CN107475275A - The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of dual anti-former regulation are modified - Google Patents
The T cell and its application that Chimeric antigen receptor and its expressing gene, the Chimeric antigen receptor of dual anti-former regulation are modified Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C07K2319/00—Fusion polypeptide
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- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C12N2510/00—Genetically modified cells
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 dual anti-former regulation and its application.The Chimeric antigen receptor expressing gene includes the first expressing fusion protein gene and the second expressing fusion protein gene.First expressing fusion protein gene includes MUC-1 antibody expressing genes, notch expression of receptor gene and the Gal4 VP64 transcription activating protein expressing genes being sequentially connected.Second expressing fusion protein gene includes the Gal4 UAS promoters expressing genes and mesothelin antibody expressing genes 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, immune response is just produced under the conditions of MUC-1 and the dual anti-original signal of mesothelin are simultaneous, 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, dual anti-original
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 dual anti-former regulation is provided and its application.
A kind of Chimeric antigen receptor expressing gene, including the first expressing fusion protein gene and the second expressing fusion protein base
Cause；
The first expressing fusion protein gene includes Mucin1 antibody expressing genes, the notch acceptors being sequentially connected
Expressing gene and Gal4-VP64 transcription activating protein expressing genes；
The second expressing fusion protein gene includes the Gal4-UAS promoters expressing gene and mesothelin being sequentially connected
Antibody expressing genes.
In one embodiment, the Mucin1 antibody expressing genes include：(a), the core 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；And/or
The notch expression of receptor gene includes：(a), the nucleotide sequence shown in SEQ ID No.2, (b) and SEQ
Nucleotide sequence shown in ID No.2 has the nucleotide sequence of at least 95% homology, or shown in (c), SEQ ID No.2
Nucleotide sequence, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence；And/or
The Gal4-VP64 transcription activating proteins expressing gene includes：(a), the nucleotides sequence shown in SEQ ID No.3
Nucleotide sequence shown in row, (b) and SEQ ID No.3 has the nucleotide sequence of at least 95% homology, or (c), SEQ
Nucleotide sequence shown in ID No.3, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.
In one embodiment, the Gal4-UAS promoters expressing gene includes：(a), shown in SEQ ID No.4
Nucleotide sequence shown in nucleotide sequence, (b) and SEQ ID No.4 has the nucleotide sequence of at least 95% homology, or
(c), the nucleotide sequence shown in SEQ ID No.4, wherein one or more bases are lacked, substituted or increased obtained nucleosides
Acid sequence；And/or
The mesothelin antibody expressing genes expressing gene includes：(a), the nucleotide sequence shown in SEQ ID No.5,
(b), there is the nucleotide sequence of at least 95% homology, or (c), SEQ ID with the nucleotide sequence shown in SEQ ID No.5
Nucleotide sequence shown in No.5, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.
In one embodiment, the second expressing fusion protein gene also includes terminator expressing gene, the end
Only sub- expressing gene is connected with the mesothelin antibody expressing genes, and the terminator expressing gene includes：(a)、SEQ ID
Nucleotide sequence shown in nucleotide sequence shown in No.6, (b) and SEQ ID No.6 has the core of at least 95% homology
Nucleotide sequence, or the nucleotide sequence shown in (c), SEQ ID No.6, wherein one or more bases are lacked, substituted or increased
Add 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 the first fusion protein and the second fusion protein；
Mucin1 antibody, notch acceptors and the Gal4-VP64 transcription that first fusion protein includes being sequentially connected swash
Living protein；
Second fusion protein includes Gal4-UAS promoters and the mesothelin antibody being sequentially connected.
In one embodiment, the Mucin1 antibody includes：(a), as the nucleotides sequence shown in SEQ ID No.1
The polypeptide that the polynucleotide encoding of row composition obtains, (b), more nucleosides with the nucleotide sequence composition shown in SEQ ID No.1
Acid has an obtained polypeptide of polynucleotide encoding of at least 98% homology, or (c), as the nucleotides shown in SEQ ID No.1
The polynucleotides of sequence composition, wherein one or more bases are lacked, substituted or increased obtained polynucleotide encoding and obtained
Polypeptide；And/or
The notch acceptors include：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.2
Obtained polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.2 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.2, its
Middle one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The Gal4-VP64 transcription activating proteins include：(a), it is made up of the nucleotide sequence shown in SEQ ID No.3
The polypeptide that polynucleotide encoding obtains；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.3 have extremely
The polypeptide that the polynucleotide encoding of few 98% homology obtains；Or (c), be made up of the nucleotide sequence shown in SEQ ID No.3
Polynucleotides, wherein one or more bases are lacked, substituted or are increased the polypeptide that obtained polynucleotide encoding obtains.
In one embodiment, second fusion protein also includes terminator, the terminator and the mesothelin
Antibody connects, in second fusion protein,
The Gal4-UAS promoters include：(a), the more nucleosides being made up of the nucleotide sequence shown in SEQ ID No.4
The polypeptide that acid encoding obtains；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.4 have at least 98%
The polypeptide that the polynucleotide encoding of homology obtains；Or (c), the multinuclear that is made up of the nucleotide sequence shown in SEQ ID No.4
Thuja acid, wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The mesothelin antibody includes：(a), the polynucleotides being made up of the nucleotide sequence shown in SEQ ID No.5 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.5
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.5,
Wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The terminator includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.6 obtains
The polypeptide arrived；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.6 have at least 98% homology
The polypeptide that polynucleotide encoding obtains；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.6, 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 dual anti-former 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, embedding described in any of the above-described
Close the T cell of the Chimeric antigen receptor modification of antigen receptor or dual anti-former regulation as claimed in claim 9 prepare it is antitumor
Medicine in application.
