CN108707199A - Target Chimeric antigen receptor T cell and its application of TEM8 - Google Patents
Target Chimeric antigen receptor T cell and its application of TEM8 Download PDFInfo
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C12N2740/00—Reverse transcribing RNA viruses
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- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The invention discloses a kind of Chimeric antigen receptor T cell for the targeting TEM8 for belonging to gene engineering technology field and its applications.The Chimeric antigen receptor T cell of the targeting TEM8 of the present invention can express SCFV-CD28-CD3 ζ fusion proteins in T cell；The cell, which passes through, obtains T cell modification and transformation, is capable of the tumour cell of specific recognition and killer T EM8 high expression, is suitable for the prevention and treatment of corresponding tumor disease.
Description
Technical field
The present invention relates to the Chimeric antigen receptor T cell of targeting TEM8 and its applications, belong to gene engineering technology field.
Background technology
The year two thousand twenty, global malignant tumour new cases and death are up to 20,000,000 and 12,000,000, and cancer becomes people
First killer of class health.Tumor class that traditional Radiotherapy chemotherapy is directed to is limited, curative effect is general and the poison of accompanied by intense is secondary makees
With.New vessels around tumour, tumor vessel are formed in tumour, growth, are played an important role in transfer process, it can be with
Nutrition is provided for tumour fast breeding.It is a step crucial in tumour progression that the new vessels of tumor inducing, which generate, is referred to as
" angiogenic switch ".Under the conditions of cancer pathology, regulation of blood vessels out of control is that the main of malignancy of tumor growth facilitates factor, according to
This takes so-called " tumour hunger cure ", passes through the suppression to tumor tissues and peripheral vessels using tumor vessel as therapy target
System makes tumour cell be unable to get sufficient nutrition supply or anoxic and death, and then controls the growth and transfer of tumour,
Through an important channel as treatment and prevention of tumour.
Chimeric antigen receptor T cell (Chimeric antigen receptor T cell, referred to as：CAR-T cell)
It is the means by genetic modification, enables the single-stranded variable region (single of the monoclonal antibody of specific recognition target antigen
Chain Fv domain, SCFV) it expresses in cell surface, while passing through the activation of transmembrane region and the cell intracellular of engineer
Proliferation signal domain mutually couples.The T cell for expressing CAR can be by SCFV Direct Recognitions and the tumour phase of combination tumor cell surface
Antigen is closed, signal is passed in T cell by CAR, to play the effect of killing tumor cell.
Tumor endothelial marker (Tumor endothelia marker, TEM8) is epicyte protein, is had apparent
Receptor and structure, express more special in tumor vascular endothelium, exist in tumor vessel, and in normal structure seldom or not
In the presence of.What is more important, TEM8 are then accredited as a kind of receptor of anthrax toxin.There are some researches prove applications to be directed to
The anti-tumour antibody sample albumen of TEM8, i.e. the fusion protein TEM8-FC of people TEM8 extracellular regions and human IgG FC segments have notable
Ground inhibits tumour growth activity and antitumous effect, illustrates that TEM8 is a potential anti-tumor target.Therefore, TEM8 conducts
The overexpression protection of Partial tumors cell is former, can be used as a promising target of CAR-T cell anti-tumors treatment.
Invention content
A kind of Chimeric antigen receptor T cell of targeting TEM8 in order to overcome the deficiencies in the prior art, the present invention provides
(Anti TEM8 CAR-T cells), the T cell are obtained by modification and transformation, being capable of specific recognition and killer T EM8 high tables
The tumour cell reached.
To achieve the goals above, first choice of the present invention provides a kind of Chimeric antigen receptor of targeting TEM8, particular technique
Scheme is as follows：
A kind of Chimeric antigen receptor of targeting TEM8, the Chimeric antigen receptor include the antigen binding domain of sequential series
ScFv and signal transduction area CD28-CD3 ζ；Its amino acid sequence is as shown in SEQ ID NO.1.
In above-mentioned Chimeric antigen receptor, the amino acid sequence of the antigen binding domain ScFv is as shown in SEQ ID NO.2.
