CN106279438A - Novel chimeric antigen receptor and application thereof - Google Patents
Novel chimeric antigen receptor and application thereof Download PDFInfo
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- CN106279438A CN106279438A CN201610720266.2A CN201610720266A CN106279438A CN 106279438 A CN106279438 A CN 106279438A CN 201610720266 A CN201610720266 A CN 201610720266A CN 106279438 A CN106279438 A CN 106279438A
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Abstract
The invention discloses the restructuring Chimeric antigen receptor with photoinduction element, including antigen binding domain, transmembrane structure district, costimulatory signal conducting region and T cell signal conducting region functional domain, at the restructuring N end of Chimeric antigen receptor, between C end or each functional domain, insert photoinduction element.The present invention uses molecular cloning method that photoinduction element LOV2 gene is cloned into plasmid Lenti EF1 α CD19CAR and builds the coexpression vector of the restructuring Chimeric antigen receptor with photoinduction element, utilizes 293T cell packaging to obtain the slow virus carrier with photoinduction CAR molecule.After restructuring Chimeric antigen receptor of the present invention is expressed in T cell by slow virus carrier, the killing toxicity of T cell is provided important channel by the reversible control of LOV2, the safety improving clinical anticancer for CAR T.
Description
Technical field
The present invention relates to biomedical conversion field, the restructuring Chimeric antigen receptor that particularly relates to there is photoinduction element and
Its encoding gene and application.
Background technology
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 eliminating internal senile cell and the cell undergone mutation, some mutant cells can become cancerous cell.Cancer immunotherapy
Being exactly based on artificial intervention, cancerous cell is killed and suppresses it to breed by the immune system transferring body self.
Adoptive cellular immunotherapy (adoptive cellular immunotherapy, ACI) refers to Activation In Vitro
Autologous or alloimmune cell infusion to patient, to kill tumor cell in the patient, be treatment malignant tumor at present
One of important means, has obtained good therapeutic effect in the clinical treatment of multiple solid tumor and neoplastic hematologic disorder.Wherein, chimeric antigen is subject to
Body (chimeric antigen receptor, CAR) T cell therapy is very fast a kind of new thin of development in recent years
Born of the same parents' immunotherapy techniques, modifies effector T cell by technique for gene engineering, overcomes tumor by local immunosuppressant microenvironment and host
Immune tolerance state, improves antineoplastic targeting, lethal and persistency.
CAR structure is made up of born of the same parents' exoantigen land, transmembrane structure district and intracellular signal transduction district.Born of the same parents' exoantigen land
The mainly heavy chain (VL) of antigenic specificity monoclonal antibody strand and the variable region of light chain (VH) forms, and the two is with hinge region even
Connect formation single-chain antibody (single chain fragment varible, scFv).CAR is by identifying the scFv of tumor antigen
With intracellular signal territory " ITAM (immunoreceptor tyrosine-based activation
Motifs, ITAM) " carry out gene recombinaton in vitro, modified the T lymphocyte of patient by Gene transfer techniques, make patient T
Expressions In Lymphocytes tumor antigen receptor, the T lymphocyte after purification and extensive amplification are modified, referred to as chimeric antigen is subject to
The T lymphocyte (CAR-T) that body is modified.
CAR technology achieve in the treatment of hematological system tumor and solid tumor clinically 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.).Have evolved to the third generation at present.First generation CAR is by strand
Antibody is connected with intracellular signal transduction district (ITAM) by transmembrane domain, and ITAM is usually CD3 ζ or Fc ε RI γ；The second filial generation
The intracellular signal transduction district of CAR introduces costimulatory molecules (costimulatory molecule, CM), and predominantly CD28 divides
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 great majority test is at cell amplification, survival time in vitro, cytokine secretion
Etc. aspect there is also deficiency, be not reaching to intended clinical effectiveness.Research shows, the activation completely of T lymphocyte depends on double
Signal and the effect of cytokine.Wherein the first signal is specific signals, TCR identify the antigen on antigen presenting cell surface
Peptide-MHC complex is started；Secondary signal is costimulatory signal, by important costimulatory moleculeses such as CD28/B7, promotes
IL-2 synthesizes, and makes T lymphocyte fully activate and avoid apoptosis.Even if T lymphocyte and antigen contact, without collaborative
Stimulus signal, cell is difficult to function of bringing into normal play.Accordingly, the Chimeric antigen receptor of CD3 ζ sequence is contained only, without association
With stimulus signal 2, it is also difficult to efficiently activate CAR-T lymphocyte.Therefore, according to the dual signal theory of T lymphocyte activation, the
Secondary and third generation CAR adds such as the costimulatory moleculeses such as CD28, CD137, to improve T lymphocyte in Chimeric antigen receptor
Cytotoxicity, proliferation activity, maintain T lymphocyte responses, extends the T lymphocyte time-to-live etc..Research confirms the second filial generation
CAR-T lymphocyte is superior to the first generation at tumor killing activity and internal time-to-live.At present, third generation CAR-T lymph is thin
Born of the same parents' clinical practice is the most fewer, therefore its safety and effectiveness are the most necessarily better than second filial generation CAR-T lymphocyte, Yi Jixuan
Select what kind of costimulatory molecules combination, in addition it is also necessary to further look at.
Although CAR-T targeting therapy on tumor cell is one of the most promising current modality of cancer treatment, but it has
The non-tumor of targeting " effect of missing the target " (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-
851) and " cytokine storm " (Wrzesinski C, Paulos CM, Kaiser A, et al.Increased
intensity lymphodepletion enhances tumor treatment efficacy of adoptively
Transferred tumor-specific T cells ［ J ］. J Immunother, 2010,33 (1): 1-7) poison
Property effect.The activity increasing CAR-T cell " switchs " important content controlling to be to solve CAR-T toxicity problem, is facing for CAR-T
The popularization and application of bed are significant.Therefore, the reversible light-operated clinical conversion to CAR-T of CAR-T killing activity has long-pending
The progradation of pole.