Above-mentioned Chimeric antigen receptor expressing gene includes the first expressing fusion protein gene and the second expressing fusion protein base
Cause.First expressing fusion protein gene include be sequentially connected Mucin1 antibody expressing genes, notch expression of receptor gene and
Gal4-VP64 transcription activating protein expressing genes.The Gal4-UAS that second expressing fusion protein gene includes being sequentially connected starts
Sub- expressing gene and mesothelin antibody expressing genes.The Chimeric antigen receptor expressing gene of this innovative design can be imported successfully
The T cell of Chimeric antigen receptor modification, the treatment for tumour etc. are formed in T cell.It can know after first expressing fusion protein
The label Mucin1 (muc-1) of other tumor cell surface is so as to being targeted to corresponding disease by Chimeric antigen receptor is specific
Stove site.And first fusion protein contacted with Mucin1 after, notch acceptors occur itself cutting, release Gal4-VP64 transcription
Activator protein.Gal4-VP64 transcription activating proteins activate the Gal4-UAS promoters of the second fusion protein, and then start mesothelin
The expression of antibody.When two kinds of tumor markers of tumor cell surface expression Mucin1 (muc-1) and mesothelin (mesothelin)
During thing, possess Mucin1 (muc-1) and mesothelin (mesothelin) dual anti-original signal, the Chimeric antigen receptor starts to be immunized
Attack, kill tumour cell.When Mucin1 (muc-1) and mesothelin (mesothelin) dual anti-original signal are not present or only
When having one of which, the Chimeric antigen receptor will not start immune attack, be closed.Therefore Mucin1 (muc-1)
It is Chimeric antigen receptor behavior " switch " in T cell with mesothelin (mesothelin) dual anti-original signal, specific dual anti-
Immune response is just produced under the conditions of original signal, 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 that the T cell of the Chimeric antigen receptor modification of the dual anti-former regulation of an embodiment exists in dual anti-original signal
Under conditions of Cellular Signaling Transduction Mediated schematic diagram；
Fig. 3 is the flow of the preparation method of the T cell of the Chimeric antigen receptor modification of the dual anti-former regulation of an embodiment
Figure；
Fig. 4 is the electrophoretogram of the first expression vector digestion products built in embodiment 1；
Fig. 5 is the electrophoretogram of the second expression vector digestion products built in embodiment 1；
Fig. 6 is the T cell (JurkatCAR) of the Chimeric antigen receptor modification obtained in micro- Microscopic observation embodiment 1
Picture；
Fig. 7 is the proliferation results contrast of the T cell of the Chimeric antigen receptor modification obtained in embodiment 1 at different conditions
Figure；
Fig. 8 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. 9 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；
Figure 10 is that the T cell of the Chimeric antigen receptor modification obtained in embodiment 1 kills to target cell at different conditions
The statistical result comparison chart of rate.
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 the first expressing fusion protein gene and the second expressing fusion protein base
Cause.First expressing fusion protein gene include be sequentially connected Mucin1 antibody expressing genes, notch expression of receptor gene and
Gal4-VP64 transcription activating protein expressing genes.The Gal4-UAS that second expressing fusion protein gene includes being sequentially connected starts
Sub- expressing gene and mesothelin antibody expressing genes.
The Chimeric antigen receptor expressing gene can express Chimeric antigen receptor (CAR), so as to the treatment for tumour.
Specifically, Mucin1 antibody expressing genes are used to express Mucin1 (muc-1).Mucin1 is in tumour cell
Particularly expression quantity is higher in solid tumor cell, and normal tissue expression amount is relatively low or hardly expresses, therefore mucoprotein-
1 (muc-1) can be as " the preferable antigen " for the treatment of tumour.Resisted by designing Mucin1 in Chimeric antigen receptor (CAR)
Body (anti muc-1), having transfected the T cell of the Chimeric antigen receptor (CAR) specific can be targeted to tumor tissues portion
Position, specificity is improved, reduces the injury to its hetero-organization, effectively reduce the side effect for the treatment of of cancer.
In one embodiment, Mucin1 antibody expressing genes include：(a), the nucleotides shown in SEQ ID No.1
Nucleotide sequence of the nucleotide sequence with least 95% homology shown in 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.Above-mentioned nucleotide sequence, which can be expressed smoothly, obtains Mucin1 antibody (anti muc-1).
Specifically, notch expression of receptor gene is used to express notch acceptors.Notch acceptors one end connection Mucin1 resists
The body other end connects Gal4-VP64 transcription activating proteins.After Mucin1 antibody is combined with Mucin1, notch acceptors occur
Itself cutting, discharges Gal4-VP64 transcription activating proteins.
In one embodiment, nucleotides sequence of the notch expression of receptor gene shown in including (a), SEQ ID No.2
Nucleotide sequence shown in row, (b) and SEQ ID No.2 has the nucleotide sequence of at least 95% homology, or (c), SEQ
Nucleotide sequence shown in ID No.2, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.
The sequence as shown in SEQ ID No.2 that inventor designs synthesis can smoothly express acquisition notch acceptors.
Specifically, Gal4-VP64 transcription activating proteins expressing gene is used to express Gal4-VP64 transcription activating proteins.
Gal4-UAS promoters in Gal4-VP64 transcription activating proteins the second fusion protein of more enough activation, so as to induce the second fusion
Mesothelin antibody expression in albumen.
In one embodiment, Gal4-VP64 transcription activating proteins expressing gene includes：(a), SEQ ID No.3 institutes
Nucleotide sequence shown in the nucleotide sequence shown, (b) and SEQ ID No.3 has the nucleotides sequence of at least 95% homology
Row, or the nucleotide sequence shown in (c), SEQ ID No.3, wherein one or more bases are lacked, substituted or increased and obtained
Nucleotide sequence.
