19 scFv, CD8
hinge,CD8 transmembrane,4-1BB intracellular,CD3ζ；
LOV2(aa 404-546 of used in the present inventionAvena sativaPhototropin 1, NPH1-1,
GenBank:AAC05083.1) from oat (Avena sativa), according to people (Homo spaice) codon preference
Codon optimization is carried out, amino acid sequence is as shown in SEQ ID No:1, and nucleotide sequence is as shown in SEQ ID No:13.
Antigen binding domain, transmembrane structure area, costimulatory signal conducting region and the T that LOV2 is inserted respectively into CD19-CAR are thin
Before and after born of the same parents' signal transduction area functional domain or between, it is specific as follows:
The liCAR5:CD19 scFv- link peptide 1-LOV2- link peptide 2-CD8 transmembrane structure hinge area-CD8 area -4-1BB is total
Stimulus signal conducting region-CD3 ζ signal transduction area (amino acid sequence as shown in SEQ ID No:8, nucleotide sequence such as SEQ ID
Shown in No:20).
The liCAR6:CD19 scFv-CD8 hinge area-link peptide 1-LOV2- transmembrane structure link peptide 2-CD8 area -4-1BB is total
Stimulus signal conducting region-CD3 ζ signal transduction area (amino acid sequence as shown in SEQ ID No:9, nucleotide sequence such as SEQ ID
Shown in No:21).
The transmembrane structure scFv-CD8 hinge area-CD8 liCAR7:CD19 area-link peptide 1-LOV2- link peptide 2-4-1BB is total
Stimulus signal conducting region-CD3 ζ signal transduction area (amino acid sequence as shown in SEQ ID No:10, nucleotide sequence such as SEQ
Shown in ID No:22).
The transmembrane structure hinge area-CD8 scFv- CD8 liCAR8:CD19 area -4-1BB costimulatory signal conducting region-connection
Peptide 1-LOV2- link peptide 2-CD3 ζ signal transduction area (amino acid sequence as shown in SEQ ID No:11, nucleotide sequence such as SEQ
Shown in ID No:23).
The transmembrane structure hinge area-CD8 liCAR9:CD19 scFv- CD8 area -4-1BB costimulatory signal conducting region-CD3 ζ
Signal transduction area-link peptide 1-LOV2- link peptide 2- human albumin (amino acid sequence as shown in SEQ ID No:12, nucleotide
Sequence is as shown in SEQ ID No:24).
1 amino acid sequence of link peptide is as shown in SEQ ID No:25, and nucleotide sequence is as shown in SEQ ID No:27；Connection
2 amino acid sequence of peptide is as shown in SEQ ID No:26, and nucleotide sequence is as shown in SEQ ID No:28.
The expression of embodiment 2, recombination Chimeric antigen receptor with photo-induction guiding element
One, coexpression vector Lenti-liCAR5, Lenti-liCAR6, Lenti-liCAR7, Lenti-liCAR8 and
The building of Lenti-liCAR9
Lenti-EF1 α-CD19CAR(CD3ZetaQQ) it is purchased from Ai Kang get biomedical technology (Suzhou) Co., Ltd
(http://www.icartab.com.cn).Plasmid pUCK-AsLOV2 is purchased from company.