Summary of the invention
It is an object of the invention to provide restructuring Chimeric antigen receptor and the encoding gene thereof of photoinduction element regulation and control.
For solving above-mentioned technical problem, the concrete technical scheme of the present invention is as follows: have the restructuring inosculating antibody of photoinduction element
Original receptor, including antigen binding domain, transmembrane structure district, costimulatory signal conducting region and T cell signal conducting region functional domain,
At the restructuring N end of Chimeric antigen receptor, between C end or each functional domain, insert photoinduction element.
Restructuring Chimeric antigen receptor of the present invention, photoinduction element is quick selected from cryptochrome (CRY), ultraviolet and blue light
One or more in sense flavoprotein (LOV and BLUF) and phytochrome (PHY), further preferred arabidopsis (Oryza sativa L., really
Fly, Fructus Lycopersici esculenti, Fructus Hordei Vulgaris, fern, lichen, algae, Africa xenopus, mice, people etc.) CRY, the LOV2 of arabidopsis (Herba bromi japonici etc.), plant
The PHY of thing.More preferably derive from the LOV2(aa 404-546 of Avena sativa Phototropin 1, NPH1-of Herba bromi japonici
1, GenBank:AAC05083.1) its aminoacid sequence is as shown in SEQ ID No:1.
Under illumination condition, owing to being caused the restructuring Chimeric antigen receptor containing photoinduction element to be able to table by light stimulation
Reach, reach the "ON" effect of light-operated property；Under the conditions of lucifuge, do not possesses the expression condition of restructuring Chimeric antigen receptor to realize light
The "Off" effect of control property.
Restructuring Chimeric antigen receptor of the present invention, described photoinduction element may be located at restructuring Chimeric antigen receptor
Between N end, C end or each functional domain, i.e. it is positioned at the N end (before antigen binding domain) of restructuring Chimeric antigen receptor, antigen
Between land and transmembrane structure district, between transmembrane structure district and costimulatory signal conducting region, between costimulatory signal conducting region
And between T cell signal conducting region or C end (after T cell signal conducting region).
Restructuring Chimeric antigen receptor of the present invention, described antigen binding domain functional domain is selected from people's antibody, Ren Yuan
Change antibody, Fab or a combination thereof, can be complete antibody, Fab, Fab', (Fab') 2, Fv fragment or strand
Variable region fragment scFv.
Preferably, antigen binding domain of the present invention functional domain combines tumor antigen.Described tumor be carcinoma of prostate,
Renal cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia (chronic lymphocytic leukemia, acute lymphoblastic
Cell leukemia, small lymphocyte leukemia, acute myeloid leukemia, multiple myeloma, adenocarcinoma, colorectal cancer,
Breast carcinoma, solid tumor, incidence cancer, glioblastoma multiforme, neuroblastoma, sarcoma, metastatic carcinoma, pleomorphism colloid are female thin
Born of the same parents' tumor etc..
Preferably, described tumor 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 its combination in any.
Preferably, costimulatory signal conducting region of the present invention is selected from CD27, CD28, CD137 (4-1BB), CD134
(OX40), the costimulatory signal conduction of CD30, CD40, PD-1, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 protein molecular
Part that district is specific binding with CD3 ζ or its combination in any.
Preferably, transmembrane structure of the present invention district is selected from the transmembrane structure district of CD3, CD4, CD8 or CD28 protein molecular.
Preferably, T cell signal conducting region functional domain of the present invention selected from CD28, CD137, Fc ε RI γ,
One or more in the T cell signal conducting region of ZAP70 or CD3 ζ protein molecular.T cell signal conducting region includes immunity
Receptor tyrosine activation motifs.
Restructuring Chimeric antigen receptor of the present invention, also has extracellular hinge between antigen binding domain and transmembrane structure district
Sequence, it is preferred that hinge region, described extracellular is the hinge region of CD8 protein molecular or immunoglobulin IgG hinge region or its group
Close.Photoinduction element may be located between antigen binding domain and hinge region, extracellular, hinge region, extracellular and transmembrane structure district it
Between, between transmembrane structure district and costimulatory signal conducting region, between costimulatory signal conducting region and T cell signal conducting region or
After T cell signal conducting region.
One preferred scheme of the present invention, time after photoinduction element is positioned at T cell signal conducting region, its end can
Having photo effect element, preferably photo effect element with connection is human albumin, and aminoacid sequence is as shown in SEQ ID No:2.
The antigen binding domain of restructuring Chimeric antigen receptor of the present invention, hinge region, extracellular, transmembrane structure district, altogether thorn
Energizing signal conducting region, each functional domain of T cell signal conducting region and photoinduction element can be joined directly together, i.e. by clone
During gene, addition restriction enzyme site or fusion DNA vaccine technology are expressed after being connected by these elements.Connection peptides can also be passed through
Connect each functional domain.The preferably flexible peptide of connection peptides or rigidity peptide, preferably flexible peptide refers to 1 ~ 20 rich in Gly and/or Ala
And/or the connection peptides of Ser amino acid residue, preferably rigidity peptide refer to that keeping forming α spiral before and after connection peptides stablizes secondary structure
Connection peptides.