In one embodiment, the first expressing fusion protein gene is included shown in the SEQ ID No.1 that are sequentially connected
The nucleotide sequence shown in nucleotide sequence and SEQ ID No.3 shown in nucleotide sequence, SEQ ID No.2.The nucleotides
The expression of polypeptides formula that the polynucleotide encoding of sequence composition obtains is anti muc-1-synNotch-Gal4VP64.
Specifically, Gal4-UAS promoters expressing gene is used to express Gal4-UAS promoters.Gal4-UAS promoters open
The expression of dynamic mesothelin antibody.
In one embodiment, Gal4-UAS promoters expressing gene includes：(a), the nucleosides shown in SEQ ID No.4
Nucleotide sequence of the nucleotide sequence with least 95% homology shown in acid sequence, (b) and SEQ ID No.4, or (c),
Nucleotide sequence shown in SEQ ID No.4, wherein one or more bases are lacked, substituted or increased obtained nucleotides sequence
Row.
Specifically, mesothelin antibody expressing genes are used to express mesothelin antibody.Mesothelin (mesothelin) is in tumour
Expression quantity is higher in cell, and normal tissue expression amount is relatively low or hardly expresses, therefore mesothelin (mesothelin) energy
Enough " preferable antigen " as treatment tumour.Mucin1 (muc-1) and mesothelin (mesothelin) form dual anti-original signal,
In the case where being had ready conditions existing for dual anti-original signal, the Chimeric antigen receptor starts immune attack, kills tumour cell.
In one embodiment, mesothelin antibody expressing genes expressing gene includes：(a), shown in SEQ ID No.5
Nucleotide sequence shown in nucleotide sequence, (b) and SEQ ID No.5 has the nucleotide sequence of at least 95% homology, or
(c), the nucleotide sequence shown in SEQ ID No.5, wherein one or more bases are lacked, substituted or increased obtained nucleosides
Acid sequence.Above-mentioned nucleotide sequence, which can be expressed smoothly, obtains mesothelin antibody.
In one embodiment, the second expressing fusion protein gene also includes terminator expressing gene, terminator expression
Gene is connected with mesothelin antibody expressing genes, and terminator expressing gene includes：(a), the nucleotides sequence shown in SEQ ID No.6
Nucleotide sequence shown in row, (b) and SEQ ID No.6 has the nucleotide sequence of at least 95% homology, or (c), SEQ
Nucleotide sequence shown in ID No.6, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.
Terminator is set further to adjust the expression of the second fusion protein.
In one embodiment, the second expressing fusion protein gene is included shown in the SEQ ID No.4 that are sequentially connected
The nucleotide sequence shown in nucleotide sequence and SEQ ID No.6 shown in nucleotide sequence, SEQ ID No5.The nucleotides sequence
The expression of polypeptides formula that the polynucleotide encoding of row composition obtains is Gal4-UAS promoter-anti mesothelin CAR-
SV40_PA_terminator。
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 tumour.
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
PLV-IRES-Puro, the first expressing fusion protein gene of above-mentioned first expressing fusion protein gene is inserted respectively into slow disease
The T cell that Chimeric antigen receptor modification is obtained into T cell is transfected in poisonous carrier again.
The Chimeric antigen receptor (chimeric antigen receptor, CAR) of one embodiment, including the first fusion
Albumen and the second fusion protein.First fusion protein includes Mucin1 antibody, notch acceptors and the Gal4- being sequentially connected
VP64 transcription activating proteins.Second fusion protein includes Gal4-UAS promoters and the mesothelin antibody being sequentially connected.
Specifically, Mucin1 antibody, notch acceptors, Gal4-VP64 transcription activating proteins, Gal4-UAS promoters and
The concrete function and property of mesothelin antibody can be found in described above, and therefore not to repeat here.
In one embodiment, Mucin1 antibody includes：(a), as the nucleotide sequence group shown in SEQ ID No.1
Into the obtained polypeptide of polynucleotide encoding, (b), have with the polynucleotides of the nucleotide sequence composition shown in SEQ ID No.1
The polypeptide that the polynucleotide encoding for having at least 98% homology obtains, or (c), as the nucleotide sequence shown in SEQ ID No.1
The polynucleotides of composition, wherein one or more bases lacked, substituted or increased obtained polynucleotide encoding obtain it is more
Peptide.
In one embodiment, notch acceptors include：(a), it is made up of the nucleotide sequence shown in SEQ ID No.2
The obtained polypeptide of polynucleotide encoding；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.2 have
The polypeptide that the polynucleotide encoding of at least 98% homology obtains；Or (c), as the nucleotide sequence group shown in SEQ ID No.2
Into polynucleotides, wherein one or more bases are lacked, substituted or are increased the polypeptide that obtained polynucleotide encoding obtains.
In one embodiment, Gal4-VP64 transcription activating proteins include：(a), as the core shown in SEQ ID No.3
The polypeptide that the polynucleotide encoding of nucleotide sequence composition obtains；(b), formed with the nucleotide sequence shown in SEQ ID No.3
Polynucleotides have the polypeptide that the polynucleotide encoding of at least 98% homology obtains；Or (c), as shown in SEQ ID No.3
The polynucleotides of nucleotide sequence composition, wherein one or more bases are lacked, substituted or increased obtained polynucleotides and compiled
The polypeptide that code obtains.
In one embodiment, Gal4-UAS promoters include：(a), as the nucleotide sequence shown in SEQ ID No.4
The polypeptide that the polynucleotide encoding of composition obtains；(b), the polynucleotides formed with the nucleotide sequence shown in SEQ ID No.4
The polypeptide that polynucleotide encoding with least 98% homology obtains；Or (c), as the nucleotides sequence shown in SEQ ID No.4
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, mesothelin antibody includes：(a), it is made up of the nucleotide sequence shown in SEQ ID No.5
The obtained polypeptide of polynucleotide encoding；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.5 have
The polypeptide that the polynucleotide encoding of at least 98% homology obtains；Or (c), as the nucleotide sequence group shown in SEQ ID No.5
Into polynucleotides, wherein one or more bases are lacked, substituted or are increased the polypeptide that obtained polynucleotide encoding obtains.