With classical CD19-CAR molecule (SEQ ID No:45) and the LOV2 molecule optimized according to people's codon Preference
(SEQ ID No:13) is template, and it is as follows to separately design primer:
LiCAR5:
CALOV (EcoRI): 5 '-TTCGAATTCGCCGCCACCATG-3 ' SEQ ID No:29；
CALOV5aR:5 '-GCTCCCACTCCCGCTTCCGCTGGACACGGTGACCAGA-3 ' SEQ ID No:33；
GSLOVF:5 '-GGAAGCGGGAGTGGGAGCCTTGCAACCACCTTGGAAAG-3 ' SEQ ID No:31；
EALOVR:5 '-CCGCTGCTTCTTTGGCCGCTGCTTCCAGCTCTTTGGCGGCCTCATC-3 ' SEQ ID
No:32；
EALOV5cF:5 '-AGCGGCCAAAGAAGCAGCGGCCAAAACCACTACCCCAGCACCGA-3 ' SEQ ID
No:34；
CALOV (BamHI): 5 '-GCGGGATCCTCACCGAGGCGGC-3 ' SEQ ID No:30；
LiCAR6:
CALOV (EcoRI): 5 '-TTCGAATTCGCCGCCACCATG-3 ' SEQ ID No:29；
CALOV6aR:5 '-GCTCCCACTCCCGCTTCCATCGCAGGCGAAGTCAA-3 ' SEQ ID No:35；
GSLOVF:5 '-GGAAGCGGGAGTGGGAGCCTTGCAACCACCTTGGAAAG-3 ' SEQ ID No:31；
EALOVR:5 '-CCGCTGCTTCTTTGGCCGCTGCTTCCAGCTCTTTGGCGGCCTCATC-3 ' SEQ ID
No:32；
EALOV6cF:5 '-AGCGGCCAAAGAAGCAGCGGCCAAAATCTACATTTGGGCCCCT-3 ' SEQ ID No:36；
CALOV (BamHI): 5 '-GCGGGATCCTCACCGAGGCGGC-3 ' SEQ ID No:30；
LiCAR7:
CALOV (EcoRI): 5 '-TTCGAATTCGCCGCCACCATG-3 ' SEQ ID No:29；
CALOV7aR:5 '-GCTCCCACTCCCGCTTCCACAGTAAAGAGTGATCA-3 ' SEQ ID No:37；
GSLOVF:5 '-GGAAGCGGGAGTGGGAGCCTTGCAACCACCTTGGAAAG-3 ' SEQ ID No:31；
EALOVR:5 '-CCGCTGCTTCTTTGGCCGCTGCTTCCAGCTCTTTGGCGGCCTCATC-3 ' SEQ ID
No:32；
EALOV7cF:5 '-AGCGGCCAAAGAAGCAGCGGCCAAAAAGCGCGGTCGGAAGA-3 ' SEQ ID No:38；
CALOV (BamHI): 5 '-GCGGGATCCTCACCGAGGCGGC-3 ' SEQ ID No:30；
LiCAR8:
CALOV (EcoRI): 5 '-TTCGAATTCGCCGCCACCATG-3 ' SEQ ID No:29；
CALOV8aR:5 '-GCTCCCACTCCCGCTTCCCAGTTCGCAGCCGCCTTCC-3 ' SEQ ID No:39；
GSLOVF:5 '-GGAAGCGGGAGTGGGAGCCTTGCAACCACCTTGGAAAG-3 ' SEQ ID No:31；
EALOVR:5 '-CCGCTGCTTCTTTGGCCGCTGCTTCCAGCTCTTTGGCGGCCTCATC-3 ' SEQ ID
No:32；
EALOV8cF:5 '-AGCGGCCAAAGAAGCAGCGGCCAAACGCGTGAAATTCAGCCGCAG-3 ' SEQ ID
No:40；
CALOV (BamHI): 5 '-GCGGGATCCTCACCGAGGCGGC-3 ' SEQ ID No:30；
LiCAR9:
CALOV (EcoRI): 5 '-TTCGAATTCGCCGCCACCATG-3 ' SEQ ID No:29；
CALOV9aR:5 '-GCTCCCACTCCCGCTTCCTCACCGAGGCGGCA-3 ' SEQ ID No:41；
GSLOVF:5 '-GGAAGCGGGAGTGGGAGCCTTGCAACCACCTTGGAAAG-3 ' SEQ ID No:31；
HSALOVR:5 '-CTTTAAACCGATGAGCAACCTCATCAATCTCCTCGGCAG-3 ' SEQ ID No:42；
LOVHSAF:5 '-AGACTGCCGAGGAGATTGATGAGGTTGCTCATCGGTTTAAAG-3 ' SEQ ID No:43；
LOVHSA (BamHI): 5 '-CGCGGATCCCTATTAAAGGCCTAAGGCAG-3 ' SEQ ID No:44；
Using plasmid Lenti-EF1 α-CD19CAR as template, (it is polymerize using NEB company Q5 high-fidelity DNA by fusion DNA vaccine
Enzyme) method amplification obtain liCAR5, liCAR6, liCAR7, liCAR8 and liCAR9 nucleotide molecule fragment.Pass through primer
The method for adding restriction enzyme adds EcoRI and BamHI restriction enzyme site at two sections of target fragment respectively.