In above-mentioned Chimeric antigen receptor, the amino acid sequence of the CD28 is as shown in SEQ ID NO.3.
In above-mentioned Chimeric antigen receptor, the amino acid sequence of the CD3 ζ is as shown in SEQ ID NO.4.
In above-mentioned Chimeric antigen receptor, the antigen binding domain ScFv is located at after birth, signal transduction area CD28-CD3 ζ across
Film and part be located at intracellular.
The present invention also provides a kind of nucleic acid molecules, encode above-mentioned Chimeric antigen receptor.
Preferably, the nucleotide sequence of the nucleic acid molecules is as shown in SEQ ID NO.5.
The present invention also provides a kind of carriers comprising above-mentioned nucleic acid molecules.
Preferably, the carrier be Lentiviral.
The present invention also provides a kind of Chimeric antigen receptor T cell of targeting TEM8, express above-mentioned chimeric antigen by
Body.
The present invention also provides above-mentioned Chimeric antigen receptor T cells in preparing detection, prevention and treatment cancer drug
Purposes.
In such use, the cancer is the cancer of TEM8 molecule height expression, it is preferred that the cancer is TEM8 molecules
The liver cancer of height expression.
The Chimeric antigen receptor T cell of the targeting TEM8 of the present invention can be prepared as follows, and specifically include following step
Suddenly：
1) synthesize, expand the Chimeric antigen receptor expressing gene of targeting TEM8, the chimeric antigen of structure expression targeting TEM8
The expression vector of receptor, the expression vector can be Lentiviral；
2) slow virus packaging plasmid and the Lentiviral is utilized to infect T cell, it is preferable that 293T cells obtain
Slow virus；
3) T lymphocytes are detached, T lymphocytes described in the slow-virus infection are used in combination, it is chimeric to obtain the targeting TEM8
Antigen receptor T cell.
The method of the Chimeric antigen receptor expressing gene of the synthesis and amplification targeting TEM8 can be according to this field routine skill
Art means obtain.
Further include being purified to the slow virus further in the above method, the slow disease after purification is used in combination
Poison infects the T lymphocytes.In general, the means of purification can be carried out using this field conventional means, such as filters, is super
Filter etc..
Beneficial effects of the present invention：
1, the present invention provides a kind of Chimeric antigen receptor of targeting TEM8, the receptor can be used for Anti TEM8 CAR-
The Chimeric antigen receptor of the structure of T cell, the targeting TEM8 is simple in structure but is effectively combined to TEM8 albumen, energy
Simply, the effective Anti TEM8 CAR-T cells for preparing with specific recognition and killing tumour.
2, Anti TEM8 CAR-T cells provided by the invention have efficient tumour for the cancer cell for expressing TEM8
Killing activity, especially for the cancer of TEM8 high expression, such as in the liver cancer of TEM8 high expression.
Description of the drawings
Fig. 1 is Anti-TEM8 CAR Lentiviral structure collection of illustrative plates.
Fig. 2 is the Wstern Blot detection figures (GAPDH is internal reference albumen) of TEM8 expressing quantities, and ADMSC is fat
Mescenchymal stem cell, Hela are cervical cancer cell, and HepG2 is liver cancer cells.
Fig. 3 is to target the Chimeric antigen receptor T cell of TEM8 and be uninfected by T cell difference effect target in 0.5h to compare
TEM8 high expresses the fragmentation effect figure of target cell HepG2.
Fig. 4 is the Chimeric antigen receptor T cell of targeting TEM8 and the T cell being uninfected by 10:It is different when 1 effect target ratio
Fragmentation effect figure of the time to TEM8 high expression target cells HepG2.
Fig. 5 is that the Chimeric antigen receptor T cell of targeting TEM8 and the T cell the being uninfected by difference in 0.5h imitate target ratio
To the fragmentation effect figure of TEM8 low expression target cells Hela.
Fig. 6 is the Chimeric antigen receptor T cell of targeting TEM8 and the T cell being uninfected by 10:It is different when 1 effect target ratio
Fragmentation effect figure of the time to TEM8 low expression target cells Hela.