The preferred version of the present invention, photoinduction components upstream is subject to restructuring chimeric antigen by flexible peptide, preferably connection peptides 1
Under the antigen binding domain of body, hinge region, extracellular, transmembrane structure district, costimulatory signal conducting region or T cell signal conducting region
Trip is connected, and the aminoacid sequence of connection peptides 1 is as shown in SEQ ID No:25.Photoinduction member downstream passes through rigidity peptide, preferably connects
Connect peptide 2 and the restructuring antigen binding domain of Chimeric antigen receptor, hinge region, extracellular, transmembrane structure district, costimulatory signal conducting region,
The upstream of T cell signal conducting region or photo effect element is connected, and the aminoacid sequence of connection peptides 2 is as shown in SEQ ID No:26.
One preferred scheme of the present invention, described restructuring Chimeric antigen receptor, photoinduction element is LOV2, and antigen is tied
Conjunction district is CD19 scFv, hinge region, extracellular is CD8 hinge region, transmembrane structure district is CD8 transmembrane structure district, costimulatory signal
Conducting region be 4-1BB costimulatory signal conducting region, T cell signal conducting region be CD3 ζ signal conducting region.When photoinduction element position
Time after T cell signal conducting region, photo effect element is human albumin.
One preferred scheme of the present invention, described restructuring Chimeric antigen receptor, CD19 scFv aminoacid 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 CD8 transmembrane structure
As shown in SEQ ID No:5,4-1BB costimulatory signal conducting region aminoacid sequence is as shown in SEQ ID No:6, photoinduction element
LOV2 aminoacid sequence such as SEQ ID No:1, CD3 ζ signal conducting region aminoacid sequence is as shown in SEQ ID No:7.Work as photo-induction
When guiding element is positioned at after T cell signal conducting region, photo effect element aminoacid sequence is as shown in SEQ ID No:2.
One preferred scheme of the present invention, described restructuring Chimeric antigen receptor aminoacid sequence such as SEQ ID No:8-
Shown in 12.
Restructuring Chimeric antigen receptor corresponding for SEQ ID No:8 is: CD19 scFv-connection peptides 1-LOV2-connection peptides 2-
-CD8 transmembrane structure, CD8 hinge region district-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor corresponding for SEQ ID No:9 is: CD19 scFv-CD8 hinge region-connection peptides 1-LOV2-
Connection peptides 2-CD8 transmembrane structure district-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor corresponding for SEQ ID No:10 is :-CD8 transmembrane structure, CD19 scFv-CD8 hinge region
District-connection peptides 1-LOV2-connection peptides 2-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor corresponding for SEQ ID No:11 is :-CD8 transmembrane structure, CD19 scFv-CD8 hinge region
District's-4-1BB costimulatory signal conducting region-connection peptides 1-LOV2-connection peptides 2-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor corresponding for SEQ ID No:12 is :-CD8 transmembrane structure, CD19 scFv-CD8 hinge region
District-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region-connection peptides 1-LOV2-connection peptides 2-human albumin.
Another object of the present invention is to provide the base encoding the above-mentioned restructuring Chimeric antigen receptor with photoinduction element
Cause, including antigen binding domain, transmembrane structure district, costimulatory signal conducting region and the base of T cell signal conducting region functional domain
Because of sequence, between two ends or each functional domain gene order of restructuring Chimeric antigen receptor gene order, insert photoinduction
Element genes.
Described photoinduction element is selected from cryptochrome (CRY), ultraviolet and sensitive to blue light flavoprotein (LOV and BLUF) and light
One or more in quick pigment (PHY), further preferred arabidopsis (Oryza sativa L., fruit bat, Fructus Lycopersici esculenti, Fructus Hordei Vulgaris, fern, lichen, algae
Class, Africa xenopus, mice, people etc.) CRY, the LOV2 of arabidopsis (Herba bromi japonici etc.), the PHY of plant.More preferably derive from Herba bromi japonici
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.
Restructuring Chimeric antigen receptor of the present invention, described antigen binding domain gene selected from people's antibody, humanized antibody,
The gene of Fab or a combination thereof, can be complete antibody, Fab, Fab', (Fab') 2, Fv fragment or strand
Variable region fragment scFv gene.
Preferably, antigen binding domain of the present invention functional domain combines tumor antigen.Described tumor be carcinoma of prostate,
Renal cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia (chronic lymphocytic leukemia, acute lymphoblastic
Cell leukemia, small lymphocyte leukemia, acute myeloid leukemia, multiple myeloma, adenocarcinoma, colorectal cancer,
Breast carcinoma, solid tumor, incidence cancer, glioblastoma multiforme, neuroblastoma, sarcoma, metastatic carcinoma, pleomorphism colloid are female thin
Born of the same parents' tumor etc..Preferably, described tumor 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 its combination in any.
Preferably, costimulatory signal conducting region gene of the present invention is selected from CD27, CD28, CD137 (4-1BB), CD134
(OX40), the costimulatory signal conduction of CD30, CD40, PD-1, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 protein molecular
Ligand gene that district's gene is specific binding with CD3 ζ or its combination in any.
Preferably, transmembrane structure of the present invention district gene is selected from the cross-film knot of CD3, CD4, CD8 or CD28 protein molecular
Structure district gene.
Preferably, T cell signal conducting region functional domain of the present invention selected from CD28, CD137, Fc ε RI γ,
One or more in the T cell signal conducting region gene of ZAP70 or CD3 ζ protein molecular.
One preferred scheme of the present invention, described restructuring Chimeric antigen receptor gene, at antigen binding domain gene and
Between transmembrane structure district gene also have hinge region, extracellular gene, hinge region, described extracellular gene, preferably CD8 protein molecular
Hinge region or the gene of immunoglobulin IgG hinge region or a combination thereof.Photoinduction element genes may be located at antigen binding domain with
Between the gene of hinge region, extracellular, between hinge region, extracellular and transmembrane structure district gene, transmembrane structure district and costimulatory signal
Between conducting region gene, between costimulatory signal conducting region and T cell signal conducting region gene or T cell signal conducting region base
Because of afterwards.
