CN107488636A - A kind of immunocyte of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch and its application - Google Patents
A kind of immunocyte of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch and its application Download PDFInfo
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Abstract
The invention discloses a kind of immunocyte of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch and its application, the safety-type Chimeric antigen receptor gene containing anti-HER2 in the immunocyte of the anti-HER2 Chimeric antigen receptors modification for carrying molecular switch, the safety-type Chimeric antigen receptor genes of anti-HER2 include HER2 single-chain antibody and have linked a molecular switch, the single-chain antibody of the HER2 includes CD8 Leader, HER2 lands, CD8 hinge area, cross-film stimulus structure domain, CD3 ζ stimulus signal conducting regions, the inventive structure stability is high, prevent because of effect or immune factor the release syndrome pathogenic injury caused by patient of missing the target, security is higher.
Description
Technical field
The present invention relates to field of biological genes, is a kind of anti-HER2 chimeric antigens for carrying molecular switch in particular
The immunocyte of acceptor modification and its application.
Background technology
In tumor therapeuticing method, in addition to the operation, radiotherapy, chemotherapy of routine, Chimeric antigen receptor (Chimeric
Antigen Receptor, CAR) T cell treatment because have it is numerous the advantages of turn into study hotspot.The original of CAR-T cell therapies
Reason is that T cell is modified, and the T cell after modification can obtain specific recognition tumor associated antigen and attack killing tumour
The ability of cell, both normal cell can will not be injured with target killing tumor cell again, and reach the purpose for removing tumour.Relatively
In Radiotherapy chemotherapy method function of immune system declines, the state of an illness easily recurs to caused by patient and drug therapy caused by drug resistance etc.
Shortcoming, this method toxic side effect are smaller.CAR-T cell therapies have been used to clinical treatment malignant tumour, and achieve some and face
Bed curative effect, the most prominent with CD19-CAR-T and CD20-CAR-T cell therapies lymphocytoma and leukaemia, some have entered
Enter II clinical trial phase.Therefore, CAR-T cells are as a kind of brand-new biological immune treatment method, in the targeted therapy of tumour
Aspect has a good application prospect.
CAR-T cells adoptive immunotherapy is effective to most of cancers, but can produce some serious adverse reactions,
CAR-T cells are there may be the attack of the mistake for normal tissue cell (effect of missing the target), or discharge substantial amounts of cell factor and to cause
Cytokine storm, the toxic side effects such as death can be caused when serious.Currently reported causes to suffer from because injecting CAR-T
The dead case of person (causes death and drawn for CD19 specific recognition CAIX antigens for lung's ponding caused by HER2
The hepatotoxicity phenomenon risen).In order to solve this safety risks, most popular method is to introduce a suicide machinery at present, is made
CAR-T cell co expression CAR and suicide gene, had both ensured the fragmentation effect to tumour, improved the security for the treatment of again.
Proto-oncogene people epithelial growth factor receptor 2 (human epidermal growth factor receptor 2,
Her-2 HER2 or c-erbB-2 genes) are also known as, transmembrane receptor protein is encoded, there is tyrosine kinase activity.Monoclonal antibody can
Suppressing tumour growth by being transferred under HER2, HER2 Overexpressions can cause cell hyperproliferation and phenotype vicious transformation,
Therefore, HER2 is tumor recurrence and the independent prognostic factor of life cycle length.HER2 genes be growth with breast cancer, invasion and attack,
Relevant important oncogene is shifted, research shows that aggressive patient with breast cancer's altimeter reaches HER2 genes, causes these cancer cells
The HER2 albumen on surface increases, also referred to as HER2 protein overexpressions.The overexpression of HER2 albumen, it will stimulate cancer cell is mad to increase
It is long, invasion increase, and then cause the formation of cancer.Correlative study finds that HER2 protein overexpressions and patient with breast cancer's is disease-free
There is certain relation the life cycle of existence summation.Breast cancer incidence has occupied the various malignant tumour forefront of women, and its incidence and
Case fatality rate has the trend risen year by year.In China, 30% patient with breast cancer is HER2 breast cancer patients with positive, and this kind of patient swells
Oncocyte grade malignancy faster, progression of disease speed faster, be more easy to occur transfer and recurrence and prognosis it is bad.Study and also found,
In addition to Her2 is overexpressed generation in breast cancer, its overexpression is also more in stomach cancer, colon cancer, lung cancer, oophoroma, cervical carcinoma etc.
Occur in the malignant cancer of kind form, significant.
