CN107475276A - The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified - Google Patents
The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified Download PDFInfo
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Abstract
The T cell modified the present invention relates to a kind of Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation and its application.The Chimeric antigen receptor expressing gene includes Tet on 3G protein-encoding genes and expressing fusion protein gene.Expressing fusion protein gene includes antigen binding regions expressing gene, hinge protein expressing gene, transmembrane protein expressing gene and the intracellular signal transduction protein-encoding gene being sequentially connected.The Chimeric antigen receptor expressing gene of this innovative design can successfully import in T cell the T cell for forming Chimeric antigen receptor modification, exist in tetracycline and just produce immune response with the conditions of specific antigen, realize that Chimeric antigen receptor immune response is controllable, treatment side effect is few, and specificity is high.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of Chimeric antigen receptor and its expressing gene, tetracycline
The T cell of the Chimeric antigen receptor modification of regulation and its application.
Background technology
Chimeric antigen receptor (chimeric antigen receptor, CAR) is what artificial constructed fusion encoded
Transmembrane molecule.Generally comprise extracellular antigen binding domain, membrane spaning domain and intracellular signal transduction domain composition.CAR-T, i.e., it is embedding
Close the T cell of antigen receptor (CAR) modification.CAR-T cells are applied to treatment of cancer, it is shown that splendid curative effect and huge latent
Power.Treated as Novartis discloses on December 5th, 2015 it in ASH meetings and targets the CAR-T (CTL019) of CD19 molecules
2 phase clinical datas in terms of refractory, relapsed acute lymphatic leukemia (ALL), 2 issues are according to the 1 issue evidence with announcement in 2014
Similar, complete remission rate (CR) distribution is up to 93% (55/59) and 92% (36/39).
Traditional CARs can be largely classified into three generations, such as Fig. 1, and first generation CARs is referred to as " letter usually using a bars chain
Number 1 " (signal 1).First generation CAR-T cells show the effect of limited in clinical test, and this is probably due to transplanting
The cell death (activation-induced cell death, AICD) of t cell activation induction, or to lack long-term T thin
Born of the same parents expand.Second generation CARs adds an extra costimulatory signal domain, is referred to as by the use of first generation CARs as a kind of pillar
" signal 2 "；Therefore, identical acceptor needs to transmit " signal 1 " and " signal 2 " is to optimize the activation of T cell.Second generation CAR-T with
First generation CAR-T is compared, and the persistence fed back in patients with non Hodgkin lymphoma and amplification property enhance.But actually which
Kind is that optimal secondary signal is also to be determined.Another needs to solve the problems, such as, when more different CAR-T cell designs
When, if the threshold value of persistence and/or amplification property is necessary to producing effective clinical result.Third generation CARs signal domain bag
Two costimulation domains, preclinical study show, third generation CAR-T cells have stronger antitumor than second generation CAR-T cells
Effect.
However, CAR-T cells are applied to treatment of cancer, and while curative effect is shown, adjoint toxic and risk, even
Cause patient death.On September 26th, 2016, Kite disclose the interim analysis results of KTE-C19 II phase clinical datas.Treatment
Complete remission rate reached 47%.However, in 62 patients of this time participation experiment, there is 1/3 patient to generate serious god
Through toxicity, 18% patient receives the influence of cytokines release syndrome, and 2 people die from the related adverse reactions of KTE-C19.
Wherein, cytokine release syndrome (cytokines release syndrome, CRS) is most significant poison
Property, it is No.1 security risk.Cytokine release syndrome is the activation based on T cell, is that one of t cell activation activity is anti-
Should, so side effect is and the positively related clinical response of CAR-T therapy mechanism.The T cell of hyperproliferation can cause CRS, table
It is now high fever and myalgia, unstable low blood pressure and respiratory failure.This is a unexpected result, because preclinical dynamic
Without the similar symptom of appearance in thing model.A crucial point is found in from CRS, except expected effector cell's factor
Outside INF- γ, IL-6 can also be lifted rapidly during the cell index level propagation that CART is treated.CRS may be directly malicious with another
Property is associated, i.e. Macrophage Activation Syndrome.
Except CRS, " targeting " toxicity as caused by the antigentic specificity of engineered T cell also be present.Such as oncolytic
Syndrome, caused by it is directly the cracking by tumour cell.When CARs targets the target spot such as CD19 of B cell surface expression
When, B cell depauperation can be caused, here it is " targeting " toxicity, but the knot for having attacked normal tissue cell of mistake
Fruit.As long as CD19CAR-T cells exist for a long time, the hypogenetic situation of B cell would not improve.B cell depauperation with
The treatment of CD20 specific monoclonal antibodies equally can cause serious hypogammag lobulinemia, it is necessary to intravenous injection of immunoglobulin.Most
The nearly T cell for reporting infusion transformation triggers 2 cases of fatal toxicity, has a patient to receive HER2-CAR and treats, and two
Example patient receives targeting MAGE-A3 TCR-T cell therapies.In this 2 cases, be because normal tissue expression these
Target spot, cause acute irreversible cardiopulmonary toxicity.All targeting toxicity is due to that the T cell of transformation cannot be distinguished from expressing target
To caused by the normal cell and tumour cell of antigen.
CAR-T treatments leukaemia can cause neurological symptom, have neurotoxicity.Several research group's reports, these
Symptom has diversity but can voluntarily disappeared, such as delirium, aphasis, dyskinesia, mutism and epileptic attack.Although with it is complete
Some temporal associations of body CRS generation, also it is present in certainly to CAR-T related in cerebrospinal fluid.The mechanism of these symptoms with
Target tissue still subject to confirmation.
In addition, CAR-T cells also have potential risks applied to treatment of cancer, the T cell of such as infusion activation, which exists, to be caused
The potential risk of anti-host's transplantation disease be present in the risk of autoimmune disease, infusion allogeneic T cells.This may cause
Once the worry of the patient of allogeneic hematopoietic stem cell transplantation was received.
To sum up, there is the problems such as more treatment side effect, poor specificity in traditional Chimeric antigen receptor.
The content of the invention
Based on this, it is necessary to provide the Chimeric antigen receptor and its expressing gene that a kind for the treatment of side effect is few, specificity is high
And application.