PCR condition:
First round PCR: 98 °C of initial denaturation, 30s；98 °C, 10s of denaturation is annealed 58 °C, 30s, extends 72 °C, 45s, 30
Circulation；Extend 72 °C eventually, 2min；4 °C of cooling.Reaction system is added jointly using this wheel PCR product as template and upstream and downstream primer
System carries out the second wheel PCR: 98 °C of initial denaturation, 30s；98 °C, 10s of denaturation is annealed 58 °C, 30s, extends 72 °C, 1min, 30
Circulation；Extend 72 °C eventually, 2min；4 °C of cooling.With EcoRI and BamHI double digestion plasmid Lenti-EF1 α-CD19CAR and recycling
Fusion DNA vaccine product (for each nucleotide molecule fragment of liCAR), plasmid reaction system be 30ul(1ul plasmid, 3 μ l's
Buffer, the EcoRI of 0.5 μ l, the BamHI of 0.5 μ l, the ddH of 25 μ l2O), PCR recovery product reaction system 30 μ l(20 μ l
PCR recovery product, the Buffer of 3 μ l, the EcoRI of 0.5 μ l, the BamHI of 0.5 μ l, the ddH2O of 6 μ l), 37 °C of water-bath 30min.
Reaction terminates to be analyzed with 1% agarose gel electrophoresis, and target fragment is separately recovered.Target fragment is cloned into plasmid with ligase
In carrier section, carrier of the reaction system for 10 μ l(0.5 μ l, the fusion DNA vaccine product of 8 μ l, the T4DNA ligase of 0.5 μ l, 1 μ l's
Buffer), 4 °C of connections are stayed overnight.Linked system is put into 70 °C of water-bath inactivator 10min, sets cooled on ice.Connection product is turned
Change DH5 α competent cell, through ice bath 30min, 42 °C of water-bath heat shock 90s, sets 5min on ice immediately again, LB liquid is added
After culture medium shakes bacterium, bacterium solution is coated in ampicillin (Amp+) resistance LB plate on, 37 °C of culture carton upside down culture cultures
Overnight.Next day observes bacterium colony growing state on plate.The monoclonal that Amp is screened on picking plate carries out bacterium colony PCR identification, uses
The analysis of 1% agarose gel electrophoresis, filters out amplified band monoclonal colonies in line.The monoclonal filtered out is expanded
Big culture, it is 15% glycerol stock in -80 °C of refrigerators that next day, which saves a part of bacterium solution,.Remaining bacterium solution extracts examination with Plasmid DNA on a small quantity
Agent box extracts plasmid.The plasmid of extraction is subjected to DNA sequencing identification.It is identified satisfactory to contain Chimeric antigen receptor gene
The lentivirus transfer carrier of segment, is respectively designated as Lenti-liCAR5, Lenti-liCAR6, Lenti-liCAR7, Lenti-
LiCAR8 and Lenti-liCAR9；Then sequencing identification.Sequencing shows encoding each gene fragment order of liCAR5 and connection just
Really, nucleotide sequence is as shown in SEQ ID No:20, and amino acid sequence is as shown in SEQ ID No:8；Encode each of liCAR6
Gene fragment order and connection are correct, and nucleotide sequence is as shown in SEQ ID No:21, amino acid sequence such as SEQ ID No:9
It is shown；Each gene fragment order and the connection for encoding liCAR7 are correct, and nucleotide sequence is as shown in SEQ ID No:22, amino
Acid sequence is as shown in SEQ ID No:10；Each gene fragment order and the connection for encoding liCAR8 are correct, and nucleotide sequence is such as
Shown in SEQ ID No:23, amino acid sequence is as shown in SEQ ID No:11；Encode each gene fragment order and the company of liCAR9
It connects correctly, nucleotide sequence is as shown in SEQ ID No:24, and amino acid sequence is as shown in SEQ ID No:12.