Fig. 7 is the liver cancer HepG2 transplanting of the Chimeric antigen receptor T cell for targeting TEM8, T cell and tumour control group
Tumor model mice life cycle curve graph.
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, right below in conjunction with the embodiment of the present invention
Technical solution in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not doing
Go out the every other embodiment obtained under the premise of creative work, shall fall within the protection scope of the present invention.
Embodiment 1 targets the preparation of the Chimeric antigen receptor T cell of TEM8
1, Lentiviral is built
Nucleic acid fragment (BamHI-Sp-EcoRI-NheI-CD28-CD3zeta- of the synthesis containing coding CD28-CD3 ζ
SalI, by Sino-U.S. calm and peaceful biotechnology (Beijing), Co., Ltd synthesizes), as shown in SEQ ID NO.6, it is named as pGSI-
seq8.Distinguish double digestion 3ug pGSI-seq8 and recombined lentivirus vector plasmid with BamHI and SalI restriction enzymes
PCDH-EF1 (Addgene companies) is connected after the recycling of digestion products glue with T4DNA ligases, and 4 DEG C of connections are stayed overnight, conversion DH5 α
Competent cell takes 100 μ L bacterium solutions to be applied on the LB plates containing ammonia benzyl resistance, and 37 DEG C are incubated overnight.Picking monoclonal carries out
Positive colony sample presentation is sequenced bacterium colony PCR, preserves sequencing result and correctly clones and extract plasmid, is named as pCDH-EF1-
emCAR。
Optimum synthesis contains the coding ScFv-anti TEM8's of 5 ' end restriction enzyme site EcoRI and 3 ' end restriction enzyme site NheI
(it is required that being free of BamHI, EcoRI, NheI and SalI restriction enzyme site, the calm and peaceful biotechnology of Sino-U.S. (Beijing) is limited for nucleotide sequence
Company synthesizes), as shown in SEQ ID NO.7, it is named as pGSI-seq9.It is double respectively with EcoRI and NheI restriction enzymes
Digestion 3ug pGSI-seq9 and skeleton plasmid pCDH-EF1-emCAR are connected after the recycling of digestion products glue with T4DNA ligases,
4 DEG C of connections overnight, convert DH5 α competent cells, take 100 μ L bacterium solutions to be applied on the LB plates containing ammonia benzyl resistance, 37 DEG C of mistakes
Night cultivates.Picking monoclonal carries out bacterium colony PCR, and positive colony sample presentation is sequenced, and preserves sequencing result and correctly clones and extract
Plasmid, is named as Anti-TEM8 CAR carriers, and Vector map is as shown in Figure 1.
2, slow virus is packed
It is put into rapidly in 37 DEG C of water-baths up to ice cube disappearance, is added dropwise from 1 293T cell frozen is taken out in liquid nitrogen
In the 15ml centrifuge tubes for preheating culture medium containing 5ml, 1200rpm centrifuges 3min, supernatant is abandoned, with 293T culture mediums (10%FBS+
DMEM it) is resuspended in cell inoculation to 150mm culture dishes, 37 DEG C, 5%CO2Saturated humidity culture.
When cell confluency degree is up to 90% or more, old culture medium is discarded, 5ml sterilizing PBS solutions are added, gently shakes, washes
PBS solution is discarded after washing cell, 2ml0.25%Trypsin-EDTA digestive juices are added, digestion 1-2min disappears completely until cell
Change is got off.The culture medium containing serum is added and terminates digestion, cell suspension 1200rpm centrifuges 3min, centrifugation gained cell culture
Base weight is outstanding, each 150mm culture dishes cell inoculation 1.2 × 107Cell is for packing slow virus, 37 DEG C, 5%CO2Saturated humidity
Culture, 20ml culture mediums/ware.