In addition, it there is a need to the T cell that a kind of Chimeric antigen receptor modification of tetracycline regulation is provided and its application.
A kind of Chimeric antigen receptor expressing gene, including Tet-on 3G protein-encoding genes and expressing fusion protein gene,
The expressing fusion protein gene includes the antigen binding regions expressing gene, hinge protein expressing gene, cross-film being sequentially connected
Protein-encoding gene and intracellular signal transduction protein-encoding gene.
In one embodiment, the Tet-on 3G protein-encoding genes include：(a), shown in SEQ ID No.1
Nucleotide sequence shown in nucleotide sequence, (b) and SEQ ID No.1 has the nucleotide sequence of at least 95% homology, or
(c), the nucleotide sequence shown in SEQ ID No.1, wherein one or more bases are lacked, substituted or increased obtained nucleosides
Acid sequence.
In one embodiment, the antigen binding regions expressing gene is used to express mesothelin antibody, the antigen
Calmodulin binding domain CaM expressing gene includes：(a) shown in, the nucleotide sequence shown in SEQ ID No.2, (b) and SEQ ID No.2
Nucleotide sequence has the nucleotide sequence of at least 95% homology, or the nucleotide sequence shown in (c), SEQ ID No.2, its
Middle one or more bases are lacked, substituted or increased obtained nucleotide sequence；And/or
The hinge protein expressing gene is used to express CD8 α, and the hinge protein expressing gene includes：(a)、SEQ ID
Nucleotide sequence shown in nucleotide sequence shown in No.3, (b) and SEQ ID No.3 has the core of at least 95% homology
Nucleotide sequence, or the nucleotide sequence shown in (c), SEQ ID No.3, wherein one or more bases are lacked, substituted or increased
Add obtained nucleotide sequence；And/or
The transmembrane protein expressing gene is used to express transmembrane protein, and the transmembrane protein includes what is be sequentially connected
CD28TM-CD28-4-1BB regions, the transmembrane protein expressing gene include：(a), the nucleotides sequence shown in SEQ ID No.4
Nucleotide sequence shown in row, (b) and SEQ ID No.4 has the nucleotide sequence of at least 95% homology, or (c), SEQ
Nucleotide sequence shown in ID No.4, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence；
And/or
The intracellular signal transduction protein-encoding gene is used to express CD3 ζ, the intracellular signal transduction protein-encoding gene
Including：(a) nucleotide sequence shown in, the nucleotide sequence shown in SEQ ID No.5, (b) and SEQ ID No.5 has extremely
The nucleotide sequence of few 95% homology, or the nucleotide sequence shown in (c), SEQ ID No.5, wherein one or more bases
Lacked, substituted or increased obtained nucleotide sequence；
A kind of expression vector, contain the Chimeric antigen receptor expressing gene described in any of the above-described in the expression vector.
A kind of Chimeric antigen receptor, including Tet-on 3G albumen and fusion protein, the fusion protein include being sequentially connected
Antigen binding regions, hinge protein, transmembrane protein and intracellular signal transduction albumen.
In one embodiment, the Tet-on 3G albumen includes：(a), as the nucleotides shown in SEQ ID No.1
The polypeptide that the polynucleotide encoding of sequence composition obtains, (b), the multinuclear with the nucleotide sequence composition shown in SEQ ID No.1
Thuja acid has an obtained polypeptide of polynucleotide encoding of at least 98% homology, or (c), as the nucleosides shown in SEQ ID No.1
The polynucleotides of acid sequence composition, wherein one or more bases are lacked, substituted or increased obtained polynucleotide encoding and obtained
The polypeptide arrived.
In one embodiment, the antigen binding regions include mesothelin antibody；And/or
The hinge protein includes CD8 α；And/or
The transmembrane protein includes the CD28-4-1BB regions being sequentially connected；And/or
The intracellular signal transduction albumen includes CD3 ζ.
In one embodiment, the antigen binding regions include：(a), as the nucleotides sequence shown in SEQ ID No.2
The polypeptide that the polynucleotide encoding of row composition obtains；(b), the more nucleosides formed with the nucleotide sequence shown in SEQ ID No.2
Acid has the polypeptide that the polynucleotide encoding of at least 98% homology obtains；Or (c), as the nucleotides shown in SEQ ID No.2
The polynucleotides of sequence composition, wherein one or more bases are lacked, substituted or increased obtained polynucleotide encoding and obtained
Polypeptide；And/or
The hinge protein includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.3
Obtained polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.3 have at least 98% homology
The obtained polypeptide of polynucleotide encoding；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.3, its
Middle one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The transmembrane protein includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.4
Obtained polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.4 have at least 98% homology
The obtained polypeptide of polynucleotide encoding；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.4, its
Middle one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The intracellular signal transduction albumen includes：(a), the multinuclear being made up of the nucleotide sequence shown in SEQ ID No.5
Thuja acid encodes obtained polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.5 have at least
The polypeptide that the polynucleotide encoding of 98% homology obtains；Or (c), be made up of the nucleotide sequence shown in SEQ ID No.5
Polynucleotides, wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains.
A kind of T cell of the Chimeric antigen receptor modification of tetracycline regulation, any of the above-described institute has been imported in the T cell
The Chimeric antigen receptor expressing gene stated, above-mentioned expression vector or the T cell energy are either transfected in the T cell
Enough express the Chimeric antigen receptor described in any of the above-described.
It is Chimeric antigen receptor expressing gene described in any of the above-described, above-mentioned expression vector, chimeric described in any of the above-described
The T cell of the Chimeric antigen receptor modification of antigen receptor or such as regulation of above-mentioned tetracycline is preparing anti-tumor drug or antiviral
Application in the medicine of infection.
Above-mentioned Chimeric antigen receptor expressing gene includes Tet-on 3G protein-encoding genes and expressing fusion protein gene.