Two, the packaging of slow virus carrier Lentivial
By the HEK 293T cell inoculation of culture in the 100mm culture dish that poly-D-lysine is handled, to cell fusion degree
When up to 70%-80%, serum-free DMEM culture solution, 37 °C, 5% CO are changed2Culture.After 2h, slow disease is packed with three plasmid calcium phosphate methods
Poison,
By taking Lenti-liCAR5 as an example, the specific steps of which are as follows:
Add slow virus carrier Lenti-liCAR5 plasmid 30 μ g, the pGP of 20 μ g, 10 μ g in 1 10ml centrifuge tube
PVSVG, 2 mol/LCaCl2124 ul add distilled water completion to 1mL, then plus 2 × Hank of 1mL, mix well, room temperature
20min is stood, is then added dropwise in above-mentioned 100mm culture dish, 37 °C, 5% CO2The 6-8 hours fresh 5%FBS of replacement of culture
DMEM culture medium continue to cultivate.Under inverted microscope observation transfection 24,48, the cell growth condition after 72h and receive daily
Collect supernatant.It is centrifuged off cell fragment, is filtered with 0.45 μm of filter.After filtering, vial supernatant is moved into Millipore concentration
Column is centrifuged 30min, is concentrated into 100 ~ 200uL, and the virus liquid of concentration is moved into EP pipe, and -80 °C save backup (or will be viral
Supernatant using ultracentrifuge with 4 °C, 7000 × g centrifugation 2h after discard supernatant, with Opti-MEM be resuspended virion, -80 °C
It saves backup).
Obtain the Lenti-liCAR6-9 of the packaging of slow virus carrier Lentivial respectively referring to the above method.
Three, lentivirus mediated photoinduction CAR transfected Jurkat cells.
By the good Jurkat cell of growth conditions with 5 × 105The density in/hole is inoculated in 6 orifice plates, adds serum-free
RPMI1640 culture medium is added 8 μ g/L Polybrene of 2ul, 100 μ l purified virus liquid is added, mix, in 37 ° to 2 ml
C、5% CO2Culture.After culture to 8h, changes the RPMI1640 culture of 10%FBS into, observe fluorescence after 48h.Contain respectively by what is obtained
There is the supernatant of slow virus carrier Lenti-liCAR5-9 after centrifugal purification, infect Jurkat cell secondary culture, is added 100
Untis/mL IL-2 promotes cell proliferation and differentiation, obtains Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-
CAR8, Jurkat-CAR9.
Embodiment 3, the recombination Chimeric antigen receptor T cell killing activity research with photo-induction guiding element
It is inoculated with 100 μ l 1 × 104The target cell (leukaemia cell Raji) in/hole is into 96 porocyte culture plates, according to 2:1
Effect target ratio is separately added into Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 cell
(cell of the Jurkat of expression liCAR5-9), mends culture solution to 200 μ l, respectively (LED light, 465nm, 30 μ under illumination condition
mol m-2 s-1, expose 4h) and 37 °C, 5% CO under the conditions of being protected from light2Incubator co-cultures 48h.
(1) one is tested
After co-culturing 48h, suspension culture is collected respectively by centrifugation and collects supernatant, user IL-2 ELISA kit
IL-2 content is detected, specific steps are referring to human IL-2's ELISA kit operation instructions.Concrete outcome is as shown in Figure 2.Fig. 2 shows
Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 cell are under illumination condition
IL-2 activity is similar to classics CAR-T cell, but under the conditions of being protected from light, and conspicuousness is lower than under each comfortable illumination condition and classical
The activity of CAR-T.Illustrate Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8 that the present invention designs,
Jurkat-CAR9 cell activity is controlled by photoinduction.