One preferred scheme of the present invention, time after photoinduction element genes is positioned at T cell signal conducting region gene,
Its end can connect photo effect element genes, the described preferred human albumin of photo effect element, nucleotide sequence such as SEQ ID
Shown in No:14.
The preferred version of the present invention, photoinduction element genes upstream is by connection peptides 1 gene and restructuring Chimeric antigen receptor
Antigen binding domain, hinge region, extracellular, transmembrane structure district, costimulatory signal conducting region or T cell signal conducting region gene
Downstream is connected, and the nucleotide sequence of connection peptides 1 is as shown in SEQ ID No:27.Connection peptides 2 is passed through in photoinduction element genes downstream
Gene and the restructuring antigen binding domain of Chimeric antigen receptor, hinge region, extracellular, transmembrane structure district, costimulatory signal conducting region, T
The upstream of cellular signal transduction district or photo effect element genes is connected, and the nucleotide of connection peptides 2 arranges as shown in SEQ ID No:28.
One preferred version of the present invention, described restructuring Chimeric antigen receptor gene, antigen binding domain is CD19
ScFv, nucleotide sequence as shown in SEQ ID No:15, hinge region, extracellular be CD8 hinge region, nucleotide sequence such as SEQ ID
No:16 is shown, transmembrane structure district is CD8 transmembrane structure district, and nucleotide sequence is as shown in SEQ ID No:17, costimulatory signal biography
Leading district is 4-1BB costimulatory signal conducting region, nucleotide sequence as shown in SEQ ID No:18, T cell signal conducting region be CD3
ζ signal conducting region, nucleotide sequence is as shown in SEQ ID No:19.After photoinduction element is positioned at T cell signal conducting region
Time, photo effect element nucleotide sequence is as shown in SEQ ID No:14.One preferred scheme of the present invention, described restructuring is embedding
Close antigen receptor nucleotide sequence as shown in SEQ ID No:20-24.
Restructuring Chimeric antigen receptor gene corresponding for SEQ ID No:20 is: CD19 scFv-connection peptides 1-LOV2-connects
-CD8 transmembrane structure, peptide 2-CD8 hinge region district-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor gene corresponding for SEQ ID No:21 is: CD19 scFv-CD8 hinge region-connection peptides 1-
LOV2-connection peptides 2-CD8 transmembrane structure district-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor gene corresponding for SEQ ID No:22 is: CD19 scFv-CD8 hinge region-CD8 cross-film
Structural area-connection peptides 1-LOV2-connection peptides 2-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor gene corresponding for SEQ ID No:23 is: CD19 scFv-CD8 hinge region-CD8 cross-film
Structural area-4-1BB costimulatory signal conducting region-connection peptides 1-LOV2-connection peptides 2-CD3 ζ signal conducting region.
Restructuring Chimeric antigen receptor gene corresponding for SEQ ID No:24 is: CD19 scFv-CD8 hinge region-CD8 cross-film
Structural area-4-1BB costimulatory signal conducting region-CD3 ζ signal conducting region-connection peptides 1-LOV2-connection peptides 2-human albumin.
Another object of the present invention is to provide the expression restructuring chimeric antigen with photoinduction element of the present invention to be subject to
The recombinant vector of body, expression cassette, slow virus carrier or cell, containing above-mentioned restructuring Chimeric antigen receptor gene.
One preferred version of the present invention, expression vector is Lenti-EF1 α, and recombinant vector is Lenti-liCAR, specifically
For Chimeric antigen receptor sequence of recombinating, nucleotide sequence, as shown in SEQ ID any one of No:20-24, inserts expression vector
Carrier obtained by between BamH I and the EcoR I restriction enzyme site of Lenti-EF1 α.
One preferred version of the present invention, described cell is selected from autologous or the T cell of transgenic, 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 restructuring Chimeric antigen receptor preparation method with photoinduction element, it may include
Following steps:
1) structure of coexpression vector
Use molecular cloning method that photoinduction element LOV2 gene is cloned into plasmid Lenti-EF1 α-CD19CAR
(CD3ZetaQQ) coexpression vector of the restructuring Chimeric antigen receptor with photoinduction element is built；
2) packaging of slow virus carrier
293T cell packaging is utilized to obtain the slow virus carrier with photoinduction CAR molecule.Described slow virus carrier is by above-mentioned
Coexpression vector and the slow virus necessary structural protein expression plasmid corotation of packaging efficiently assemble gained in the cell of slow virus
There is the slow virus expressing recombination.One preferred version of the present invention, by above-mentioned coexpression vector and slow virus structure egg
White expression vector pGP and pVSVG jointly transfects the cultivation of HEK 293T cell and obtains slow virus carrier；
3) lentivirus mediated has the restructuring Chimeric antigen receptor transfectional cell of photoinduction element.
The method that the present invention provides comprises the steps: to build genes of interest slow virus package carrier, packs slow virus, slowly
Virus-mediated T cell transgenic, it is special that recombinant C AR-T cell has reversible photocontrol to the specific killing activity of target cell
Property, the most only there is under illumination condition killing ability.
The invention have the advantages that
The invention provides a kind of restructuring Chimeric antigen receptor with photoinduction element, it is demonstrated experimentally that this recombiant protein passes through
After slow virus carrier is expressed in T cell, the killing toxicity of T cell is by the reversible control of LOV2.Of the present invention have
The restructuring Chimeric antigen receptor of photoinduction element maintains the targeting of CAR-T and anti-personnel increases these two kinds of characteristics simultaneously
Controlling switch, the safety improving clinical anticancer for CAR-T provides important channel.
Accompanying drawing explanation
Fig. 1 is the restructuring Chimeric antigen receptor structural representation with photoinduction element of the present invention.