Expressing fusion protein gene includes antigen binding regions expressing gene, hinge protein expressing gene, the transmembrane protein being sequentially connected
Expressing gene and intracellular signal transduction protein-encoding gene.The Chimeric antigen receptor expressing gene of this innovative design can succeed
Import in T cell and form the T cell of Chimeric antigen receptor modification, the treatment for cancer or virus infection etc..At no Fourth Ring
Under conditions of element, Tet-on 3G albumen can not be combined with promoter so that fusion protein can not be expressed, the chimeric antigen by
Body will not start immune attack, be closed.And under the conditions of existing for tetracycline, Tet-on 3G albumen and tetracycline
With reference to starting the gene expression of fusion protein.The antigen binding regions of fusion protein and specific antigen binding, so as to be fitted together to
Antigen receptor specificity is targeted to corresponding lesion site.Chimeric antigen receptor starts immune attack, and it is corresponding anti-to kill expression
Former target cell.Therefore tetracycline is Chimeric antigen receptor behavior " switch " in T cell, in tetracycline presence and specifically
Immune response is just produced under antigen existence condition, realizes that Chimeric antigen receptor immune response is controllable, treatment side effect is few, specificity
It is high.
Brief description of the drawings
Fig. 1 is an embodiment CARs structural representations；
Fig. 2 is the T cell for the Chimeric antigen receptor modification that the tetracycline of an embodiment is adjusted with or without tetracycline
Under the conditions of Cellular Signaling Transduction Mediated schematic diagram；
Fig. 3 is the flow of the preparation method of the T cell for the Chimeric antigen receptor modification that the tetracycline of an embodiment is adjusted
Figure；
Fig. 4 is the electrophoretogram of the Lentiviral digestion products built in embodiment 1；
Fig. 5 is the T cell (JurkatCAR) of the Chimeric antigen receptor modification obtained in micro- Microscopic observation embodiment 1
Picture；
Fig. 6 is the proliferation results contrast of the T cell of the Chimeric antigen receptor modification obtained in embodiment 1 at different conditions
Figure；
Fig. 7 is the IFN γ secretory volume of the T cell of the Chimeric antigen receptor modification obtained in embodiment 1 at different conditions
Comparative result figure；
Fig. 8 is that the T cell of the Chimeric antigen receptor modification obtained in embodiment 1 kills the stream of target cell at different conditions
Formula scatterplot result comparison chart；
Fig. 9 is the T cell of the Chimeric antigen receptor modification obtained in embodiment 1 at different conditions to target cell killing rate
Statistical result comparison chart.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, with reference to specific embodiment and
Accompanying drawing is described in detail to the embodiment of the present invention.Elaborate in the following description many details in order to
Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology
Personnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is not by following public specific implementation
Limitation.
A kind of Chimeric antigen receptor expressing gene, including Tet-on 3G protein-encoding genes and expressing fusion protein gene,
The expressing fusion protein gene includes the antigen binding regions expressing gene, hinge protein expressing gene, cross-film being sequentially connected
Protein-encoding gene and intracellular signal transduction protein-encoding gene.
The Chimeric antigen receptor expressing gene can express Chimeric antigen receptor (CAR), so as to for cancer or virus sense
The treatment of dye etc..
Specifically, Tet-on 3G protein-encoding genes are used to express Tet-on 3G albumen.
In one embodiment, Tet-on 3G protein-encoding genes include：(a), the nucleosides shown in SEQ ID No.1
Nucleotide sequence of the nucleotide sequence with least 95% homology shown in acid sequence, (b) and SEQ ID No.1, or (c),
Nucleotide sequence shown in SEQ ID No.1, wherein one or more bases are lacked, substituted or increased obtained nucleotides sequence
Row.
Specifically, antigen binding regions expressing gene is used to express antigen binding regions.Chimeric antigen receptor gene expression
Afterwards, antigen binding regions be normally at it is extracellular so as to corresponding antigen binding.Antigen binding regions can be by chimeric antigen
Receptor-specific is targeted to corresponding lesion site, improves specificity.Antigen for example can be that tumor associated antigen can also
For the related antigen of other diseases, antigen binding regions are then antibody corresponding to antigen.
Specifically, antigen binding regions are in tumor associated antigen calmodulin binding domain CaM and virus-associated antigen calmodulin binding domain CaM
It is at least one.
In one embodiment, antigen binding regions expressing gene is used to express mesothelin antibody (anti-
mesothelin).Mesothelin (mesothelin) expression quantity in tumour cell is higher, and normal tissue expression amount it is relatively low or
Hardly express, therefore mesothelin (mesothelin) can be as " the preferable antigen " for the treatment of tumour.By in chimeric antigen
Design mesothelin (mesothelin) calmodulin binding domain CaM, has transfected the T cell energy of the Chimeric antigen receptor (CAR) in acceptor (CAR)
It is enough specific to be targeted to tumor tissues position, specificity is improved, the injury to its hetero-organization is reduced, effectively reduces oncotherapy
Side effect.
Further, antigen binding regions expressing gene includes：(a), the nucleotide sequence shown in SEQ ID No.2,
(b), there is the nucleotide sequence of at least 95% homology, or (c), SEQ ID with the nucleotide sequence shown in SEQ ID No.2
Nucleotide sequence shown in No.2, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.This is anti-
Former calmodulin binding domain CaM expressing gene, which can be expressed smoothly, obtains mesothelin antibody (anti-mesothelin).
Specifically, hinge protein expressing gene is used to express hinge protein, hinge protein one end connection antigen knot of expression
Close region other end connection transmembrane protein.Hinge protein is set to be advantageous to antigen between antigen binding regions and transmembrane protein
The identification signal conduction of calmodulin binding domain CaM enters intracellular, the ability of raising antigen binding regions identification antigen.
In one embodiment, hinge protein includes CD8 α.CD8 alpha signal conducting powers are strong, improve antigen binding regions
Identify the ability of antigen.Certainly, in other embodiments, hinge protein can also be other molecules such as CD8 β etc..
Further, hinge protein expressing gene includes：(a), the nucleotide sequence shown in SEQ ID No.3, (b), with
Nucleotide sequence shown in SEQ ID No.3 has the nucleotide sequence of at least 95% homology, or (c), SEQ ID No.3 institutes
The nucleotide sequence shown, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.The hinge protein
Expressing gene, which can be expressed smoothly, obtains CD8 α.