The content of the invention
In order to make up above deficiency, the invention provides a kind of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch
Immunocyte and its application.
The solution of the present invention is：
A kind of immunocyte for the anti-HER2 Chimeric antigen receptors modification for carrying molecular switch, the carrying molecular switch
The safety-type Chimeric antigen receptor gene containing anti-HER2 in the immunocyte of anti-HER2 Chimeric antigen receptors modification, the anti-HER2 peaces
Holotype Chimeric antigen receptor gene includes HER2 single-chain antibody and has linked a molecular switch, the single-chain antibody of the HER2
Including CD8 Leader, HER2 lands, CD8 hinge area, cross-film-stimulus structure domain, CD3 ζ stimulus signal conducting regions.
As preferable technical scheme, the cross-film-stimulus structure domain be selected from CD8, CD27, CD28, CD137/4-1BB,
All or part of fragment of CD134/OX40, ICOS molecule.
As preferable technical scheme, the molecular switch is the FKBP12-F36V suicide switch members based on Fas genes
Part.
As preferable technical scheme, the molecular switch includes FKBP12-dimerization chemical induction subsystem
Unite (FK506binding protein 12-chemical inducers of dimerizations, FKBP12-CID),
Fkbp ligand body dimer AP1903 (has specificity) to endogenous protein, and Fas genes (neoplasm necrosis-nerve growth
The member of factor acceptor superfamily), the molecular switch is located at N-terminal or C-terminal in the single-chain antibody of the HER2；FKBP12-
F36V includes a FKBP12 domain, and Phe instead of Val on the 36th acid residues sites, and it has high selectivity
With sub- nanomole affinity.Dimer AP1903 can activate the compound that Fas protein-crosslinkings form inducing death, so as to mediate target
Apoptosis.Its operation principle is shown in Fig. 1.
As preferable technical scheme, the immunocyte is that autologous or transgenosis T cell, NK cells, middle grain are thin
Born of the same parents, monocyte, cytotoxic T lymphocyte, regulatory T-cell, memory t cell, bispecific T cell, CIK cell are wherein
One kind.
As preferable technical scheme, the anti-safety-type Chimeric antigen receptor genes of HER2 are by FKBP12-linker-
Fas-T2A-Leader-scFv (HER2)-CD8 α-CD28-CD137-CD3 ζ nucleotide sequence links together, and forms restructuring base
Because of sequence, gene order is obtained by chemical synthesis, forms complete specific chimeric antigen receptor gene DNA.
As preferable technical scheme, FKBP12-linker-Fas-T2A-Leader-scFv (the HER2)-CD8 α-
CD28-CD137-CD3 ζ recombinations are the nucleotide sequence shown in sequence table SEQ ID NO.1.
The invention also discloses a kind of medicine for the treatment of cancer, the anti-HER2 chimeric antigens containing the carrying molecular switch
The immunocyte of acceptor modification.
It is pernicious that the immunocyte of the anti-HER2 Chimeric antigen receptors modification of described carrying molecular switch is used for preparation treatment
The purposes of the medicine of cancer.
Preferable technical scheme, the malignant cancer are breast cancer, stomach cancer, colon cancer, lung cancer, oophoroma, cervical carcinoma etc..
By adopting the above-described technical solution, a kind of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch is immune
Cell, it is described carry molecular switch anti-HER2 Chimeric antigen receptors modification immunocyte in the safety-type inosculating antibody containing anti-HER2
Original receptor gene, the anti-safety-type Chimeric antigen receptor genes of HER2 include HER2 single-chain antibody and have linked a molecule
Switch, the single-chain antibody of the HER2 include CD8 Leader, HER2 lands, CD8 hinge area, cross-film-stimulus structure domain,
CD3 ζ stimulus signal conducting regions.
The invention advantage：
The present invention on the basis of third generation CAR-T receptor technologies, be prepared for it is a kind of common contain HER2 as antigen by
The CAR-T cells of the new suicide gene systems of FKBP of body and Fas genes based on people, the cell had both enhanced thin to breast cancer
The fragmentation effect of born of the same parents, it can stop in time again because miss the target effect or cytokine storm are the potential safety hazard that patient brings.
Of the present invention is the new suicide gene systems of FKBP12-F36V of the Fas genes based on people, with other suicides
Genic system is compared, and its genome structure is relatively stable, can be fast and effectively under FKBP part dimer AP1903 effects
Target cell is killed, prevents from injuring because miss the target effect or immune factor release syndrome cause a disease caused by patient, security is higher.