In one embodiment, the second fusion protein also includes terminator, and terminator is connected with mesothelin antibody.Terminate
Attached bag includes：(a), the polypeptide that the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.6 obtains；(b), with
The polynucleotide encoding that the polynucleotides of nucleotide sequence composition shown in SEQ ID No.6 have at least 98% homology obtains
Polypeptide；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.6, wherein one or more base quilts
Missing, substitute or increase the polypeptide that obtained polynucleotide encoding obtains.
Further, the first fusion protein includes Mucin1 antibody, notch acceptors and the Gal4-VP64 being sequentially connected
Transcription activating protein.The expression formula of first fusion protein is anti muc-1-synNotch-Gal4VP64.
Further, the second fusion protein includes Gal4-UAS promoters, mesothelin antibody and the terminator being sequentially connected.
The expression formula of second fusion protein is Gal4-UAS promoter-anti mesothelin CAR-SV40_PA_
terminator。
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 tumour etc..When tumor cell surface expression glues
When albumen -1 (muc-1) and mesothelin (mesothelin) two kinds of tumor markers, possess Mucin1 (muc-1) and mesothelin
(mesothelin) dual anti-original signal, the Chimeric antigen receptor start immune attack, kill tumour cell.Work as Mucin1
(muc-1) and mesothelin (mesothelin) dual anti-original signal be not present or only one of which when, the Chimeric antigen receptor
Immune attack will not be started, be closed.Therefore Mucin1 (muc-1) and the dual anti-former letter of mesothelin (mesothelin)
Number it is Chimeric antigen receptor behavior " switch " in T cell, immune response is just produced under the conditions of specific dual anti-original signal, it is real
Existing Chimeric antigen receptor immune response is controllable, and treatment side effect is few, and specificity is high.
Above-mentioned Chimeric antigen receptor (CAR) can be used in anti-tumor drug.
In the application of one embodiment, Chimeric antigen receptor (CAR) is used in the medicine of anti-breast cancer.
The T cell (CAR-T) of the Chimeric antigen receptor modification of the dual anti-former regulation of one embodiment, T cell in the T cell
It is middle to have imported above-mentioned Chimeric antigen receptor expressing gene.Or above-mentioned expression vector is transfected in the T cell.Or the T is thin
Born of the same parents 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, Chimeric antigen receptor modification T cell in Mucin1 antibody with
Mucin1 on tumour cell combines, and itself cutting occurs for notch acceptors, discharges Gal4-VP64 transcription activating proteins.
Gal4-VP64 transcription activating proteins activate the Gal4-UAS promoters of the second fusion protein, and then start the table of mesothelin antibody
Reach.When tumor cell surface expression Mucin1 (muc-1) and mesothelin (mesothelin) two kinds of tumor markers, possess
Mucin1 (muc-1) and mesothelin (mesothelin) dual anti-original signal, respectively with Mucin1 antibody and mesothelin antibody
With reference to the T cell of Chimeric antigen receptor modification is activated, and then starts immune attack, kills tumour cell.
When Mucin1 (muc-1) and mesothelin (mesothelin) dual anti-original signal are not present or only one of which
When, the Chimeric antigen receptor will not start immune attack, be closed.
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 the T cell (CAR-T) of the Chimeric antigen receptor modification of above-mentioned dual anti-former regulation is specific to expressing
The tumour cell of Mucin1 (muc-1) and mesothelin (mesothelin) dual anti-original signal has specific Efficient killing effect to make
With.And to not expressing the cell of Mucin1 (muc-1) and mesothelin (mesothelin) or only expressing one of which antigen
Cell, the Chimeric antigen receptor will not start immune attack, be closed, to normal killing functions of immunocytes very little.Cause
This, the T cell (CAR-T) of Chimeric antigen receptor modification is expected to apply in anti-tumor drug, and one is provided for oncotherapy
Kind thinking.The T cell of Chimeric antigen receptor modification realizes that Chimeric antigen receptor immune response is controllable, and treatment side effect is few.
The T cell (CAR-T) of the Chimeric antigen receptor modification of above-mentioned dual anti-former 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 of the Chimeric antigen receptor modification of dual anti-former regulation is used in anti-breast cancer
Medicine in.
Referring to Fig. 3, the preparation method of the T cell of the Chimeric antigen receptor modification of the dual anti-former regulation of an embodiment,
Comprise the following steps S110~S130.
S110, Chimeric antigen receptor expressing gene is provided, the Chimeric antigen receptor expressing gene includes the first fusion protein
Expressing gene and the second expressing fusion protein gene, the first expressing fusion protein gene include the Mucin1 antibody being sequentially connected
Expressing gene, notch expression of receptor gene and Gal4-VP64 transcription activating protein expressing genes, the second expressing fusion protein base
Because of the Gal4-UAS promoters expressing gene and mesothelin antibody expressing genes that include being sequentially connected.
Specifically, the structure of Chimeric antigen receptor (CAR) refers to described above, and therefore not to repeat here.
In one embodiment, the second fusion protein also includes terminator, and terminator is connected with mesothelin antibody.
In one embodiment, the expression formula of the first fusion protein of coding is anti muc-1-synNotch-
Gal4VP64.The expression formula of second fusion protein of coding is Gal4-UAS promoter-anti mesothelin CAR-
SV40_PA_terminator。
Specifically, synthesized by way of gene chemical synthesis anti muc-1-synNotch-Gal4VP64 nucleotide sequence with
And Gal4-UAS promoter-anti mesothelin CAR-SV40_PA_terminator nucleotide sequence.It is fitted together to
Antigen receptor expressing gene.