Specifically, transmembrane protein expressing gene is used to express transmembrane protein.After Chimeric antigen receptor gene expression, cross-film egg
Cell membrane is passed through in vain, expression of the transmembrane protein to Chimeric antigen receptor (CAR) plays an important role.
In one embodiment, transmembrane protein includes the CD28TM-CD28-4-1BB regions being sequentially connected, wherein
CD28TM regions are connected with hinge protein, and 4-1BB regions are connected with intracellular signal transduction albumen.
Further, transmembrane protein expressing gene includes：(a), the nucleotide sequence shown in SEQ ID No.4, (b), with
Nucleotide sequence shown in SEQ ID No.4 has the nucleotide sequence of at least 95% homology, or (c), SEQ ID No.4 institutes
The nucleotide sequence shown, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.The transmembrane protein
Expressing gene, which can be expressed smoothly, obtains CD28TM-CD28-4-1BB.
Specifically, intracellular signal transduction protein-encoding gene is used to express intracellular signal transduction albumen.Intracellular signal transduction
Albumen can instruct intracellular signal transduction, the conjunction of modulin to intracellular delivery Chimeric antigen receptor (CAR) signal
Into expression.
In one embodiment, intracellular signal transduction albumen is CD3 ζ.CD3 ζ molecular weight is small, and conduction efficiency is high.Certainly,
Intracellular signal transduction albumen can also be CD3 α or CD3 γ etc. in other embodiments.
Further, intracellular signal transduction protein-encoding gene includes：(a), the nucleotides sequence shown in SEQ ID No.5
Nucleotide sequence shown in row, (b) and SEQ ID No.5 has the nucleotide sequence of at least 95% homology, or (c), SEQ
Nucleotide sequence shown in ID No.5, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.
In one embodiment, expressing fusion protein gene includes the nucleosides shown in the SEQ ID No.2 that are sequentially connected
Nucleotide sequence shown in nucleotide sequence, SEQ ID No.4 and SEQ ID No.5 institutes shown in acid sequence, SEQ ID No.3
The nucleotide sequence shown.The expression of polypeptides formula that the polynucleotide encoding of nucleotide sequence composition obtains is anti-
mesothelin-CD8αhinge-CD28TM-CD28-4-1BB-CD3ζ。
The Chimeric antigen receptor expressing gene of this innovative design, which can be imported successfully, forms Chimeric antigen receptor in T cell
The T cell of modification, the treatment for cancer or virus infection etc..
A kind of expression vector, contain above-mentioned Chimeric antigen receptor expressing gene in the expression vector.
Specifically, expression vector for example can be the slow virus carrier for inserting above-mentioned Chimeric antigen receptor expressing gene,
Above-mentioned Chimeric antigen receptor expressing gene is inserted into slow virus carrier and transfected again into T cell, obtains Chimeric antigen receptor
The T cell of modification.
In one embodiment, Tet-on 3G protein-encoding genes can be inserted into slow virus carrier, pass through base
Because technique construction obtains the carrier containing Tet-on 3G protein-encoding genes, then expressing fusion protein gene is inserted into and contained
In the carrier for having Tet-on 3G protein-encoding genes.
In another embodiment, the carrier containing Tet-on 3G protein-encoding genes also can be directly commercially available.
The Chimeric antigen receptor (chimeric antigen receptor, CAR) of one embodiment, including Tet-on 3G
Albumen and fusion protein, fusion protein include antigen binding regions, hinge protein, transmembrane protein and the intracellular signal being sequentially connected
Conductive protein.
Specifically, Tet-on 3G albumen, antigen binding regions, hinge protein, transmembrane protein and intracellular signal transduction albumen
Concrete function and property can be found in described above, therefore not to repeat here.
In one embodiment, Tet-on 3G albumen includes：(a), as the nucleotide sequence shown in SEQ ID No.1
The polypeptide that the polynucleotide encoding of composition obtains, (b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.1
The polypeptide that polynucleotide encoding with least 98% homology obtains, or (c), as the nucleotides sequence shown in SEQ ID No.1
The polynucleotides of composition are arranged, wherein one or more bases are lacked, substituted or increased what obtained polynucleotide encoding obtained
Polypeptide.
In one embodiment, antigen binding regions are selected from tumor associated antigen calmodulin binding domain CaM and virus-associated antigen knot
Close at least one of region.
Specifically, antigen binding regions include mesothelin antibody.Antigen binding regions include：(a), by SEQ ID No.2
The polypeptide that the polynucleotide encoding of shown nucleotide sequence composition obtains, the nucleotides sequence shown in (b) and SEQ ID No.2
The polynucleotides of row composition have an obtained polypeptide of polynucleotide encoding of at least 98% homology, or (c), by SEQ ID
The polynucleotides of nucleotide sequence composition shown in No.2, wherein one or more bases are lacked, substituted or increased what is obtained
The polypeptide that polynucleotide encoding obtains.
In one embodiment, hinge protein includes CD8 α.Certainly, in other embodiments, hinge protein may be used also
To be other molecules such as CD8 β etc..
Specifically, hinge protein includes：(a), the polynucleotides being made up of the nucleotide sequence shown in SEQ ID No.3 are compiled
The polypeptide that code obtains；(b) it is, homologous with least 98% with the polynucleotides of the nucleotide sequence composition shown in SEQ ID No.3
The polypeptide that the polynucleotide encoding of property obtains；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.3,
Wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains.
In one embodiment, memebrane protein includes the CD28-4-1BB regions being sequentially connected.
Specifically, transmembrane protein includes：(a), the polynucleotides being made up of the nucleotide sequence shown in SEQ ID No.4 are compiled
The polypeptide that code obtains；(b) it is, homologous with least 98% with the polynucleotides of the nucleotide sequence composition shown in SEQ ID No.4
The polypeptide that the polynucleotide encoding of property obtains；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.4,
Wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains.
In one embodiment, intracellular signal transduction albumen includes CD3 ζ.
Specifically, intracellular signal transduction albumen includes：(a), it is made up of the nucleotide sequence shown in SEQ ID No.5 more
The polypeptide that nucleotide coding obtains；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.5 have at least
The polypeptide that the polynucleotide encoding of 98% homology obtains；Or (c), be made up of the nucleotide sequence shown in SEQ ID No.5
Polynucleotides, wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains.