The new suicide gene systems of FKBP12-F36V of Fas genes of the present invention based on people with the addition of autothermic cracking
Peptide T 2A is connected with CAR structures, T2A by self cleavage function CAR enter cell after both altimeter up to the specificity it is embedding
Antigen receptor gene is closed, expresses molecular switch again, both functions are separate, do not interfere with each other.
Brief description of the drawings
FKBP12-F36V molecular switch schematic diagrams of the Fig. 1 based on Fas genes.
Fig. 2 contains the Anti-HER2 of molecular switch CAR module maps.
Fig. 3 Flow cytometries have expression of the HER2-CAR carriers on T cell surface of molecular switch.Fig. 4
Tumor size change curve after mouse model T lymphocytes have been treated.
Fig. 5 is FKBP12-linker-Fas-T2A-Leader-scFv of the present invention (HER2)-CD8 α-CD28-
CD137-CD3 ζ recombination design drawings.
Embodiment
In order that the technical means, the inventive features, the objects and the advantages of the present invention are easy to understand, tie below
Specific embodiment is closed, the present invention is expanded on further.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
Commercially obtain.
Embodiment 1：Carry the structure of the HER2 specific C AR expression vectors of molecular switch
The CAR modules signal of Anti-HER2 containing molecular switch is shown in that (complete nucleic-acid sequences are shown in annex SEQ ID to Fig. 2
NO.1)。
Carry the Anti-HER2 of molecular switch each module nucleotide sequences of CAR
(1) FKBP-F36V nucleic acid artificial sequence (SEQ ID NO.2)
(2) linker nucleic acid artificial sequence (SEQ ID NO.3)
(3) Fas nucleic acid artificial sequence (SEQ ID NO.4)
(4) autothermic cracking peptide T 2A nucleic acid artificial sequence (SEQ ID NO.5)
(5) sub- Leader nucleic acid artificial sequence (SEQ ID NO.6) is guided
(6) Anti-HER2single chain antibody fragment (scFv) nucleic acid artificial sequence (SEQ ID
NO.7)
(7) CD8Hinge areas nucleic acid artificial sequence (SEQ ID NO.8)
(8) CD28 transmembrane regions nucleic acid artificial sequence (SEQ ID NO.9)
(9) CD137 intracellular regions nucleic acid artificial sequence (SEQ ID NO.10)
(10) CD3 ζ intracellular regions nucleic acid artificial sequence (SEQ ID NO.11)
Respectively by SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ
ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 nucleic acid coding sequence student on commission
Work bioengineering (Shanghai) Co., Ltd. synthesizes its whole expression cassette, is inserted into Lentiviral plent-C-GFP
(Invitrogen) NotI-AsiSI sites (see Fig. 2), E.coli (DH5 α) is transformed into, after being sequenced correctly, used
The plasmid purification kit extraction of Invitrogen companies and plasmid purification, obtain the high-quality plasmid of recombinant expression carrier.
Embodiment 2：Slow virus is packed and titre detection
Slow virus package cell line 293T is inoculated in containing in DMEM+10%FBS 10cm culture dishes, 37 DEG C, 5%
Cultivated under the conditions of CO2, adherent rate is transfected after being 70%-80%.By the recombinant plasmid (about 10 μ g) in embodiment 1 and zero load
Plasmid (about 10 μ g) uses calcium phosphate transfection method cotransfection 293T cells with slow virus packaging plasmid respectively, gently mixes, is placed in
37 DEG C, cultivate 12h in 5%CO2 incubators, add the DMEM fluid nutrient medium 8mL containing 10%FBS, continue culture to 48 hours.
Observe there is egfp expression into the cell under inverted fluorescence microscope after 48h.After 72h, supernatant is collected, removes cell
Fragment, concentrated after harvest is viral, obtain the HER2-CAR virus liquids of concentrated carrying molecular switch, -70 DEG C of Low-temperature Ices
Saved backup in case.According to Lenti-XTMGo StixTMKit (ocean Science and Technology Ltd. of Beijing China product) measure disease
Malicious titre, the results showed that, the titre 3.26 × 10 of recombinant slow virus6pfu/mL。
Embodiment 3：The preparation of HER2 CAR-T cells with molecular switch and the killing detection to tumour cell
After viral concentration carry out T cell transfection, with TBD sample rates separating liquid (purchased from Tianjin Hao oceans China Tech biology) from
Isolated PBMC in peripheral blood.After T cell is activated, 1 × 10 is taken out6Individual cell, add concentrated carrying molecular switch
HER2-CAR virus liquids, mix, MOI=8.The detection of related destination protein is carried out to the T cell of transfection after one week.