(2) two are tested
After co-culturing 48h, suspension culture is collected, cell activity is detected by lactic dehydrogenase (LDH) method for releasing.It connects
100 μ l 1x10 of kind4The target cell Raji in/hole into 96 porocyte culture plates, according to different effect target ratios (E/T value 0.5:1,1:1,
2:1,4:1,8:1,16:1) it is separately added into Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8,
Jurkat-CAR9 cell, mends culture solution to 200 μ l, respectively in illumination and 37 °C, 5% CO under the conditions of be protected from light2Incubator is trained altogether
Support 48h.After co-culturing 48h, tissue culture plate is taken out in cell incubator, is added in the culture hole for being only inoculated with Raji cell
Lactic dehydrogenase (LDH) releasing agent that kit provides, additional amount are 10%(20 μ l of original nutrient solution volume).LDH is added to release
It after putting agent, blows and beats and mixes for several times repeatedly, then proceed to be incubated in cell incubator.After 1h, cell plates porous plate will be cultivated
400 × 0g of centrifuge is centrifuged 5min.The 120 μ l of supernatant for taking each hole respectively is added in a 96 new orifice plate corresponding apertures, immediately
Carry out sample measurement.Each hole is separately added into the LDH detection working solution of 60 μ l.It mixes, room temperature, which is protected from light, is incubated for 30min, then exists
OD value is measured at 490nm.Detailed step is referring to lactic dehydrogenase citotoxicity detection kit operation instructions.Specific knot
Fruit as shown in fig. 3 to 7, as the result is shown as effect target ratio increases, is overexpressed the Jurkat-CAR5 of recombination Chimeric antigen receptor,
Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 Execution are also promoted therewith.In illumination item
Under part, dosage effect similar with classic CD19-CAR T cell shows these and is overexpressed recombination Chimeric antigen receptor
The specific killing ability of Jurkat cell because of modification without changing；And the Jurkat-CAR5 under the conditions of being protected from light,
The more classic CAR T of the killing ability of Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 cell is low
Under show its activity completely display.These results indicate that photocontrol recombination Chimeric antigen receptor of the present invention swashs
Access living is activated T cell killing activity by light induction.Illumination, which becomes in CAR-T treatment, as a result, controls cell killing energy
The activation condition of power.
Claims (14)
1. the recombination Chimeric antigen receptor with photo-induction guiding element, including antigen binding domain, extracellular hinge area, transmembrane structure
Area, costimulatory signal conducting region and T cell signal transduction area functional domain, it is characterised in that in recombination Chimeric antigen receptor
Photo-induction guiding element is inserted between N-terminal, C-terminal or each functional domain, the photo-induction guiding element is LOV2, and amino acid sequence is such as
Shown in SEQ ID No:1, the antigen binding domain is the scFv of CD19 protein molecular, amino acid sequence such as SEQ ID No:3 institute
Show, extracellular hinge area is the hinge area of CD8 protein molecular, and as shown in SEQ ID No:4, transmembrane structure area is amino acid sequence
The transmembrane structure area of CD8 protein molecular, for amino acid sequence as shown in SEQ ID No:5, costimulatory signal conducting region is 4-1BB egg
The costimulatory signal conducting region of white molecule, for amino acid sequence as shown in SEQ ID No:6, T cell signal transduction area is CD3 ζ egg
The signal transduction area of white molecule, amino acid sequence is as shown in SEQ ID No:7.
2. recombination Chimeric antigen receptor as described in claim 1, it is characterised in that the photo-induction guiding element is located at antigen binding
Between area and extracellular hinge area, between extracellular hinge area and transmembrane structure area, transmembrane structure area and costimulatory signal conduction
Between area, between costimulatory signal conducting region between T cell signal transduction area or after T cell signal transduction area.