It is appreciated that polymorphism and variation due to the expressing gene of Chimeric antigen receptor, coding identical albumen may
There can be the expressing gene of diversified forms.If base is lacked, substituted or increased in expressing gene, or the missing of amino acid,
Insertion, substitution or other variations, so as to cause the one or more amino acid of the amino acid sequence of protein appearance to be lacked, substituted
Or increase.Express obtained protein and be different from corresponding protein, but there is no the more of obvious function difference with the protein
Peptide or protein is referred to as functional equivalent variant.There is no obvious function difference with Chimeric antigen receptor therefore, it is possible to express to obtain
The expressing genes of more peptide or proteins will be understood that the expressing gene being equal with the Chimeric antigen receptor expressing gene in the application.
With more peptide or proteins that Chimeric antigen receptor does not have obvious function difference all will be understood that with the chimeric antigen in the application by
The equivalent albumen of body.
By continuous research and probe, successful design goes out the Chimeric antigen receptor (CAR) of above-mentioned functional structure, the inosculating antibody
Original receptor (CAR) specific in T cell can be expressed so as to the treatment for cancer or virus infection etc..At no Fourth Ring
Under conditions of element, Tet-on 3G albumen can not be combined with promoter so that fusion protein can not be expressed, the chimeric antigen by
Body will not start immune attack, be closed.And under the conditions of existing for tetracycline, Tet-on 3G albumen and tetracycline
With reference to starting the gene expression of fusion protein.The antigen binding regions of fusion protein and specific antigen binding, so as to be fitted together to
Antigen receptor specificity is targeted to corresponding lesion site.Chimeric antigen receptor starts immune attack, and it is corresponding anti-to kill expression
Former target cell.Therefore tetracycline is Chimeric antigen receptor behavior " switch " in T cell, under the conditions of specific tetracycline
Immune response is produced, realizes that Chimeric antigen receptor immune response is controllable, treatment side effect is few, and specificity is high.
Above-mentioned Chimeric antigen receptor (CAR) can be used in the medicine of anti-tumor drug or viral infection resisting.
In the application of one embodiment, Chimeric antigen receptor (CAR) has mesothelin antibody, the Chimeric antigen receptor
(CAR) can be used in the medicine of anti-breast cancer.
The T cell (CAR-T) of the Chimeric antigen receptor modification of the tetracycline regulation of one embodiment, is imported in the T cell
Above-mentioned Chimeric antigen receptor expressing gene.Or above-mentioned expression vector is transfected in the T cell.Or the T cell can
Express Chimeric antigen receptor described above.
The expressing gene and structure of Chimeric antigen receptor (CAR) refer to described above, and therefore not to repeat here.
In one embodiment, referring to Fig. 2, under conditions of no tetracycline, Tet-on 3G albumen can not be with opening
Mover is combined so that fusion protein can not be expressed, and intracellular signal transduction is closed.In bar existing for tetracycline
Under part, Tet-on 3G albumen is combined with tetracycline, starts the gene expression of fusion protein, plays immunization.Therefore tetracycline
It is Chimeric antigen receptor behavior " switch " in T cell, immune response is just produced under the conditions of specific tetracycline, realizes chimeric
Antigen receptor immune response is controllable, and treatment side effect is few.
In one embodiment, T cell is Jurkat cell.One kind that Jurkat cell belongs in T lymphocytes, its
Multiplication capacity is strong, suitably the host cell as Chimeric antigen receptor (CAR).
Test result indicates that T cell (CAR-T) tetracycline of the Chimeric antigen receptor modification of above-mentioned tetracycline regulation is present
Under the conditions of there is specific Efficient killing effect to act on the tumour cell of expressing specific antigen, and without tetracycline or even if having Fourth Ring
Element but without express specific antigen killing functions of immunocytes very little.Therefore, the T cell (CAR-T) of Chimeric antigen receptor modification
It is expected to apply in anti-tumor drug, a kind of thinking is provided for oncotherapy.The T cell of Chimeric antigen receptor modification is four
Ring element exists and specifically just produces immune response under antigen existence condition, realizes that Chimeric antigen receptor immune response is controllable,
Treatment side effect is few.
The T cell (CAR-T) of the Chimeric antigen receptor modification of above-mentioned tetracycline regulation can be used in anti-tumor drug
Or in the medicine of viral infection resisting.
In the application of one embodiment, the T cell (CAR-T) of the Chimeric antigen receptor modification of tetracycline regulation can transport
In the medicine of anti-breast cancer.
Referring to Fig. 3, the preparation method of the T cell of the Chimeric antigen receptor modification of the tetracycline regulation of an embodiment,
Comprise the following steps S110~S120.
S110, by expressing fusion protein gene insert the slow virus carrier containing Tet-on 3G protein-encoding genes in, obtain
To expression vector, wherein expressing fusion protein gene includes antigen binding regions expressing gene, the hinge protein table being sequentially connected
Up to gene, transmembrane protein expressing gene and intracellular signal transduction protein-encoding gene.
Specifically, the structure of Chimeric antigen receptor (CAR) refers to described above, and therefore not to repeat here.
In one embodiment, the slow virus carrier containing Tet-on 3G protein-encoding genes is pLVX-TetOne,
The slow virus carrier contains Tet-on 3G protein-encoding genes, and pLVX-TetOne is tetracycline-controlled expression system carrier.
Specifically, the sequence of Tet-on 3G protein-encoding genes is as shown in SEQ ID No.1.
Specifically, the carrier containing Tet-on 3G protein-encoding genes can insert Tet-on 3G protein-encoding genes
Into slow virus carrier, build to obtain by gene technology, also can directly be commercially available.
In one embodiment, the expression formula of the fusion protein of coding is anti-mesothelin-CD8 α hinge-
CD28TM-CD28-4-1BB-CD3ζ。
Specifically, anti-mesothelin-CD8 α hinge-CD28TM-CD28-4- are synthesized by way of gene chemical synthesis
1BB-CD3 ζ nucleotide sequence.