S120, the first expressing fusion protein gene obtained in S110 is connected in slow virus carrier, obtains the first table
It is connected to up to carrier, and by the second expressing fusion protein gene obtained in S110 in slow virus carrier, obtains the second expression load
Body.
Specifically, the first expressing fusion protein gene of acquisition, the second expressing fusion protein gene are passed through restricted interior
Enzyme cutting double digestion, it is then attached in the slow virus carrier handled by identical restriction enzymes double zyme cutting.
In one embodiment, restriction enzyme is, for example, to include Nde I, Xho I, EcoR I or BamH I etc..
In one embodiment, slow virus carrier pLV-IRES-Puro.
S130, the first expression vector obtained in S120 and the second expression vector are packaged as slow virus and transfected thin to T
In born of the same parents, the T cell of the Chimeric antigen receptor modification of dual anti-former regulation is obtained.
Specifically, the first expression vector obtained in S120 and the second expression vector are first transfected into host cell such as 239T
In cell, amplification obtains substantial amounts of slow virus.Then by the slow-virus transfection of certain virus titer into T cell, obtain dual anti-
The T cell (CAR-T) of the Chimeric antigen receptor modification of original regulation.
In one embodiment, expression vector is packaged as slow virus and transfected into the operation in T cell, viral MOI
Value about 10.
The method of the T cell of the above-mentioned Chimeric antigen receptor modification for preparing dual anti-former regulation, it is easy to operate, prepare
The T cell of Chimeric antigen receptor modification just produces immune response under specific dual anti-former existence condition, realize chimeric antigen by
Body 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 dual anti-former regulation
The first expressing fusion protein gene that the first fusion protein of coding is respectively synthesized by genome company (including connects successively
Shown in the nucleotide sequence shown in SEQ ID No.1 connect, nucleotide sequence and SEQ ID No.3 shown in SEQ ID No.2
Nucleotide sequence.The expression formula of first fusion protein is anti muc-1-synNotch-Gal4VP64.
By EcoR I and BamH I double digestion the first expressing fusion protein genes, and clone into slow virus carrier pLV-
In IRES-Puro, the first expression vector is obtained.The first expression vector EcoR I and BamH the I double digestions built are reflected
Fixed, (2.4kb fragments are purpose fragment to digestion products electrophoretogram in wherein Fig. 4, and 8.1kb fragments are carrier piece in itself as shown in Figure 4
Section).The size of purpose fragment is consistent with expection, illustrates vector construction success.
The second expressing fusion protein gene that the second fusion protein of coding is respectively synthesized by genome company (including connects successively
The nucleotide sequence shown in SEQ ID No.4 connect, nucleotide sequence and SEQ ID No.6 shown in SEQ ID No.5).The
The expression formula of two fusion proteins is Gal4-UAS promoter-anti mesothelin CAR-SV40_PA_terminator.
By Nde I and Xho I double digestion the second expressing fusion protein genes, and clone into slow virus carrier pLV-
In IRES-Puro, the second expression vector is obtained.The second expression vector Nde I and Xho the I double digestions built are reflected
Fixed, (2kb fragments are purpose fragment to digestion products electrophoretogram in wherein Fig. 5, and 9.6kb fragments are carrier piece in itself as shown in Figure 5
Section).The size of purpose fragment is consistent with expection, illustrates vector construction success.
It is slow virus by the first expression vector of above-mentioned acquisition and the second expression vector, and (viral MOI values are distinguished by virus
It is 10) to transfect into Jurkat E6.1 cells (T lymphocyte strains, purchased from U.S. typical case thing collection, ATCC), obtains double
The Jurkat cell (JurkatCAR) of the Chimeric antigen receptor modification of antigen 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, the dual anti-former regulation with puromycin-resistant is established
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 fig. 6, Jurkat E6.1 growth conditions are good.
Test one
The T cell proliferative conditions measure of the Chimeric antigen receptor modification of dual anti-former regulation
By 5 × 105The JurkatCAR cells prepared in individual embodiment 1 are separately added into containing U-2OS, U-2OS/muc-1+、
U-2OS/muc-1+/mesothelin+、A431、A431/mesothelin+, MCF7 cells 6 orifice plates (5 × 105/ hole) in.Its
In, U-2OS is human osteosarcoma cell, and A431 is people's epidermis cancer cell, and MCF7 is human breast cancer cell, U-2OS, A431 and
MCF7 is purchased from ATCC.U-2OS/muc-1+Represent to make U-2OS cross table by muc-1 genetic modifications on the basis of U-2OS cells
Up to muc-1 antigens.U-2OS/muc-1+/mesothelin+Represent to pass through muc-1 and mesothelin on the basis of U-2OS cells
Genetic modification, U-2OS is set to be overexpressed muc-1 antigens and mesothelin antigens.A431/mesothelin+Represent thin in A431
The basis of born of the same parents makes A431 be overexpressed mesothelin antigens by mesothelin genetic modifications.In above-mentioned cell, U-2OS is not
Express muc-1 antigens and mesothelin antigens.U-2OS/muc-1+Muc-1 antigens can only be expressed with A431.U-2OS/muc-1+/mesothelin+、A431/mesothelin+Muc-1 antigens and mesothelin antigens can be expressed with MCF7.