Fig. 3 is that the HER2-CAR carriers of the carrying molecular switch built using the present invention of Flow cytometry are thin in T
Expression on cellular surface.As a result the cell for showing to have 29.2% is GFP (slow virus carrier expresses GFP albumen) sun in itself
Property, the HER2-CAR for the carrying molecular switch for illustrating to build using the present invention can be in T cell surface expression, and transfection efficiency is
29.2%.
Embodiment 4：HER2 CAR-T cells control efficiency analysis with molecular switch
By HER2 CAR-T (hereinafter referred to as CAR (HER2)-T) cell with molecular switch according to density 1 × 105Individual/
Ml is inoculated with 96 orifice plates, per hole 100ul, is placed in 5%CO2,37 DEG C of incubator culture 24h；Add the 10nM AP1903 (U.S.
Apexbio products), 20 μ L CCK-8 (MCE Products) are added after 12h per hole, continue to be incubated upper ELIASA detection after 2h, in
OD values are read at 450nm wavelength.CAR (the HER2)-T cell control group for not adding AP1903 is set and only adds AP1903 acellular
Blank control group.The death rate of T cell=[1- (adding AP1903 groups OD values-blank control group OD values)/(not plus AP1903 groups OD
Value-blank control group OD values)] × 100%.The death rate of T cell is controls of 29.4%, the AP1903 to CAR-T cytoactives
Positive rate=99.36% of the rate=death rate/recombinant slow virus infection T cell；What as a result the explanation present invention designed has molecule
The HER2 CAR-T cytoactives of switch are controlled by AP1903, greatly improve the Clinical efficacy and security of CAR technologies.
Embodiment 5：HER2 CAR-T cells in vitro killing experiments with molecular switch
The inoculation 100 μ L present invention is prepared into CAR-T cells and unloaded slow-virus infection T cell is killed as effector cell
Hinder determination of activity, target cell is the positive breast cancer cell line T47D of HER2 expression.According to effector cell and target cell numbers ratio
Example is 5:20 μ L CCK-8 are added per hole after being placed in 5%CO2,37 DEG C of incubator culture 24h in 1 96 well culture plates of addition, are continued
After being incubated 2h, ELIASA detection 450nm wavelength, OD values, killing rate=[1- (experimental group OD values-effector cell's control group OD are read
Value)/target cell control group OD values] × 100%.With unloaded slow-virus infection T as a control group.CAR-T cells are expressed HER2
The killing-efficiency of positive breast cancer cell line T47D cells is 93.06%-96.18%.Prepared by experimental group of the present invention
The specific killing activity that CAR-T cells express HER2 positive breast cancer cell T47D is significantly higher than control group.Illustrate this
HER2 CAR-T cells prepared by invention have the function of killing HER2 positive cells.
Embodiment 6：HER2 CAR-T cells with molecular switch are to internal killing experiments
Choose nude mice model (be purchased from Traditional Chinese Medicine University Of Guangzhou) in Animal House raise (23 ± 2 DEG C of room temperature, humidity 50% ±
10%) the breast cancer cell line T47D cells of logarithmic phase, are collected, phosphate buffer (PBS) is diluted to 2 × 105Individual/mL.Nothing
Under the conditions of bacterium, in the left oxter inoculation 0.2mL T47D cell suspending liquids of mouse, 20d is observed, it is big to treat that 10cm (diameter) occurs in oxter
Small harder tubercle is divided into 4 groups as successful standard is modeled.A groups are the HER2 with molecular switch prepared by embodiment 3
CAR-T cell therapy groups；B groups are the T lymphocytes treatment group of untransfected prepared by embodiment 3；C groups are prepared by embodiment 3
HER2 CAR-T cells and injection AP1903 treatment groups with molecular switch；D groups are saline control group.A groups are in HER2
CAR-T cell culture is to the 14th day and the 16th day, tail vein injection 2 × 106Cell, the T lymphocyte cultures of B group untransfecteds are arrived
14th day and the 16th day tail vein injection 2 × 106Cell, C groups are in HER2 CAR-T cell culture to the 14th day and the 16th day, tail
Intravenous injection 2 × 106Cell, and inject AP1903, the treatment time of D groups and A groups, B groups and C groups after treatment injection 2h every time
It is identical, the physiological saline of tail vein injection same volume.Draw neck to put to death respectively at 7d, 14d, 21d and 28d after treatment, take breast cancer
Mouse model tumor tissues, calculate mouse model tumor size.