In one embodiment, expressing fusion protein gene includes the nucleosides shown in the SEQ ID No.2 that are sequentially connected
Nucleotide sequence shown in nucleotide sequence, SEQ ID No.4 and SEQ ID No.5 institutes shown in acid sequence, SEQ ID No.3
The nucleotide sequence shown.
Specifically, expressing fusion protein gene is then attached to by identical limit by restriction enzymes double zyme cutting
In the slow virus carrier of property enzymes double zyme cutting processing processed.
In one embodiment, restriction enzyme includes AgeI and BamH I.
S120, the expression vector obtained in S110 is packaged as slow virus and transfected into T cell, obtain tetracycline regulation
Chimeric antigen receptor modification T cell.
Specifically, the expression vector obtained in S110 is first transfected into host cell such as 239T cells, amplification obtains big
The slow virus of amount.Then by the slow-virus transfection of certain virus titer into T cell, obtain tetracycline regulation chimeric antigen by
The T cell (CAR-T) of body modification.
In one embodiment, expression vector is packaged as slow virus and transfected into the operation in T cell, the disease of transfection
Malicious MOI values are about 10.
The method of the T cell of the above-mentioned Chimeric antigen receptor modification for preparing tetracycline regulation, it is easy to operate, prepare
The T cell of Chimeric antigen receptor modification exists in tetracycline and specifically produces immune response under antigen existence condition, realizes
Chimeric antigen receptor immune response is controllable, and treatment side effect is few.
It is specific embodiment part below.
In following examples, unless otherwise instructed, the experimental method of unreceipted actual conditions, generally according to normal condition,
For example, see Pehanorm Brooker, EF, not the written molecular cloning of Ritchie, T Mannies A Disi etc. (Jin Dongyan, Li Mengfeng etc. are translated) is real
Test guide [M] (Beijing：Science Press, 1992) method that condition or kit manufacturer described in are recommended is realized.
Reagent used in embodiment is commercially available.
Embodiment 1
Prepare the T cell of the Chimeric antigen receptor modification of tetracycline regulation
Slow virus carrier pLVX-TetOne is provided by Clontech companies, and Tet-on 3G eggs are contained in pLVX-TetOne
White expressing gene, Tet-on3G protein-encoding genes are as shown in SEQ ID No.1.
Expressing fusion protein gene (including the nucleosides shown in the SEQ ID No.2 being sequentially connected is synthesized by genome company
Nucleotide sequence shown in nucleotide sequence, SEQ ID No.4 and SEQ ID No.5 institutes shown in acid sequence, SEQ ID No.3
The nucleotide sequence shown).The expression formula of fusion protein is anti-mesothelin-CD8 α hinge-CD28TM-CD28-4-
1BB-CD3ζ。
Synthesis expressing fusion protein gene by AgeI and BamH I double digestions and is cloned into slow virus carrier pLVX-
In TetOne, expression vector is obtained.
The Lentiviral built is identified by AgeI and BamH I double digestions, digestion products electrophoretogram
(1.6kb fragments are purpose fragment in wherein Fig. 4, and 8.2kb fragments are carrier fragment itself) as shown in Figure 4.Purpose fragment it is big
It is small to be consistent with expection, illustrate vector construction success.The expression plasmid of above-mentioned acquisition is packaged as slow virus, and virus is (viral
MOI values into Jurkat E6.1 cells (T lymphocyte strains, purchased from U.S. typical case thing collection, ATCC), obtain for 10) transfection
To the Jurkat cell (JurkatCAR) of the Chimeric antigen receptor modification of tetracycline regulation.
After 2 days, the Jurkat E6.1 cells after transfection are transferred in the culture mediums of RPMI 1640 with puromycin,
And by limiting dilution assay by Cell-cloned.Through the screening of 21 days, establish what the tetracycline with puromycin-resistant was adjusted
The Jurkat E6.1 cell lines (JurkatCAR) of Chimeric antigen receptor modification.Jurkat after micro- Microscopic observation transfection
The growth photo of E6.1 (JurkatCAR) cell is transfected successfully as shown in figure 5, Jurkat E6.1 growth conditions are good.
Test one
The T cell proliferative conditions measure of the Chimeric antigen receptor modification of tetracycline regulation
By 5 × 105The JurkatCAR cells prepared in individual embodiment 1 are separately added into containing K562, K562/
mesothelin+, MCF7 cells 6 orifice plates (5 × 105/ hole) in.Wherein, K562 is human erythroleukemia cell, and MCF7 is people
Breast cancer cell, K562 and MCF7 are purchased from ATCC.K562/mesothelin+Expression passes through on the basis of K562 cells
Mesothelin genetic modifications, K562 is set to be overexpressed mesothelin genes.In above-mentioned cell, K562 is not expressed
Mesothelin, K562/mesothelin+Mesothelin can be expressed with MCF7.
Experiment is divided into two groups, and A groups are handled without tetracycline, and B groups add tetracycline, concentration 1mg/mL.Respectively at the 3rd day
(D3), the 7th day (D7), cell count is carried out to the JurkatCAR cells of suspension.
As a result as shown in fig. 6, K562/mesothelin in B groups+, the JurkatCAR in MCF7 holes can largely breed,
And other hole inner cells are hardly bred.Result above shows, when bar being present in tetracycline induction and mesothelin antigens
Under part, could largely it breed after the T cell of CAR modifications combines with corresponding antigens (mesothelin).And when tetracycline is not deposited
In the presence of specific antigen, JurkatCAR (T cell of the Chimeric antigen receptor modification of tetracycline regulation) does not increase substantially
Grow.
Test two
IFN γ secretory volume determines in the T cell of the Chimeric antigen receptor modification of tetracycline regulation
By 5 × 105The JurkatCAR cells prepared in individual embodiment 1 in 24 orifice plates with K562, K562/
mesothelin+, MCF7 cells (5 × 105/ hole) co-culture.
Experiment is divided into two groups, and A groups are handled without tetracycline, and B groups add tetracycline, concentration 1mg/mL.Received after 72 hours
Collect supernatant, the secretory volume of IFN γ is detected by the ELISA detection kit (BD Biosciences) of IFN γ.