2h before transfection, is changed to 18ml DMEM culture mediums by 293T cell culture mediums, is added into A sterile centrifugation tubes
The DMEM culture mediums of 1ml preheatings, be then added prepared anti-TEM8 CAR vector plasmids, pHelper1 plasmids and
PHelper2 plasmids (anti-TEM8 CAR carriers：pHelper1：Mass ratio=1 pHelper2：1：1, totally 54 μ g,
PHelper1 and pHelper2 plasmids are the helper plasmid of slow virus packaging), it is uniformly mixed.It is added into B sterile centrifugation tubes
The DMEM culture mediums of 1ml preheatings, are then added 108 μ l Lipofectamin2000 (liposome) solution, are uniformly mixed.A manage and
B pipes incubate 5 minutes at room temperature.It is added to the liquid in B pipes is droplets of in A pipes, is uniformly mixed, be incubated at room temperature 20min,
Obtain DNA- liposome transfection complexes.
DNA- liposome transfection complexes are transferred to and are changed in the 293T cells of liquid in advance, mixing, 37 DEG C, 5%CO2It is full
With humidity culture.It is inhaled after culture 6-8h and abandons the culture medium containing transfection mixture, 20ml preheatings are added per ware cell contains 5%
The DMEM culture mediums of FBS, 37 DEG C, 5%CO2Saturated humidity culture obtains the slow virus for carrying Anti-TEM8 CAR sequences
Grain.
3, slow virus purifies
It changes after liquid respectively that supernatant is temporary to be stored in 4 DEG C for 24 hours and 48h, collecting, and changes 20ml fresh cultures.It will collect
4 DEG C of the liquid arrived, 3500rpm centrifuge 15min, abandon precipitation, supernatant are carried out with Millipore albumen ultrafiltration columns (10KD) dense
Contracting is carried out at the same time virus titer measurement, and slow virus (Anti-TEM8 CAR) is diluted to 1*10 according to measurement result8TU/ml,
Slow virus (Anti-TEM8 CAR) after packing is placed in -80 DEG C of preservations.
4, the separation of CD3+T cells
Whole blood is poured into 50ml centrifuge tubes, room temperature centrifuges 20 minutes, and control centrifugal force is 800g；Take centrifugation rear lower thin
Born of the same parents' ingredient adds injection physiological saline to 50ml；25ml aforesaid liquids are taken to be added to 25ml human lymphocyte separating liquids respectively, it will
Centrifuge tube room temperature centrifuges 20 minutes, and control centrifugal force is 800g；Tunica albuginea confluent monolayer cells are taken, injection physiological saline is added, complements to
50ml；Centrifugation 8 minutes, control centrifugal force are 600g, abandon supernatant, obtain peripheral blood mononuclear cells (peripheral blood
Mononuclear cell, referred to as：PBMC).
5, slow virus carrier infects T lymphocytes
The single core of peripheral blood that the RPMI1640 complete medium culture of isolated for being 10%FBS with mass fraction obtains is thin
CD 3-resisting monoclonal antibody activation is added for first day in born of the same parents；Slow virus (the Anti-TEM8 after purification of 80MOI is added in third day
CAR), 1000g centrifuges 1h, and culture medium is then changed to the training of the X-Vivo15 serum-frees containing 700IU/ml recombinant human il-2s
Base is supported, culture 7 days is continued, obtains targeting TEM8 Chimeric antigen receptor T cells.
Embodiment 2 targets the fragmentation effect of the cancer cell to expressing TEM8 of TEM8 Chimeric antigen receptor T cells
Made with the cervical cancer tumer line Hela of the liver cancer cell lines HepG2 cells and low expression TEM8 of stablizing expression TEM8
For target cell, the Anti-TEM8 CAR for targeting TEM8 Chimeric antigen receptors T cell made from embodiment 1 respectively and being uninfected by
Effector cell is made in the T cell (being known as being uninfected by T cell) of slow virus, and target cell is inoculated with 96 holes according to 5000/ml of density
Plate, per 100 μ l of hole, according to 5:1,10:1,20:Target cell is added in effector cell by 1 effect target ratio, is placed in 5%CO2, 37 DEG C of cultures
Case culture 4h detects cell viability using CCK8, calculates killing-efficiency.As a result as seen in figures 3-6, the results showed that, express TEM8
Chimeric antigen receptor T cell cell to TEM8 masculine liver cancer cells have very strong and special lethal effect.