Tumor size change curve after Fig. 4 mouse model T lymphocytes have been treated, physiological saline group D and is opened with molecule
HER2 CAR-T cell therapy group A, the T lymphocytes treatment group B of pass is compared, and tumor size reduces in treatment group A and B, but controls
Treatment group A tumor sizes compared with treatment group B reduce substantially, and HER2 CAR-T cells can trigger powerful to breast cancer cell
Cytotoxicity；Treatment group C compares with physiological saline group D treatment groups, A, after HER2 CAR-T cells of the injection with molecular switch
AP1903 is injected again, and tumour becomes larger, and tumour growth trend is consistent with physiological saline group D, illustrates that the molecular switch exists
In the presence of AP1903, effect has been played in vivo, and the Fas suicides approach induced by FKBP-F36V is thin by HER2 CAR-T
Born of the same parents remove, and have ceased the killing activity of CAR-T cells against tumor in time.
Therefore, the HER2 CAR-T cells provided by the invention for carrying molecular switch both had stronger killing energy to tumour
Power, CAR-T lethal effect can be terminated in time again in the presence of AP1903.
Embodiment 5：CAR-T cell clinical application methods
For the treatment of HER2 tumour patients, treatment method is local tumor injection and venous re-transfusion 2 × 106Individual CAR-T
Cell.If there is the generation of adverse reaction, local tumor injection and venous re-transfusion AP1903 (0.01-100mg/kg), you can eventually
Only.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent circle.
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tatgtgaaca ctgtgaccct tgcaccaaat gtgaacatgg aatcatcaag gaatgcacac 540
tcaccagcaa caccaagtgc aaagaggaag gatccagatc taacttgggg tggctttgtc 600
ttcttctttt gccaattcca ctaattgttt gggtgaagag aaaggaagta cagaaaacat 660
gcagaaagca cagaaaggaa aaccaaggtt ctcatgaatc tccaacctta aatcctgaaa 720
cagtggcaat aaatttatct gatgttgact tgagtaaata tatcaccact attgctggag 780
tcatgacact aagtcaagtt aaaggctttg ttcgaaagaa tggtgtcaat gaagccaaaa 840
tagatgagat caagaatgac aatgtccaag acacagcagg acagaaagtt caactgcttc 900
gtaattggca tcaacttcat ggaaagaaag aagcgtatga cacattgatt aaagatctca 960
aaaaagccaa tctttgtact cttgcagaga aaattcagac tatcatcctc aaggacatta 1020
ctagtgactc agaaaattca aacttcagaa atgaaatcca aagcttggtc tacccaactt 1080
tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaac 1137
<210> 5
<211> 54
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 5
gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg acca 54
<210> 6
<211> 63
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 6
atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60
ccc 63
<210> 7
<211> 795
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 7
atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60
cccgaggtgc agctggtgga gtctggggga ggcttggtac agccagggcg gtccctgaga 120
ctctcctgta cagcttctgg atctgacatt aatgattatc ctattagctg gttccgccag 180
gctccaggga aggggctgga gtgggtaggt ttcattaata gcggtgggtc tacatggtac 240
gcctcgtggg tgaaaggcag attcaccatc tcaagagatg attccaaaag catcgcctat 300
ctgcaaatga acagcctgaa aaccgatgac acagccactt atttctgtgc tagaggatac 360
tccacgtatt atggtgattt taatatctgg ggccaaggga caatggtcac cgtctcgagc 420
ggaggaggag gaagcggagg aggaggaagc ggaggaggag gaagcgacgt tgtgatgacc 480
cagtctcctt cctctctgtc tgcatctgta ggagacagag tcaccatcac ttgccaagcc 540
agtcagagta ttgatagcaa tttggcctgg tttcagcaga aaccagggaa agcccctaac 600
ctcctgatct atagggcgtc taatttagct agtggggtcc cgtcaaggtt cagcggcagt 660
ggatctggga cagaattcac tctcaccatc agcagcctgg gaagagaaga tgctgcaact 720
tattactgtc ttggaggagt tggaaatgtt tcttacagaa cttcgttcgg ccaagggacc 780
aaggtggaaa tcaaa 795
<210> 8
<211> 135
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 8
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 9
<211> 79
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 9
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgtc tagtaacagt 60
ggcctttatt ttcgtgagg 79
<210> 10
<211> 126
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 10
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 11
<211> 336
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 11
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
Claims (10)
- A kind of 1. immunocyte for the anti-HER2 Chimeric antigen receptors modification for carrying molecular switch, it is characterised in that：The carrying The safety-type Chimeric antigen receptor gene containing anti-HER2 in the immunocyte of the anti-HER2 Chimeric antigen receptors modification of molecular switch, institute Stating the safety-type Chimeric antigen receptor genes of anti-HER2 includes HER2 single-chain antibody and has linked a molecular switch, described HER2 single-chain antibodies include CD8Leader, HER2 land, CD8 hinge area, cross-film-stimulus structure domain, CD3 ζ stimulus signals Conducting region.