Fig. 2 be transfect the restructuring Chimeric antigen receptor with photoinduction element of the present invention Jurkat-CAR5～
Jurkat-CAR9 cytoactive photoinduction controls result of study.
Fig. 3 Jurkat-CAR5 cellkilling capacity experiment knot of different effect target ratios under the conditions of illumination and lucifuge respectively
Really.
Fig. 4 Jurkat-CAR6 cellkilling capacity experiment knot of different effect target ratios under the conditions of illumination and lucifuge respectively
Really.
Fig. 5 Jurkat-CAR7 cellkilling capacity experiment knot of different effect target ratios under the conditions of illumination and lucifuge respectively
Really.
Fig. 6 Jurkat-CAR8 cellkilling capacity experiment knot of different effect target ratios under the conditions of illumination and lucifuge respectively
Really.
Fig. 7 Jurkat-CAR9 cellkilling capacity experiment knot of different effect target ratios under the conditions of illumination and lucifuge respectively
Really.
Detailed description of the invention
The concrete steps of the present invention are described by the following examples, but are not limited by the example.
The term used in the present invention, except as otherwise noted, typically has those of ordinary skill in the art and generally manages
The implication solved.
Below in conjunction with specific embodiment and with reference to data, the present invention is described in further detail.Should be understood that this embodiment is simply
In order to demonstrate the invention, rather than limit the scope of the present invention by any way.
In the examples below, the various processes not described in detail and method are conventional methods as known in the art.
Below in conjunction with specific embodiment, the present invention is further described.
Material used in following example, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1, there is the design of the restructuring Chimeric antigen receptor of photoinduction element
The present invention selects clinical effectiveness preferable CD19-CAR gene, and composed component is as follows: CD19 scFv, CD8
hinge、CD8 transmembrane、4-1BB intracellular、CD3ζ；
LOV2(aa 404-546 of used by the present inventionAvena sativaPhototropin 1, NPH1-1, GenBank:
AAC05083.1) derive from Herba bromi japonici (Avena sativa), according to people (Homo spaice) codon preference carries out codon
Optimizing, aminoacid sequence is as shown in SEQ ID No:1, and nucleotide sequence is as shown in SEQ ID No:13.
LOV2 is inserted respectively into the antigen binding domain of CD19-CAR, transmembrane structure district, costimulatory signal conducting region and T thin
Before and after born of the same parents' signal conducting region functional domain or between, specific as follows:
LiCAR5:CD19 scFv-connection peptides 1-LOV2-connection peptides 2-CD8 hinge region-CD8 transmembrane structure district-4-1BB stimulates altogether
Signal conducting region-CD3 ζ signal conducting region (aminoacid sequence as shown in SEQ ID No:8, nucleotide sequence such as SEQ ID No:
Shown in 20).
District-4-1BB is common in liCAR6:CD19 scFv-CD8 hinge region-connection peptides 1-LOV2-connection peptides 2-CD8 transmembrane structure
Stimulus signal conducting region-CD3 ζ signal conducting region (aminoacid sequence as shown in SEQ ID No:9, nucleotide sequence such as SEQ ID
Shown in No:21).
-CD8 transmembrane structure district, liCAR7:CD19 scFv-CD8 hinge region-connection peptides 1-LOV2-connection peptides 2-4-1BB is altogether
Stimulus signal conducting region-CD3 ζ signal conducting region (aminoacid sequence as shown in SEQ ID No:10, nucleotide sequence such as SEQ
Shown in ID No:22).
-CD8 transmembrane structure district, liCAR8:CD19 scFv-CD8 hinge region-4-1BB costimulatory signal conducting region-connection
Peptide 1-LOV2-connection peptides 2-CD3 ζ signal conducting region (aminoacid sequence as shown in SEQ ID No:11, nucleotide sequence such as SEQ
Shown in ID No:23).
-CD8 transmembrane structure, liCAR9:CD19 scFv-CD8 hinge region district-4-1BB costimulatory signal conducting region-CD3 ζ
Signal conducting region-connection peptides 1-LOV2-connection peptides 2-human albumin (aminoacid sequence as shown in SEQ ID No:12, nucleotide
Sequence is as shown in SEQ ID No:24).
Connection peptides 1 aminoacid sequence is as shown in SEQ ID No:25, and nucleotide sequence is as shown in SEQ ID No:27；Connect
Peptide 2 aminoacid sequence is as shown in SEQ ID No:26, and nucleotide sequence is as shown in SEQ ID No:28.
Embodiment 2, there is the expression of the restructuring Chimeric antigen receptor of photoinduction element
One, coexpression vector Lenti-liCAR5, Lenti-liCAR6, Lenti-liCAR7, Lenti-liCAR8 and Lenti-
The structure of liCAR9
Lenti-EF1 α-CD19CAR(CD3ZetaQQ) purchased from Ai Kang get biomedical technology (Suzhou) company limited (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 according to the optimization of people's codon preference
(SEQ ID No:13) is template, separately designs primer as follows:
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；
With plasmid Lenti-EF1 α-CD19CAR as template, by fusion DNA vaccine (using NEB company Q5 high-fidelity DNA polymerase)
Method amplification obtain liCAR5, liCAR6, liCAR7, liCAR8 and liCAR9 nucleotide molecule fragment.Added by primer
The method adding restricted enzyme adds EcoRI and BamHI restriction enzyme site at two sections of purpose fragment respectively.