Embodiment 3 targets fragmentation effect of the TEM8 Chimeric antigen receptors T cell to in-vivo tumour
Take 6 week old female SCID mouse (buying from Beijing Vital River Experimental Animals Technology Co., Ltd.) 30, left oxter
5*10 is subcutaneously injected6HepG2 (liver cancer) cell, waits for that tumour grows to 50-100mm3Size, tumor model are randomly divided into 3 groups：Control
Group, normal T-cell group and anti-TEM8 Chimeric antigen receptors T cell (preparation of embodiment 1) group；Control group tail vein injection physiology
Brine 200ul/ times, 2 times a week；T cell group tail vein injection T cell 1*107A/time, 2 times a week；Anti- TEM8 chimeric antigens
The anti-TEM8 Chimeric antigen receptors T cell 1*10 of recipient T cells group tail vein injection7A/time, 2 times a week；In statistics 100 days
Mouse survival state does survival rate curve.Fig. 7 the result shows that, target the Chimeric antigen receptor T cell of TEM8 compared to not feeling
The T cell and control group of dye can postpone the life cycle of the transplanted human hepatocellular carcinoma mouse model of TEM8 high expression.
The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；Although with reference to aforementioned each reality
Applying example, invention is explained in detail, it will be understood by those of ordinary skill in the art that：It still can be to aforementioned each
Technical solution recorded in embodiment is modified, and either carries out equivalent replacement to which part or all technical features；And
These modifications or replacements, the range for various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution.
Sequence table
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<120>Target Chimeric antigen receptor T cell and its application of TEM8
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Thr Ser Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn
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Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
210 215 220
Tyr Tyr Cys Ala Arg Gly Gly Tyr Phe Leu Asp Tyr Trp Gly Gln Gly
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Thr Leu Val Thr Val Ser Ser Ala Ser Lys Ile Glu Val Met Tyr Pro
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Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys
275 280 285
Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
290 295 300
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
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Ser Arg Leu Leu His Ser Asp Tyr Met Gln Met Thr Pro Arg Arg Pro
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<213> Artificial Sequence
<220>
<223>Antigen binding domain ScFv
<400> 2
Glu Phe Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro
1 5 10 15
Gly Gln Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Ile Gly Gly Ile
20 25 30
Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
35 40 45
Ile Tyr Ala Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
50 55 60
Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln
65 70 75 80
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ile Thr Ser
85 90 95
Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val Ala
100 105 110
Ala Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro
130 135 140
Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser
145 150 155 160
Thr Ser Gly Gly Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala
165 170 175
Leu Glu Trp Leu Ala His Ile Tyr Ser Asn Asp Asp Lys Ser Tyr Ser
180 185 190
Thr Ser Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn
195 200 205
Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
210 215 220
Tyr Tyr Cys Ala Arg Gly Gly Tyr Phe Leu Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Ala Ser
245
<210> 3
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223>The areas CD28
<400> 3
Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
1 5 10 15
Gln Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro
20 25 30
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly
35 40 45
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
50 55 60
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
65 70 75 80
Gln Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
85 90 95
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
100 105
<210> 4
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223>The areas CD3 ζ
<400> 4
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
Arg
<210> 5
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223>The nucleic acid molecules of Chimeric antigen receptor
<400> 5
gaattcgaca tcgagctgac tcaacctcca agtgtcagcg tcgctccagg acagactgct 60
cggatcagct gctccggtga taacattgga ggaatctacg ttcactggta tcagcagaaa 120
cccggtcagg cacccgtcct ggtgatctat gctgactcca agaggccttc tggaatccct 180
gagaggttta gcggaagtaa cagcggtaac accgctaccc tgacaatctc cggaacccag 240
gctgaagatg aggccgatta ctactgtcag tcttatgaca tcacctccct ggtctttggt 300
ggcggaacta agctgactgt gctgcggact gttgctgctc ctagtggagg cggagggagc 360
ggaggtggtg gtagtggagg tggtggcagc caggtccagc tcaaggagtc tggacccgca 420
ctggtgaaac caacccagac actgacactg acctgcacct ttagcggctt ctctctgagt 480
acttctggtg gaggagtgag ctggattagg cagcctcctg gcaaagctct ggagtggctc 540
gcacacatct atagcaatga cgacaagtct tattccacaa gtctcaagac tagactgact 600
atctctaagg ataccagtaa gaaccaggtt gttctgacca tgaccaatat ggacccagtt 660
gacacagcaa catactactg cgcacgcgga ggttacttcc tggattactg gggacagggt 720
acactggtga ctgtgagttc tgctagcaag attgaggtca tgtatcctcc tccttacctg 780
gacaatgaga agagtcaggg caccatcatt catgtgaagg gcaagcacct gtgccctagt 840
cctctgtttc ctggcccaag taagcctttc tgggtcctgg tggtggttgg tggagtgctg 900
gcttgttata gtctgctggt gactgtggcc ttcattatct tctgggtgcg cagcaaacgc 960
tctcggctgc tccattccga ctacatgcag atgacaccac gcagacccgg tcctaccaga 1020
aagcattacc agccctacgc acctccacgg gatttcgcag catacagaag agtgaagttc 1080
tccaggtccg cagatgctcc tgcataccag cagggacaga atcagctgta caacgagctg 1140
aacctcggca gacgcgagga atacgatgtt ctggataagc ggagaggtcg cgacccagaa 1200
atgggtggga aacctcagcg gagaaagaac cctcaggagg gtctgtacaa cgaactccag 1260
aaggataaga tggccgaggc ctactctgag atcggtatga agggcgagcg gagaagaggg 1320
aaagggcacg atggactgta tcaaggactg agtaccgcca caaaggacac atacgatgca 1380
ctgcacatgc aggccctgcc accacgc 1407
<210> 6
<211> 753
<212> DNA
<213> Artificial Sequence
<220>
<223>Encode the nucleic acid of CD28-CD3 ζ
<400> 6
ggatccgcca ccatggagca caaggaggtt gtgctcctcc tcctcctgtt tctgaagtct 60
ggccaagggg aattcgctag caagattgag gtcatgtatc ctcctcctta cctggacaat 120
gagaagagtc agggcaccat cattcatgtg aagggcaagc acctgtgccc tagtcctctg 180
tttcctggcc caagtaagcc tttctgggtc ctggtggtgg ttggtggagt gctggcttgt 240
tatagtctgc tggtgactgt ggccttcatt atcttctggg tgcgcagcaa acgctctcgg 300
ctgctccatt ccgactacat gcagatgaca ccacgcagac ccggtcctac cagaaagcat 360
taccagccct acgcacctcc acgggatttc gcagcataca gaagagtgaa gttctccagg 420
tccgcagatg ctcctgcata ccagcaggga cagaatcagc tgtacaacga gctgaacctc 480
ggcagacgcg aggaatacga tgttctggat aagcggagag gtcgcgaccc agaaatgggt 540
gggaaacctc agcggagaaa gaaccctcag gagggtctgt acaacgaact ccagaaggat 600
aagatggccg aggcctactc tgagatcggt atgaagggcg agcggagaag agggaaaggg 660
cacgatggac tgtatcaagg actgagtacc gccacaaagg acacatacga tgcactgcac 720
atgcaggccc tgccaccacg ctaataggtc gac 753
<210> 7
<211> 747
<212> DNA
<213> Artificial Sequence
<220>
<223>The nucleotide of ScFv-anti TEM8
<400> 7
gaattcgaca tcgagctgac tcaacctcca agtgtcagcg tcgctccagg acagactgct 60
cggatcagct gctccggtga taacattgga ggaatctacg ttcactggta tcagcagaaa 120
cccggtcagg cacccgtcct ggtgatctat gctgactcca agaggccttc tggaatccct 180
gagaggttta gcggaagtaa cagcggtaac accgctaccc tgacaatctc cggaacccag 240
gctgaagatg aggccgatta ctactgtcag tcttatgaca tcacctccct ggtctttggt 300