Respectively in the 3rd day (D3), the 7th day (D7), cell count is carried out to the JurkatCAR cells of suspension.As a result such as Fig. 7
It is shown, with muc-1 (Mucin1) and the U-2OS/muc-1+/mesothelin of mesothelin (mesothelin) double antigen presentations
+, after A431/mesothelin+, MCF7 cells contacting, JurkatCAR can largely breed.And the JurkatCAR of other groups is basic
Do not breed.Result above shows, when simultaneously two kinds of antigen signals of muc-1 (Mucin1) and mesothelin (mesothelin) are present
When, JurkatCAR (T cell of the Chimeric antigen receptor modification of dual anti-former regulation) can largely breed.Be not present when dual signal or
When only existing a kind of signal, JurkatCAR (T cell of the Chimeric antigen receptor modification of dual anti-former regulation) does not breed substantially.
Test two
IFN γ secretory volume determines in the T cell of the Chimeric antigen receptor modification of dual anti-former regulation
By 5 × 105The JurkatCAR cells prepared in individual embodiment 1 in 24 orifice plates with U-2OS, U-2OS/muc-1+、
U-2OS/muc-1+/mesothelin+、A431、A431/mesothelin+, MCF7 cells (5 × 105/ hole) co-culture.
Supernatant is collected after 72 hours, IFN γ is detected by the ELISA detection kit (BD Biosciences) of IFN γ
Secretory volume.
As a result as shown in figure 8, with muc-1 (Mucin1) and the U- of mesothelin (mesothelin) two kinds of antigen presentations
2OS/muc-1+/mesothelin+、A431/mesothelin+, MCF7 cells co-culture after, JurkatCAR can largely secrete
IFN-γ；Other group not secretion of gamma-IFN substantially.Result above shows, when muc-1 (Mucin1) and mesothelin (mesotheliums
Element) two kinds of antigens exist simultaneously, and JurkatCAR (T cell that the Chimeric antigen receptor of dual anti-former regulation is modified) can identify swollen
Oncocyte target spot simultaneously can secrete more IFN-γs after being activated.When dual anti-original signal is not present or only exists a kind of signal,
JurkatCAR (T cell of the Chimeric antigen receptor modification of dual anti-former regulation) only secretes a small amount of or not secretion of gamma-IFN substantially.
Test three
The T cell killing effect in vitro measure of the Chimeric antigen receptor modification of dual anti-former regulation
By the JurkatCAR cells prepared in embodiment 1 and target cell U-2OS, U-2OS/muc-1+、U-2OS/muc-1+/
mesothelin+、A431、A431/mesothelin+, MCF7 cells co-culture (effect target than 1:1).Using the double mark methods of CFSE/PI
Detect Cytotoxicity in vitro ability of the JurkatCAR cells after genetic modification to different type tumour cell.
Method is as follows：With final concentration of 0.05 μM of CFSE to 1 × 106Individual target cell carries out living cells dye marker, by 1
×106Individual JurkatCAR cells and 1 × 106The target cell of individual CFSE marks mixes, and 200g, 1min centrifugation make cell phase mutual connection
Touch, in 5%CO2, 4h is incubated in 37 DEG C of incubators altogether, after reaction terminates, 1 μ g/ml PI dye liquors is added, mixes, room temperature lucifuge is incubated
Flow cytometer detection is carried out after educating 15min.
As a result as shown in Figure 9 and Figure 10, right upper quadrant cell mass is killed cell proportion in Fig. 9.JurkatCAR energy
The effectively U-2OS/muc-1 of killing muc-1 (Mucin1) and mesothelin (mesothelin) double antigen presentations+/
mesothelin+、A431/mesothelin+, MCF7 cells, and other group of cell does not kill substantially.Result above shows, when
Two kinds of antigens of muc-1 (Mucin1) and mesothelin (mesothelin) exist simultaneously, and (dual anti-original adjusts embedding JurkatCAR
Close the T cell of antigen receptor modification) it can identify and effectively kill target cell.Dual anti-original signal is not present or only existed a kind of letter
Number when, JurkatCAR (T cell of the Chimeric antigen receptor modification of dual anti-former regulation) does not kill target cell substantially.
To sum up, the T cell (JurkatCAR) of the Chimeric antigen receptor modification of above-mentioned dual anti-former regulation is (viscous to expression muc-1
Albumen -1) and the dual anti-former tumour cells of mesothelin (mesothelin) there is specific Efficient killing effect to act on, and mucoprotein -
1 (muc-1) and mesothelin (mesothelin) dual anti-original signal be not present or only one of which when, to killing functions of immunocytes
Very little.Therefore, the T cell of Chimeric antigen receptor modification can be used as anti-tumor drug, and a kind of think of is provided for oncotherapy
Road.The T cell of Chimeric antigen receptor modification is in specific muc-1 (Mucin1) and mesothelin (mesothelin) dual anti-original
Immune response is just produced under existence condition, realizes that Chimeric antigen receptor immune response is controllable, treatment side effect is few.