3. recombination Chimeric antigen receptor as claimed in claim 2, it is characterised in that the photo-induction guiding element is located at T cell signal
After conducting region, end is connected with luminous effect element, and the luminous effect element is human albumin.
4. recombination Chimeric antigen receptor as claimed in claim 3, it is characterised in that the luminous effect element amino acid sequence is such as
Shown in SEQ ID No:2.
5. recombination Chimeric antigen receptor according to any one of claims 1-4, it is characterised in that antigen binding domain, extracellular hinge
Sequence, transmembrane structure area, costimulatory signal conducting region, each functional domain in T cell signal transduction area and photo-induction guiding element can
To be connected directly, each functional domain can also be connected by link peptide.
6. recombination Chimeric antigen receptor as claimed in claim 5, it is characterised in that the amino acid sequence of the link peptide such as SEQ
Shown in ID No:25-26.
7. recombination Chimeric antigen receptor as described in claim 1, it is characterised in that the recombination Chimeric antigen receptor amino
Acid sequence is as shown in SEQ ID No:8-12.
8. a kind of recombination Chimeric antigen receptor gene with photo-induction guiding element, it is characterised in that coding such as claim 1-7 appoints
Recombination Chimeric antigen receptor described in one, including antigen binding domain, extracellular hinge area, transmembrane structure area, costimulatory signal
The gene order of conducting region and T cell signal transduction area functional domain, at the both ends of recombination Chimeric antigen receptor gene order
Or photoinduction element genes are inserted between each functional domain gene order, the photo-induction guiding element is LOV2, gene order
As shown in SEQ ID No:13, the photoinduction element genes are located between antigen binding domain and extracellular hinge area gene, are thin
Between extracellular hinge area and transmembrane structure area gene, between transmembrane structure area and costimulatory signal conducting region gene, costimulation letter
Between number conducting region between T cell signal transduction area gene or after T cell signal transduction area gene, the antigen binding
Area is CD19 scFv, and nucleotide sequence is as shown in SEQ ID No:15, extracellular hinge area is CD8 hinge area, nucleotide sequence
As shown in SEQ ID No:16, transmembrane structure area be the transmembrane structure CD8 area, nucleotide sequence as shown in SEQ ID No:17, altogether
Stimulus signal conducting region is 4-1BB costimulatory signal conducting region, and nucleotide sequence is as shown in SEQ ID No:18, T cell signal
Conducting region is CD3 ζ signal transduction area, and nucleotide sequence is as shown in SEQ ID No:19.
9. recombination Chimeric antigen receptor gene as claimed in claim 8, it is characterised in that the photoinduction element genes are located at T
After cellular signal transduction area gene, end is connected with luminous effect element genes, the luminous effect element genes sequence such as SEQ
Shown in ID No:14.
10. recombination Chimeric antigen receptor gene as claimed in claim 8 or 9, it is characterised in that antigen binding domain, extracellular hinge
Sequence, transmembrane structure area, costimulatory signal conducting region, each functional structure domain gene in T cell signal transduction area and photoinduction member
Part gene can be connected directly, and can also connect each functional domain by connection peptide gene.
11. recombination Chimeric antigen receptor gene as claimed in claim 10, it is characterised in that the nucleotides sequence of the link peptide
Column are as shown in SEQ ID No:27-28.
12. recombination Chimeric antigen receptor gene as claimed in claim 8, it is characterised in that the recombination Chimeric antigen receptor
Gene order is as shown in SEQ ID No:20-24.
13. expressing recombinant vector, expression cassette or the cell with the recombination Chimeric antigen receptor of photo-induction guiding element, it is characterised in that
Gene containing the described in any item recombination Chimeric antigen receptors of such as claim 8-12.
14. expression as claimed in claim 13 has the recombinant vector of the recombination Chimeric antigen receptor of photo-induction guiding element, expression
Frame or cell, it is characterised in that the cell is selected from self or transgenosis T cell, NK cell, CIK cell.
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