PCR condition:
First round PCR: denaturation 98 ° of C, 30s；Degeneration 98 ° of C, 10s, anneal 58 ° of C, 30s, extends 72 ° C, 45s, 30 circulation；
Extend 72 ° of C, 2min eventually；Lower the temperature 4 ° of C.This is taken turns PCR primer and jointly adds response system as template and upstream and downstream primer, enter
Row second takes turns PCR: denaturation 98 ° of C, 30s；Degeneration 98 ° of C, 10s, anneal 58 ° of C, 30s, extends 72 ° C, 1min, 30 circulation；
Extend 72 ° of C, 2min eventually；Lower the temperature 4 ° of C.With melting of EcoRI with BamHI double digestion plasmid Lenti-EF1 α-CD19CAR and recovery
Closing PCR primer (for each nucleotide molecule fragment of liCAR), plasmid reaction system is the plasmid of 30ul(1ul, 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 reclaims product reaction system 30 μ l(20 μ l's
PCR reclaims 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 ° of C water-bath 30min.
Reaction terminates with 1% agarose gel electrophoresis analysis, is separately recovered purpose fragment.With ligase, purpose fragment is cloned into plasmid
In carrier section, reaction system is the carrier of 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 ° of C connect overnight.Linked system is put into 70 ° of C water-bath inactivator 10min, puts cooled on ice.Connection product is turned
Change DH5 α competent cell, through ice bath 30min, 42 ° of C water-bath heat shocks 90s, put 5min on ice the most immediately, add LB liquid
After culture medium shakes bacterium, bacterium solution is coated in ampicillin (Amp+) resistance LB flat board on, 37 ° of C incubators are inverted and are cultivated
Overnight.Observe colony growth situation on flat board next day.On picking flat board, the monoclonal of Amp screening, carries out bacterium colony PCR qualification, uses
1% agarose gel electrophoresis analysis, filters out amplified band monoclonal in line bacterium colony.The monoclonal filtered out is expanded
Big cultivation, it is that 15% glycerol stock is in-80 ° of C refrigerators that next day preserves a part of bacterium solution.Residue bacterium solution plasmid DNA extracts examination on a small quantity
Agent box extracts plasmid.The plasmid of extraction is carried out DNA sequencing qualification.Identified satisfactory containing Chimeric antigen receptor gene
The lentivirus transfer carrier of fragment, is respectively designated as Lenti-liCAR5, Lenti-liCAR6, Lenti-liCAR7, Lenti-
LiCAR8 and Lenti-liCAR9；Then check order qualification.Order-checking shows, each gene fragment order of coding liCAR5 and connection are just
Really, its nucleotide sequence is as shown in SEQ ID No:20, and aminoacid sequence is as shown in SEQ ID No:8；Coding liCAR6's is each
Gene fragment order and connect correct, its nucleotide sequence as shown in SEQ ID No:21, aminoacid sequence such as SEQ ID No:9
Shown in；Each gene fragment order of coding liCAR7 and connect correct, its nucleotide sequence 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 of coding liCAR8 are correct, and its nucleotide sequence is such as
Shown in SEQ ID No:23, aminoacid sequence is as shown in SEQ ID No:11；Each gene fragment order of coding liCAR9 and company
Connecing correct, its nucleotide sequence is as shown in SEQ ID No:24, and aminoacid sequence is as shown in SEQ ID No:12.
Two, the packaging of slow virus carrier Lentivial
The HEK 293T cell of cultivation is inoculated in the 100mm culture dish that poly-D-lysine processes, treats that cell degrees of fusion reaches
During 70%-80%, change serum-free DMEM culture fluid, 37 ° of C, 5% CO2Cultivate.After 2h, pack slow virus with three plasmid calcium phosphate methods,
As a example by Lenti-liCAR5, it specifically comprises the following steps that
Slow virus carrier Lenti-liCAR5 plasmid 30 μ g, the pGP of 20 μ g is added in 1 10ml centrifuge tube, 10 μ g's
PVSVG, 2 mol/LCaCl2124 ul, add distilled water completion to 1mL, then add 1mL 2 × Hank, fully mix, and room temperature is quiet
Put 20min, be then added dropwise in above-mentioned 100mm culture dish, 37 ° of C, 5% CO2Cultivate and within 6-8 hour, change fresh 5%FBS's
DMEM culture medium continues to cultivate.Observe under inverted microscope transfection 24,48, cell growth condition after 72h collecting every day
Supernatant.It is centrifuged off cell debris, filters with 0.45 μm filter.After filtration, vial supernatant is moved into Millipore and concentrates
Post, centrifugal 30min, it is concentrated into 100 ~ 200uL, the virus liquid that will concentrate moves into EP pipe, and-80 ° of C save backup (or by virus
Supernatant uses supercentrifuge to be centrifuged supernatant discarded after 2h with 4 ° of C, 7000 × g, with the resuspended virion of Opti-MEM ,-80 ° of C
Save backup).
The Lenti-liCAR6-9 of the packaging of slow virus carrier Lentivial is obtained respectively with reference to said method.
Three, lentivirus mediated photoinduction CAR transfected Jurkat cells.
By Jurkat cell good for growth conditions with 5 × 105The density in/hole is inoculated in 6 orifice plates, adds serum-free
RPMI1640 culture medium, to 2 ml, adds 2ul 8 μ g/L Polybrene, adds 100 μ l purified virus liquid, and mixing, in 37 °
C、5% CO2Cultivate.Cultivating to 8h, the RPMI1640 changing 10%FBS into cultivates, and observes fluorescence after 48h.Respectively by containing of obtaining
There is the supernatant of slow virus carrier Lenti-liCAR5-9 after centrifugal purification, infect Jurkat cell Secondary Culture, add 100
Untis/mL IL-2 promotes cell proliferation and differentiation, it is thus achieved that Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-
CAR8, Jurkat-CAR9.