As a result as shown in fig. 7, K562/mesothelin in B groups+, the JurkatCAR cells in MCF7 holes can largely divide
IFN γ is secreted, and other hole inner cells hardly secrete IFN γ.Result above shows, when in tetracycline induction and
Under mesothelin antigen existence conditions, can largely it divide after the T cell of CAR modifications combines with specific antigen (mesothelin)
Secrete IFN γ.And when tetracycline be not present or without specific antigen in the presence of, JurkatCAR (tetracycline regulation chimeric antigen by
The T cell of body modification) IFN γ is not secreted substantially.
Test three
The T cell killing effect in vitro measure of the Chimeric antigen receptor modification of tetracycline regulation
By the JurkatCAR cells prepared in embodiment 1 and target cell K562, K562/mesothelin+, MCF7 cells
Co-culture and (imitate target than 1:1).Different type is swollen using the JurkatCAR cells after the double mark method detection genetic modifications of CFSE/PI
The Cytotoxicity in vitro ability of oncocyte.
Experiment is divided into two groups, and group does not have to tetracycline and handled, and B groups add tetracycline, concentration 1mg/mL.Method is as follows：With
Final concentration of 0.05 μM of CFSE is to 1 × 106Individual target cell carries out living cells dye marker, by 1 × 106Individual JurkatCAR cells
With 1 × 106The target cell of individual CFSE marks mixes, and 200g, 1min centrifugation make cell contact with each other, in 5%CO2, 37 DEG C of incubators
In be incubated 4h altogether, after reaction terminates, add 1 μ g/ml PI dye liquors, mix, room temperature lucifuge carries out flow cytometer detection after being incubated 15min.
As a result as shown in Figure 8 and Figure 9, right upper quadrant cell mass is killed cell proportion in Fig. 8.Induced in tetracycline
Under, JurkatCAR can effectively kill K562/mesothelin+, MCF7 cancer cells；And other group of cell does not kill substantially.More than
As a result show, induced when in tetracycline, can effectively be killed after the T cell of CAR modifications combines with corresponding antigens (mesothelin)
Hinder target cell；And in the absence of tetracycline situation or without specific antigen in the presence of, JurkatCAR (tetracycline regulation inosculating antibody
The T cell of original receptor modification) target cell is not killed substantially.
To sum up, in tetracycline there is bar in the T cell (JurkatCAR) of the Chimeric antigen receptor modification of above-mentioned tetracycline regulation
There is specific Efficient killing effect to act on the tumour cell for expressing specific antigen under part, and without tetracycline or even if having tetracycline
But without the killing functions of immunocytes very little of expression specific antigen.Therefore, the T cell of Chimeric antigen receptor modification can be used as anti-
The medicine of tumour, a kind of thinking is provided for oncotherapy.The T cell of Chimeric antigen receptor modification is in tetracycline and specifically
Immune response is just produced under antigen existence condition, realizes that Chimeric antigen receptor immune response is controllable, treatment side effect is few.
In addition, above test data is illustrated using mesothelin as antigen, in other embodiments, when on fusion protein
When designing different antigen binding regions, under tetracycline induction, the T cell pair of the Chimeric antigen receptor modification of tetracycline regulation
Good lethal effect can also be produced by expressing the target cell of corresponding antigen.
Embodiment described above only expresses one or more of embodiments of the present invention, and it describes more specific and detailed
Carefully, but the limitation to the scope of the claims of the present invention therefore can not be interpreted as.It should be pointed out that the common skill for this area
For art personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to this hair
Bright protection domain.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Shenzhen University
<120>The T cell and its answer that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified
With
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 747
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atgtctagac tggacaagag caaagtcata aactctgctc tggaattact caatggagtc 60
ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc 120
ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg 180
gacaggcatc atacccactc ctgccccctg gaaggcgagt catggcaaga ctttctgcgg 240
aacaacgcca agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat 300
ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg 360
tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt 420
acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca 480
cctaccaccg attctatgcc cccacttctg aaacaagcaa ttgagctgtt cgaccggcag 540
ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag 600
ctaaagtgcg aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc 660
ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat 720
tttgaccttg acatgctccc cgggtaa 747
<210> 2
<211> 807
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gccaccatgg ccttaccagt gaccgccttg ctcctgccgc tgccgctggc cttgctgctc 60
cacgccgcca ggccgggatc ccaggtacaa ctgcagcagt ctgggcctga gctggagaag 120
cctggcgctt cagtgaagat atcctgcaag gcttctggtt actcattcac tggctacacc 180
atgaactggg tgaagcagag ccatggaaag agccttgagt ggattggact tattactcct 240
tacaatggtg cttctagcta caaccagaag ttcaggggca aggccacatt aactgtagac 300
aagtcatcca gcacagccta catggacctc ctcagtctga catctgaaga ctctgcagtc 360
tatttctgtg caaggggggg ttacgacggg aggggttttg actactgggg ccaagggacc 420
acggtcaccg tctcctcagg tgtaggcggt tcaggcggcg gtggctctgg cggtggcgga 480
tcggacatcg agctcactca gtctccagca atcatgtctg catctccagg ggagaaggtc 540
accatgacct gcagtgccag ctcaagtgta agttacatgc actggtacca gcagaagtca 600
ggcacctccc ccaaaagatg gatttatgac acatccaaac tggcttctgg agtcccaggt 660
cgcttcagtg gcagtgggtc tggaaactct tactctctca caatcagcag cgtggaggct 720
gaagatgatg caacttatta ctgccagcag tggagtggtt accctctcac gttcggtgct 780
gggacaaagt tggaaatcaa agctagc 807
<210> 3
<211> 135
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 4
<211> 330
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60
gcctttatta ttttctgggt gaggagtaag aggagcaggg gagggcacag tgactacatg 120
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180
cgcgacttcg cagcctatcg ctccaaacgg ggcagaaaga aactcctgta tatattcaaa 240
caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 300
ccagaagaag aagaaggagg atgtgaactg 330
<210> 5
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300
acctacgacg cccttcacat gcaggccctg ccccctcgct aa 342
Claims (10)
1. a kind of Chimeric antigen receptor expressing gene, it is characterised in that including Tet-on 3G protein-encoding genes and fusion protein
Expressing gene, the expressing fusion protein gene include antigen binding regions expressing gene, the hinge protein expression being sequentially connected
Gene, transmembrane protein expressing gene and intracellular signal transduction protein-encoding gene.