- 2. a kind of immunocyte of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch as claimed in claim 1, its It is characterised by：The cross-film-stimulus structure domain is selected from CD8, CD27, CD28, CD137/4-1BB, CD134/OX40, ICOS molecule The all or part of fragment of one of them.
- 3. a kind of immunocyte of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch as claimed in claim 1, its It is characterised by：The molecular switch is the FKBP12-F36V suicide switch elements based on Fas genes.
- 4. a kind of immunocyte of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch as claimed in claim 1, its It is characterised by：The molecular switch includes FKBP12-dimerization chemical induction subsystem, fkbp ligand body dimer AP1903, and Fas genes, the molecular switch are located at N-terminal or C-terminal in the single-chain antibody of the HER2.
- 5. a kind of immunocyte of anti-HER2 Chimeric antigen receptors modification for carrying molecular switch as claimed in claim 1, its It is characterised by：The immunocyte is autologous or transgenosis T cell, NK cells, middle granulocyte, monocyte, cell toxicant Property T lymphocytes, regulatory T-cell, memory t cell, bispecific T cell, CIK cell one kind therein.
- A kind of 6. anti-HER2 Chimeric antigen receptors T cell for carrying molecular switch as claimed in claim 1, it is characterised in that： The safety-type Chimeric antigen receptor genes of anti-HER2 are by FKBP12-linker-Fas-T2A-Leader-scFv (HER2)-CD8 α-CD28-CD137-CD3 ζ nucleotide sequence is linked together, and forms recombination sequence, and gene order is obtained by chemical synthesis , form complete specific chimeric antigen receptor gene DNA.
- A kind of 7. anti-HER2 Chimeric antigen receptors T cell for carrying molecular switch as claimed in claim 6, it is characterised in that： FKBP12-linker-Fas-T2A-Leader-scFv (the HER2)-CD8 α-CD28-CD137-CD3 ζ recombinations are sequence Nucleotide sequence shown in list SEQ ID NO.1.
- A kind of 8. medicine for the treatment of cancer, it is characterised in that：Anti- HER2 containing the carrying molecular switch described in claim 1 The immunocyte of Chimeric antigen receptor modification.
- 9. the immunocyte of the anti-HER2 Chimeric antigen receptors modification of the carrying molecular switch described in claim 1 is controlled for preparation Treat the purposes of the medicine of malignant cancer.
- 10. the purposes of the medicine as claimed in claim 9 for preparing treatment malignant tumour, it is characterised in that：The malignant cancer For breast cancer, stomach cancer, colon cancer, lung cancer, oophoroma, cervical carcinoma etc..
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| CN109824781A (en) * | 2019-01-21 | 2019-05-31 | 卢英 | Specific chimeric antigen receptor, encoding gene and the expression vector of anti-human 2 antigen of HER and application |
| CN110396131A (en) * | 2019-08-23 | 2019-11-01 | 北京鼎成肽源生物技术有限公司 | A kind of Chimeric antigen receptor, recombinant vector, recombinant cell and the application of ErbB2 single-chain antibody, targeting people ErbB2 |
| CN110484507A (en) * | 2018-01-31 | 2019-11-22 | 温州医科大学 | A kind of technology of preparing of the Novel chimeric antigen receptor T cell of target tumor Her2 |
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| CN116672442A (en) * | 2023-07-27 | 2023-09-01 | 吉林大学 | Preparation of medicine for treating osteosarcoma by combining ligustilide and HER2-CAR-T cells |
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| WO2021036244A1 (en) * | 2019-08-26 | 2021-03-04 | 深圳宾德生物技术有限公司 | Chimeric antigen receptor t cell carrying safety switch and targeting her2, preparation method therefor, and application thereof |
| CN116672442A (en) * | 2023-07-27 | 2023-09-01 | 吉林大学 | Preparation of medicine for treating osteosarcoma by combining ligustilide and HER2-CAR-T cells |
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