Embodiment 3, have photoinduction element restructuring Chimeric antigen receptor T cell killing activity research
Inoculate 100 μ l 1 × 104The target cell (leukaemia Raji) in/hole, in 96 porocyte culture plates, imitates target according to 2:1
Ratio is separately added into Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 cell (table
Reach the cell of the Jurkat of liCAR5-9), mend culture fluid to 200 μ l, (LED, 465nm, 30 μm ol under illumination condition respectively
m-2 s-1, expose 4h) and lucifuge under the conditions of 37 ° of C, 5% CO2Incubator co-cultures 48h.
(1) experiment one
After co-culturing 48h, collect suspension culture respectively and collect supernatant through centrifugal, use human IL-2 ELISA kit inspection
Surveying IL-2 content, concrete steps see 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 is under illumination condition
IL-2 activity is similar to classical CAR-T cell, but under the conditions of lucifuge, significance is less than under each comfortable illumination condition and classical
The activity of CAR-T.Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8 that the present invention designs are described,
Jurkat-CAR9 cytoactive is by photoinduction control.
(2) experiment two
After co-culturing 48h, collect suspension culture, detect cytoactive by lactic acid dehydrogenase (LDH) method for releasing.Inoculation
100μl 1x104In target cell Raji to the 96 porocyte culture plate in/hole, according to difference effect target ratio (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, mend culture fluid to 200 μ l, 37 ° of C, 5% CO under the conditions of illumination and lucifuge respectively2Incubator is trained altogether
Support 48h.After co-culturing 48h, from cell culture incubator, take out Tissue Culture Plate, add in the culture hole only inoculating Raji cell
Lactic acid dehydrogenase (LDH) releasing agent that test kit provides, addition is 10%(20 μ l of original culture fluid volume).Add LDH to release
After putting agent, repeatedly blow and beat and mix for several times, then proceed to hatch in cell culture incubator.After 1h, by cultivation cell plates porous plate
Centrifuge 400 × 0g is centrifuged 5min.Take the supernatant 120 μ l in each hole respectively, join in 96 new orifice plate respective aperture, immediately
Carry out sample determination.Each hole is separately added into the LDH of 60 μ l and detects working solution.Mixing, room temperature lucifuge is hatched 30min, is then existed
Optical density value is measured at 490nm.Detailed step sees lactic acid dehydrogenase citotoxicity detection kit operation instructions.Concrete knot
As shown in fig. 3 to 7, result shows along with effect target ratio increases fruit, the Jurkat-CAR5 of process LAN restructuring Chimeric antigen receptor,
Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 Execution promotes the most therewith.At illumination bar
Under part, the dosage effect similar to classic CD19-CAR T cell indicates these process LAN restructuring Chimeric antigen receptor
The specific killing ability of Jurkat cell does not change because of modifying；And the Jurkat-CAR5 under the conditions of lucifuge,
The killing ability more classic CAR T of Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 cell is low
Under show that its activity shows the most completely.These results indicate that photocontrol restructuring Chimeric antigen receptor of the present invention is sharp
Path of living is activated T cell killing activity by light induction.Thus, illumination controls cell killing energy in becoming CAR-T treatment
The activation condition of power.
Claims (30)
1. there is the restructuring Chimeric antigen receptor of photoinduction element, pass including antigen binding domain, transmembrane structure district, costimulatory signal
Lead district and T cell signal conducting region functional domain, it is characterised in that in the restructuring N end of Chimeric antigen receptor, C end or each merit
Photoinduction element can be inserted between domain.
Recombinate the most as claimed in claim 1 Chimeric antigen receptor, it is characterised in that described photoinduction element selected from LOV, CRYs,
One or more in BLUF or PHY.
Recombinate the most as claimed in claim 2 Chimeric antigen receptor, it is characterised in that described photoinduction element is LOV2, aminoacid
Sequence is as shown in SEQ ID No:1.
Recombinate the most as claimed in claim 1 Chimeric antigen receptor, it is characterised in that described antigen binding domain functional domain selects
From people's antibody, humanized antibody, Fab and its combination in any.
Recombinate the most as claimed in claim 1 Chimeric antigen receptor, it is characterised in that antigen binding domain functional domain be Fab,
Fab', (Fab') 2, Fv or scFv.
Recombinate the most as claimed in claim 1 Chimeric antigen receptor, it is characterised in that antigen binding domain functional domain combines swollen
Tumor antigen.
Recombinate the most as claimed in claim 6 Chimeric antigen receptor, it is characterised in that described tumor antigen is selected from CD19, CD20,
CD22, CD23, ROR1, CD30, CD56, kappa light chain, CD44, mesothelin, Penn/UCSF, NKG2D part, CD33/
IL3Ra, c-Met, PAMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGEA3TCR or its combination in any.
Recombinate the most as claimed in claim 1 Chimeric antigen receptor, it is characterised in that described costimulatory signal conducting region is selected from
CD27, CD28,4-1BB, CD134, CD30, CD40, PD-1, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 protein molecular
The costimulatory signal conducting region part specific binding with CD3 ζ or its combination in any.
Recombinate the most as claimed in claim 1 Chimeric antigen receptor, it is characterised in that described T cell signal conducting region functional structure
Territory is selected from T cell signal conducting region or its combination in any of Fc ε RI γ, ZAP70 or CD3 ζ protein molecular.
Recombinate the most as claimed in claim 1 Chimeric antigen receptor, it is characterised in that described transmembrane structure district selected from CD3, CD4,
The transmembrane structure district of CD8 or CD28 protein molecular or its combination in any.
11. recombinate Chimeric antigen receptor as claimed in claim 1, it is characterised in that in antigen binding domain and transmembrane structure district it
Between also have hinge region, extracellular.
12. recombinate Chimeric antigen receptor as claimed in claim 11, it is characterised in that hinge region, described extracellular is immunity ball
Protein I gG hinge region or the hinge region of CD8 protein molecular or a combination thereof.