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 dual anti-former regulation are modified
With
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 795
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atggcgctcc ctgtcaccgc actgcttctt ccgctggcac tgctgctgca cgctgcacgg 60
cctgaggtgc agctgcagca gagcggcggc ggcctggtgc agcccggcgg cagcatgaag 120
ctgagctgcg tggccagcgg cttcaccttc agcaactact ggatgaactg ggtgcgccag 180
agccccgaga agggcctgga gtgggtggcc gagatccgcc tgaagagcaa caactacgcc 240
acccactacg ccgagagcgt gaagggccgc ttcaccatca gccgcgacga cagcaagagc 300
agcgtgtacc tgcagatgaa caacctgcgc gccgaggaca ccggcatcta ctactgcacc 360
ttcggcaaca gcttcgccta ctggggccag ggcaccaccg tgaccgtgag cagcggcggc 420
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatcgtggt gacccaggag 480
agcgccctga ccaccagccc cggcgagacc gtgaccctga cctgccgcag cagcaccggc 540
gccgtgacca ccagcaacta cgccaactgg gtgcaggaga agcccgacca cctgttcacc 600
ggcctgatcg gcggcaccaa caaccgcgcc cccggcgtgc ccgcccgctt cagcggcagc 660
ctgatcggcg acaaggccgc cctgaccatc accggcgccc agaccgagga cgaggccatc 720
tacttctgcg ccctgtggta cagcaaccac tgggtgttcg gcggcggcac caagctgacc 780
gtgctgggca gcgag 795
<210> 3
<211> 1008
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atcctggact acagcttcgg gggtggggcc gggcgcgaca tccccccgcc gctgatcgag 60
gaggcgtgcg agctgcccga gtgccaggag gacgcgggca acaaggtctg cagcctgcag 120
tgcaacaacc acgcgtgcgg ctgggacggc ggtgactgct ccctcaactt caatgacccc 180
tggaagaact gcacgcagtc tctgcagtgc tggaagtact tcagtgacgg ccactgtgac 240
agccagtgca actcagccgg ctgcctcttc gacggctttg actgccagcg tgcggaaggc 300
cagtgcaacc ccctgtacga ccagtactgc aaggaccact tcagcgacgg gcactgcgac 360
cagggctgca acagcgcgga gtgcgagtgg gacgggctgg actgtgcgga gcatgtaccc 420
gagaggctgg cggccggcac gctggtggtg gtggtgctga tgccgccgga gcagctgcgc 480
aacagctcct tccacttcct gcgggagctc agccgcgtgc tgcacaccaa cgtggtcttc 540
aagcgtgacg cacacggcca gcagatgatc ttcccctact acggccgcga ggaggagctg 600
cgcaagcacc ccatcaagcg tgccgccgag ggctgggccg cacctgacgc cctgctgggc 660
caggtgaagg cctcgctgct ccctggtggc agcgagggtg ggcggcggcg gagggagctg 720
gaccccatgg acgtccgcgg ctccatcgtc tacctggaga ttgacaaccg gcagtgtgtg 780
caggcctcct cgcagtgctt ccagagtgcc accgacgtgg ccgcattcct gggagcgctc 840
gcctcgctgg gcagcctcaa catcccctac aagatcgagg ccgtgcagag tgagaccgtg 900
gagccgcccc cgccggcgca gctgcacttc atgtacgtgg cggcggccgc ctttgtgctt 960
ctgttcttcg tgggctgcgg ggtgctgctg tcccgcaagc gccggcgg 1008
<210> 2
<211> 636
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atgaagctgc tgagcagcat cgagcaggcc tgtgacatct gccggctgaa gaaactgaag 60
tgcagcaaag aaaagcccaa gtgcgccaag tgcctgaaga acaactggga gtgccggtac 120
agccccaaga ccaagagaag ccccctgacc agagcccacc tgaccgaggt ggaaagccgg 180
ctggaaagac tggaacagct gtttctgctg atcttcccac gcgaggacct ggacatgatc 240
ctgaagatgg acagcctgca ggacatcaag gccctgctga ccggcctgtt cgtgcaggac 300
aacgtgaaca aggacgccgt gaccgacaga ctggccagcg tggaaaccga catgcccctg 360
accctgcggc agcacagaat cagcgccacc agcagcagcg aggaaagcag caacaagggc 420
cagcggcagc tgacagtgtc tgctgctgca ggcggaagcg gaggctctgg cggatctgat 480
gccctggacg acttcgacct ggatatgctg ggcagcgacg ccctggatga ttttgatctg 540
gacatgctgg gatctgacgc tctggacgat ttcgatctcg acatgttggg atcagatgca 600
ctggatgact ttgacctgga catgctcgga tcatga 636
<210> 4
<211> 243
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
catatgggcc taactggccg gtaccgagtt tctagacgga gtactgtcct ccgagcggag 60
tactgtcctc cgactggagc ggagtactgt cctccgatcg gagtactgtc ctccgcgaat 120
tccggagtac tgtcctccga agacgctagc ggggggctat aaaagggggt gggggcgttc 180
gtcctcactc tagatctgcg atctaagtaa gcttggcaat ccggtactgt tggtaaagcc 240
acc 243
<210> 6
<211> 1608
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
atggccttac cagtgaccgc cttgctcctg ccgctgccgc tggccttgct gctccacgcc 60
gccaggccgg gatcccaggt acaactgcag cagtctgggc ctgagctgga gaagcctggc 120
gcttcagtga agatatcctg caaggcttct ggttactcat tcactggcta caccatgaac 180
tgggtgaagc agagccatgg aaagagcctt gagtggattg gacttattac tccttacaat 240
ggtgcttcta gctacaacca gaagttcagg ggcaaggcca cattaactgt agacaagtca 300
tccagcacag cctacatgga cctcctcagt ctgacatctg aagactctgc agtctatttc 360
tgtgcaaggg ggggttacga cgggaggggt tttgactact ggggccaagg gaccacggtc 420
accgtctcct caggtgtagg cggttcaggc ggcggtggct ctggcggtgg cggatcggac 480
atcgagctca ctcagtctcc agcaatcatg tctgcatctc caggggagaa ggtcaccatg 540
acctgcagtg ccagctcaag tgtaagttac atgcactggt accagcagaa gtcaggcacc 600
tcccccaaaa gatggattta tgacacatcc aaactggctt ctggagtccc aggtcgcttc 660
agtggcagtg ggtctggaaa ctcttactct ctcacaatca gcagcgtgga ggctgaagat 720
gatgcaactt attactgcca gcagtggagt ggttaccctc tcacgttcgg tgctgggaca 780
aagttggaaa tcaaagctag caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgtgattttt gggtgctggt ggtggttggt 960
ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1020
agtaagagga gcaggggagg gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1200
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1260
gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1320
cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1380
cgtggccggg accctgagat ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc 1440
ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1500
ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1560
aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctaa 1608
<210> 5
<211> 171
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ggccgcgact ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg 60
atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt 120
gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacctcga g 171
Claims (10)
1. a kind of Chimeric antigen receptor expressing gene, it is characterised in that merged including the first expressing fusion protein gene and second
Protein-encoding gene；
The first expressing fusion protein gene includes Mucin1 antibody expressing genes, the notch expression of receptor being sequentially connected
Gene and Gal4-VP64 transcription activating protein expressing genes；
The second expressing fusion protein gene includes the Gal4-UAS promoters expressing gene and mesothelin antibody being sequentially connected
Expressing gene.