2. conjunction antigen receptor expressing gene according to claim 1, it is characterised in that the Tet-on 3G protein expressions
Gene includes：(a) nucleotide sequence shown in, the nucleotide sequence shown in SEQ ID No.1, (b) and SEQ ID No.1
There are the nucleotide sequence of at least 95% homology, or the nucleotide sequence shown in (c), SEQ ID No.1, wherein one or more
Base is lacked, substituted or increased obtained nucleotide sequence.
3. conjunction antigen receptor expressing gene according to claim 1, it is characterised in that the antigen binding regions express base
Because being used for expressing mesothelin antibody, the antigen binding regions expressing gene includes：(a), the nucleotides shown in SEQ ID No.2
Nucleotide sequence of the nucleotide sequence with least 95% homology shown in sequence, (b) and SEQ ID No.2, or (c),
Nucleotide sequence shown in SEQ ID No.2, wherein one or more bases are lacked, substituted or increased obtained nucleotides sequence
Row；And/or
The hinge protein expressing gene is used to express CD8 α, and the hinge protein expressing gene includes：(a)、SEQ ID No.3
Nucleotide sequence shown in shown nucleotide sequence, (b) and SEQ ID No.3 has the nucleotides of at least 95% homology
Sequence, or the nucleotide sequence shown in (c), SEQ ID No.3, wherein one or more bases are lacked, substituted or increased
The nucleotide sequence arrived；And/or
The transmembrane protein expressing gene is used to express transmembrane protein, and the transmembrane protein includes the CD28TM- being sequentially connected
CD28-4-1BB regions, the transmembrane protein expressing gene include：(a), the nucleotide sequence shown in SEQ ID No.4, (b),
There is the nucleotide sequence of at least 95% homology, or (c), SEQ ID No.4 with the nucleotide sequence shown in SEQ ID No.4
Shown nucleotide sequence, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence；And/or
The intracellular signal transduction protein-encoding gene is used to express CD3 ζ, the intracellular signal transduction protein-encoding gene bag
Include：(a) nucleotide sequence shown in, the nucleotide sequence shown in SEQ ID No.5, (b) and SEQ ID No.5 has at least
The nucleotide sequence of 95% homology, or the nucleotide sequence shown in (c), SEQ ID No.5, wherein one or more base quilts
Missing, substitute or increase obtained nucleotide sequence.
4. a kind of expression vector, it is characterised in that containing embedding as described in any one of claims 1 to 3 in the expression vector
Close antigen receptor expressing gene.
A kind of 5. Chimeric antigen receptor, it is characterised in that including Tet-on 3G albumen and fusion protein, the fusion protein bag
Include the antigen binding regions being sequentially connected, hinge protein, transmembrane protein and intracellular signal transduction albumen.
6. Chimeric antigen receptor according to claim 5, it is characterised in that the Tet-on 3G albumen includes：(a), by
The polypeptide that the polynucleotide encoding of nucleotide sequence composition shown in SEQ ID No.1 obtains, shown in (b) and SEQ ID No.1
The polynucleotides of nucleotide sequence composition there is the obtained polypeptide of polynucleotide encoding of at least 98% homology, or (c), by
The polynucleotides of nucleotide sequence composition shown in SEQ ID No.1, wherein one or more bases are lacked, substituted or increased
The polypeptide that obtained polynucleotide encoding obtains.
7. Chimeric antigen receptor according to claim 5, it is characterised in that
The antigen binding regions include mesothelin antibody；And/or
The hinge protein includes CD8 α；And/or
The transmembrane protein includes the CD28-4-1BB regions being sequentially connected；And/or
The intracellular signal transduction albumen includes CD3 ζ.
8. Chimeric antigen receptor according to claim 5, it is characterised in that the antigen binding regions include：(a), by
The polypeptide that the polynucleotide encoding of nucleotide sequence composition shown in SEQ ID No.2 obtains；(b), and shown in SEQ ID No.2
The polynucleotides of nucleotide sequence composition there is the obtained polypeptide of polynucleotide encoding of at least 98% homology；Or (c), by
The polynucleotides of nucleotide sequence composition shown in SEQ ID No.2, wherein one or more bases are lacked, substituted or increased
The polypeptide that obtained polynucleotide encoding obtains；And/or
The hinge protein includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.3 obtains
Polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.3 have the more of at least 98% homology
The polypeptide that nucleotide coding obtains；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.3, wherein one
Individual or multiple bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The transmembrane protein includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.4 obtains
Polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.4 have the more of at least 98% homology
The polypeptide that nucleotide coding obtains；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.4, wherein one
Individual or multiple bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The intracellular signal transduction albumen includes：(a), the polynucleotides being made up of the nucleotide sequence shown in SEQ ID No.5
Encode obtained polypeptide；(b) it is, same with least 98% with the polynucleotides of the nucleotide sequence composition shown in SEQ ID No.5
The polypeptide that the polynucleotide encoding of source property obtains；Or (c), more nucleosides for being made up of the nucleotide sequence shown in SEQ ID No.5
Acid, wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains.
A kind of 9. T cell of the Chimeric antigen receptor modification of tetracycline regulation, it is characterised in that imported in the T cell as
Chimeric antigen receptor expressing gene described in any one of claims 1 to 3, or transfected such as claim 4 in the T cell
Described expression vector, or the T cell can express the Chimeric antigen receptor as described in any one of claim 5~8.
10. Chimeric antigen receptor expressing gene, expression as claimed in claim 4 carry as described in any one of claims 1 to 3
Body, the Chimeric antigen receptor as described in any one of claim 5~8 or tetracycline as claimed in claim 9 are adjusted chimeric
Application of the T cell of antigen receptor modification in the medicine of anti-tumor drug or viral infection resisting is prepared.
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|---|---|---|---|
| CN201710792923.9A CN107475276A (en) | 2017-09-05 | 2017-09-05 | The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified |
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| CN201710792923.9A CN107475276A (en) | 2017-09-05 | 2017-09-05 | The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of tetracycline regulation are modified |
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