13. recombinate Chimeric antigen receptor as claimed in claim 11, it is characterised in that described photoinduction element is positioned at antigen knot
Close between district and hinge region, extracellular, between hinge region, extracellular and transmembrane structure district, transmembrane structure district passes with costimulatory signal
Lead between district, between costimulatory signal conducting region and between T cell signal conducting region or after T cell signal conducting region.
14. recombinate Chimeric antigen receptor as claimed in claim 13, it is characterised in that described photoinduction element is positioned at T cell letter
After number conducting region, its end connects that to have photo effect element, described photo effect element be human albumin.
15. recombinate Chimeric antigen receptor as claimed in claim 13, it is characterised in that described photo effect element aminoacid sequence
As shown in SEQ ID No:2.
The 16. restructuring Chimeric antigen receptor as described in any one of claim 11-15, it is characterised in that antigen binding domain, cell
Outer hinge region, transmembrane structure district, costimulatory signal conducting region, each functional domain of T cell signal conducting region and photoinduction unit
Part can be joined directly together, it is also possible to connects each functional domain by connection peptides.
The Chimeric antigen receptor 17. recombinate as claimed in claim 16, it is characterised in that the aminoacid sequence of described connection peptides is such as
Shown in SEQ ID No:25-26.
The 18. restructuring Chimeric antigen receptor as described in any one of claim 11-15, it is characterised in that antigen binding domain is CD19
The scFv of protein molecular, hinge region, extracellular be the hinge region of CD8 protein molecular, transmembrane structure district be CD8 protein molecular across
Membrane structure district, costimulatory signal conducting region are that the costimulatory signal conducting region of 4-1BB protein molecular, T cell signal conducting region are
The signal conducting region of CD3 ζ protein molecular.
19. recombinate Chimeric antigen receptor as claimed in claim 18, it is characterised in that described CD19 scFv aminoacid 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 CD8 transmembrane structure
As shown in SEQ ID No:5,4-1BB costimulatory signal conducting region aminoacid sequence is as shown in SEQ ID No:6, CD3 ζ signal
Conducting region aminoacid sequence is as shown in SEQ ID No:7.
20. recombinate Chimeric antigen receptor as claimed in claim 1, it is characterised in that described restructuring Chimeric antigen receptor amino
Acid sequence is as shown in SEQ ID No:8-12.
21. 1 kinds of restructuring Chimeric antigen receptor genes with photoinduction element, it is characterised in that coding is such as claim 1-10
Restructuring Chimeric antigen receptor described in any one, thin including antigen binding domain, transmembrane structure district, costimulatory signal conducting region and T
The gene order of born of the same parents' signal conducting region functional domain, at the two ends of restructuring Chimeric antigen receptor gene order or each function knot
Photoinduction element genes is inserted between structure domain gene sequence.
22. restructuring Chimeric antigen receptor genes as claimed in claim 21, it is characterised in that described photoinduction element is LOV2,
Gene order is as shown in SEQ ID No:13.
23. restructuring Chimeric antigen receptor genes as claimed in claim 21, it is characterised in that tie in antigen binding domain and cross-film
Also having hinge region, extracellular gene between structure district, described photoinduction element genes is positioned at antigen binding domain and hinge region, extracellular
Between gene, between hinge region, extracellular and transmembrane structure district gene, transmembrane structure district and costimulatory signal conducting region gene it
Between, between costimulatory signal conducting region and between T cell signal conducting region gene or after T cell signal conducting region gene.
24. restructuring Chimeric antigen receptor genes as claimed in claim 23, it is characterised in that described photoinduction element genes position
After T cell signal conducting region gene, its end connects photo effect element genes, and described photo effect element genes sequence is such as
Shown in SEQ ID No:14.
The 25. restructuring Chimeric antigen receptor genes as described in any one of claim 21-24, it is characterised in that antigen binding domain,
Hinge region, extracellular, transmembrane structure district, costimulatory signal conducting region, T cell signal conducting region each functional structure domain gene and
Photoinduction element genes can be joined directly together, it is also possible to connects each functional domain by connection peptides gene.
The Chimeric antigen receptor 26. recombinate as claimed in claim 25, it is characterised in that the nucleotide sequence of described connection peptides is such as
Shown in SEQ ID No:27-28.
27. restructuring Chimeric antigen receptor genes as claimed in claim 23, it is characterised in that described antigen binding domain is CD19
ScFv, nucleotide sequence as shown in SEQ ID No:15, hinge region, extracellular be CD8 hinge region, nucleotide sequence such as SEQ ID
No:16 is shown, transmembrane structure district is CD8 transmembrane structure district, and nucleotide sequence is as shown in SEQ ID No:17, costimulatory signal biography
Leading district is 4-1BB costimulatory signal conducting region, nucleotide sequence as shown in SEQ ID No:18, T cell signal conducting region be CD3
ζ signal conducting region, nucleotide sequence is as shown in SEQ ID No:19.
28. restructuring Chimeric antigen receptor genes as claimed in claim 21, it is characterised in that described restructuring chimeric antigen is subject to
Body gene order is as shown in SEQ ID No:20-24.
29. expression have the recombinant vector of restructuring Chimeric antigen receptor of photoinduction element, expression cassette, slow virus carrier or thin
Born of the same parents, it is characterised in that containing the gene of Chimeric antigen receptor of recombinating as described in any one of claim 21-28.
30. recombinant vectors expressing the restructuring Chimeric antigen receptor with photoinduction element as claimed in claim 29, it is special
Levy be described cell selected from autologous or the T cell of transgenic, NK cell, cytotoxic T lymphocyte or regulatory T-cell,
Memory t cell, bispecific T cell, CIK cell.
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