ggcggaacta agctgactgt gctgcggact gttgctgctc ctagtggagg cggagggagc 360
ggaggtggtg gtagtggagg tggtggcagc caggtccagc tcaaggagtc tggacccgca 420
ctggtgaaac caacccagac actgacactg acctgcacct ttagcggctt ctctctgagt 480
acttctggtg gaggagtgag ctggattagg cagcctcctg gcaaagctct ggagtggctc 540
gcacacatct atagcaatga cgacaagtct tattccacaa gtctcaagac tagactgact 600
atctctaagg ataccagtaa gaaccaggtt gttctgacca tgaccaatat ggacccagtt 660
gacacagcaa catactactg cgcacgcgga ggttacttcc tggattactg gggacagggt 720
acactggtga ctgtgagttc tgctagc 747
Claims (10)
1. a kind of Chimeric antigen receptor of targeting TEM8, which is characterized in that the Chimeric antigen receptor includes the anti-of sequential series
Former combined area ScFv and signal transduction area CD28-CD3 ζ；Its amino acid sequence is as shown in SEQ ID NO.1.
2. Chimeric antigen receptor according to claim 1, which is characterized in that the amino acid sequence of the antigen binding domain ScFv
Row are as shown in SEQ ID NO.2.
3. Chimeric antigen receptor according to claim 1, which is characterized in that the amino acid sequence of the CD28 such as SEQ ID
Shown in NO.3.
4. Chimeric antigen receptor according to claim 1, which is characterized in that the amino acid sequence of the CD3 ζ such as SEQ ID
Shown in NO.4.
5. encoding the nucleic acid molecules of any one of the claim 1-4 Chimeric antigen receptors.
6. including the carrier of nucleic acid molecules described in claim 5.
7. carrier according to claim 6, which is characterized in that the carrier be Lentiviral.
8. a kind of Chimeric antigen receptor T cell of targeting TEM8, which is characterized in that it is expressed described in claim any one of 1-4
Chimeric antigen receptor.
9. purposes of the Chimeric antigen receptor T cell described in claim 8 in preparing detection, prevention and treatment cancer drug.
10. purposes according to claim 9, which is characterized in that the cancer is the cancer of TEM8 molecule height expression.
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| CN201810527514.0A CN108707199A (en) | 2018-05-29 | 2018-05-29 | Target Chimeric antigen receptor T cell and its application of TEM8 |
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| CN108707199A true CN108707199A (en) | 2018-10-26 |
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| CN201810527514.0A Pending CN108707199A (en) | 2018-05-29 | 2018-05-29 | Target Chimeric antigen receptor T cell and its application of TEM8 |
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| WO2021224438A1 (en) | 2020-05-07 | 2021-11-11 | Institut Curie | Antxr1 as a biomarker of immunosuppressive fibroblast populations and its use for predicting response to immunotherapy |
| WO2023077000A1 (en) * | 2021-10-28 | 2023-05-04 | University Of Southern California | Inhibitory chimeric antigen receptor and uses thereof |
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| US9181340B2 (en) * | 2011-06-14 | 2015-11-10 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | TEM8 antibodies, conjugates thereof, and their use |
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| US9181340B2 (en) * | 2011-06-14 | 2015-11-10 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | TEM8 antibodies, conjugates thereof, and their use |
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| WO2020116686A1 (en) * | 2018-12-06 | 2020-06-11 | 고려대학교 산학협력단 | Human anti-antxr chimeric antigen receptor and uses thereof |
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| WO2021224438A1 (en) | 2020-05-07 | 2021-11-11 | Institut Curie | Antxr1 as a biomarker of immunosuppressive fibroblast populations and its use for predicting response to immunotherapy |
| WO2023077000A1 (en) * | 2021-10-28 | 2023-05-04 | University Of Southern California | Inhibitory chimeric antigen receptor and uses thereof |
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