2. Chimeric antigen receptor expressing gene according to claim 1, it is characterised in that the Mucin1 antibody expression
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；And/or
The notch expression of receptor gene includes：(a), the nucleotide sequence shown in SEQ ID No.2, (b) and SEQ ID
Nucleotide sequence shown in No.2 has the nucleotide sequence of at least 95% homology, or the core shown in (c), SEQ ID No.2
Nucleotide sequence, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence；And/or
The Gal4-VP64 transcription activating proteins expressing gene includes：(a), the nucleotide sequence shown in SEQ ID No.3,
(b), there is the nucleotide sequence of at least 95% homology, or (c), SEQ ID with the nucleotide sequence shown in SEQ ID No.3
Nucleotide sequence shown in No.3, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.
3. Chimeric antigen receptor expressing gene according to claim 1, it is characterised in that the Gal4-UAS starts sublist
Include up to gene：(a) nucleotide sequence shown in, the nucleotide sequence shown in SEQ ID No.4, (b) and SEQ ID No.4
Nucleotide sequence with least 95% homology, or the nucleotide sequence shown in (c), SEQ ID No.4, one of them or it is more
Individual base is lacked, substituted or increased obtained nucleotide sequence；And/or
The mesothelin antibody expressing genes expressing gene includes：(a), the nucleotide sequence shown in SEQ ID No.5, (b), with
Nucleotide sequence shown in SEQ ID No.5 has the nucleotide sequence of at least 95% homology, or (c), SEQ ID No.5 institutes
The nucleotide sequence shown, wherein one or more bases are lacked, substituted or increased obtained nucleotide sequence.
4. Chimeric antigen receptor expressing gene according to claim 1, it is characterised in that second expressing fusion protein
Gene also includes terminator expressing gene, and the terminator expressing gene is connected with the mesothelin antibody expressing genes, described
Terminator expressing gene includes：(a) core shown in, the nucleotide sequence shown in SEQ ID No.6, (b) and SEQ ID No.6
Nucleotide sequence has the nucleotide sequence of at least 95% homology, or the nucleotide sequence shown in (c), SEQ ID No.6, wherein
One or more bases are lacked, substituted or increased obtained nucleotide sequence.
5. a kind of expression vector, it is characterised in that containing embedding as described in any one of Claims 1 to 4 in the expression vector
Close antigen receptor expressing gene.
6. a kind of Chimeric antigen receptor, it is characterised in that including the first fusion protein and the second fusion protein；
First fusion protein includes Mucin1 antibody, notch acceptors and the Gal4-VP64 transcriptional activation eggs being sequentially connected
In vain；
Second fusion protein includes Gal4-UAS promoters and the mesothelin antibody being sequentially connected.
7. Chimeric antigen receptor according to claim 6, it is characterised in that
The Mucin1 antibody includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.1
Obtained polypeptide, (b), the polynucleotides formed with the nucleotide sequence shown in SEQ ID No.1 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.1, its
Middle one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The notch acceptors include：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.2 obtains
Polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.2 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.2, wherein one
Individual or multiple bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The Gal4-VP64 transcription activating proteins include：(a), the multinuclear being made up of the nucleotide sequence shown in SEQ ID No.3
Thuja acid encodes obtained polypeptide；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.3 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.3
Polynucleotides, wherein one or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains.
8. Chimeric antigen receptor according to claim 6, it is characterised in that second fusion protein also includes terminating
Son, the terminator are connected with the mesothelin antibody, in second fusion protein,
The Gal4-UAS promoters include：(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；And/or
The mesothelin antibody includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.5 obtains
The polypeptide arrived；(b), the polynucleotides with the nucleotide sequence composition shown in SEQ ID No.5 have at least 98% homology
The polypeptide that polynucleotide encoding obtains；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.5, wherein
One or more bases are lacked, substituted or increased the polypeptide that obtained polynucleotide encoding obtains；And/or
The terminator includes：(a), the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.6 obtains
Polypeptide；(b), there is the multinuclear of at least 98% homology with the polynucleotides of the nucleotide sequence composition shown in SEQ ID No.6
Thuja acid encodes obtained polypeptide；Or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.6, one of them
Or multiple 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 dual anti-former 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 4, or transfected such as claim 5 in the T cell
Described expression vector, or the T cell can express the Chimeric antigen receptor as described in any one of claim 6~8.
10. Chimeric antigen receptor expressing gene, expression as claimed in claim 5 carry as described in any one of Claims 1 to 4
Body, the Chimeric antigen receptor as described in any one of claim 6~8 or dual anti-original as claimed in claim 9 adjust chimeric
Application of the T cell of antigen receptor modification in anti-tumor drug is prepared.
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| CN113388643A (en) * | 2021-06-18 | 2021-09-14 | 廖文强 | Gene sequence with molecular switch, plasmid and immune cell |
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| CN113248619A (en) * | 2020-02-10 | 2021-08-13 | 上海帝鹤思医疗科技有限公司 | Double-targeting chimeric antigen receptor, coding gene and recombinant expression vector |
| CN113388643A (en) * | 2021-06-18 | 2021-09-14 | 廖文强 | Gene sequence with molecular switch, plasmid and immune cell |
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