CN110526992A - A kind of Chimeric antigen receptor of targeting EGFR, Chimeric antigen receptor T cell and its preparation method and application - Google Patents

A kind of Chimeric antigen receptor of targeting EGFR, Chimeric antigen receptor T cell and its preparation method and application Download PDF

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CN110526992A
CN110526992A CN201810560790.7A CN201810560790A CN110526992A CN 110526992 A CN110526992 A CN 110526992A CN 201810560790 A CN201810560790 A CN 201810560790A CN 110526992 A CN110526992 A CN 110526992A
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egfr
cell
chimeric antigen
antigen receptor
car
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万晓春
刘绿艳
唐超
李欣
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Shenzhen Benta Biological Technology Co Ltd
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Shenzhen Benta Biological Technology Co Ltd
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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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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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

Chimeric antigen receptor CAR-EGFR, the CAR-EGFR the present invention provides a kind of targeting EGFR include the amino acid sequence as shown in SEQ ID NO:1.The present invention also provides a kind of Chimeric antigen receptor T cells including the CAR-EGFR, the Chimeric antigen receptor CAR-EGFR of the targeting EGFR can be with the targeting EGFR positive tumor cell of specificity, it activates T cell to play immunization of cell and realizes killing efficient to EGFR positive tumor cell and specific, with lasting cell viability and lethality, and damage is not will cause to normal cell.The present invention also provides a kind of preparation method and application of the Chimeric antigen receptor T cell of targeting EGFR.

Description

A kind of Chimeric antigen receptor of targeting EGFR, Chimeric antigen receptor T cell and its preparation Methods and applications
Technical field
The present invention relates to field of medical biotechnology, in particular to a kind of Chimeric antigen receptor of targeting EGFR, chimeric antigen by Body T cell and its preparation method and application.
Background technique
Prevention and control of cancer difficulty is big, Disease Spectrum weight, it has also become seriously endanger human health great public health problem it One.EGF-R ELISA (epidermal growth factor receptor, EGFR) is epidermal growth factor (EGF) The receptor of cell Proliferation and signal transduction belongs to one kind of tyrosine kinase I receptor subfamily.Studies have shown that in many realities High expression or unconventionality expression in body tumour there are EGFR.The proliferation of EGFR and tumour cell, tumor invasion, turns angiogenesis It moves and the inhibition of Apoptosis is related.Therefore in oncotherapy, EGFR can become a very important medication target spot.
Chimeric antigen receptor T cell technology (Chimeric Antigen Receptor-Modified T Cells, CAR- T) as one of current newest immunocyte technology, because it can activate self immune system in vivo, routinely target It is killed to tumour cell, is finally reached fully erased malignant cell, is widely paid close attention to and studied.But The current application of CART technology is also limited to blood tumor, does not have correlative study also for the application of a variety of EGFR positive solid tumors.
Summary of the invention
To solve the above problems, the present invention provides the Chimeric antigen receptor CAR-EGFR and target of a kind of targeting EGFR To the Chimeric antigen receptor T cell of EGFR.The Chimeric antigen receptor CAR-EGFR of the targeting EGFR can be with the targeting of specificity EGFR positive tumor cell, activation T cell play immunization of cell and realize and specificity efficient to EGFR positive tumor cell Killing, there is lasting cell viability and lethality, and not will cause damage to normal cell.The present invention also provides one The preparation method and application of the Chimeric antigen receptor T cell of kind targeting EGFR.
In a first aspect, the present invention provides the Chimeric antigen receptor CAR-EGFR, the CAR-EGFR of a kind of targeting EGFR Including the amino acid sequence as shown in SEQ ID NO:1.
Optionally, the encoding gene of the Chimeric antigen receptor CAR-EGFR of the targeting EGFR includes such as SEQ ID NO:2 Shown in nucleotide sequence.
Optionally, the Chimeric antigen receptor encoding gene of the targeting EGFR should consider degeneracy base, i.e., such as SEQ ID The encoding gene of amino acid sequence shown in NO:1 includes the nucleotide sequence as shown in SEQ ID NO:2, and protection scope is also answered The protection and SEQ ID NO:2 have the nucleotide sequence of base degeneracy matter, the corresponding amino acid sequence of these nucleotide sequences Column remain as SEQ ID NO:1.
First aspect present invention provide the CAR-EGFR can specific recognition EGFR antigen protein, with EGFR antigen Albumen is specifically bound, and has stronger affine activity to the solid tumor cell of tumour cell, especially expression EGFR.
Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells of targeting EGFR, including the embedding of targeting EGFR Antigen receptor CAR-EGFR is closed, the CAR-EGFR includes the amino acid sequence as shown in SEQ ID NO:1.
Optionally, the amino acid sequence of the CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:2.
Optionally, the Chimeric antigen receptor CAR-EGFR of the targeting EGFR includes costimulatory signal area and the first signal Area, the costimulatory signal area and the first signaling zone belong to intracellular signal area；The costimulatory signal area includes CD27 signal Area, first signaling zone include CD3 ζ signaling zone.
CD27 signaling zone of the present invention can mention for the complete activation of the Chimeric antigen receptor T cell of the targeting EGFR For costimulatory signal, promote T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific, preferably Maintain the vigor and lethality of Chimeric antigen receptor T cell.
The Chimeric antigen receptor T cell of targeting EGFR of the present invention can include neuroglia with efficient identification and killing The expression such as tumor, cancer of pancreas and lung cancer have the cancer cell of EGFR；It is suitable for the solid tumor cell or tissue of the EGFR positive simultaneously.Due to There is expression, therefore, the Chimeric antigen receptor T of herein described targeting EGFR in a variety of cancer cells or tumour cell in EGFR Cell it is applied widely, a variety of EGFR positive tumor cells can be killed.
The Chimeric antigen receptor T cell for the targeting EGFR that second aspect of the present invention provides, including the embedding of targeting EGFR Close antigen receptor CAR-EGFR, this receptor for T cell targeted expression EGFR in specific manner tumour cell, in CAR- After EGFR is in conjunction with EGFR, the intracellular signal area of the T cell is activated, and promotes T cell in the amplification of patient's body, and efficiently And the killing tumor cell of specificity, and normal cell is hardly caused to damage, to reach removing tumour cell, realize Antitumor purpose.
The third aspect, the present invention provides a kind of recombinant viral vector, the recombinant viral vector includes such as first aspect The CAR-EGFR or as described in second aspect the CAR-EGFR of the Chimeric antigen receptor T cell of targeting EGFR encoding gene.
Optionally, the encoding gene of the CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:3.
Optionally, the viral vectors in the recombinant viral vector includes slow virus carrier, adenovirus vector or reverse transcription Viral vectors.
Still optionally further, the viral vectors is slow virus carrier.Further optionally, the slow virus carrier packet Include at least one of pWPXLD carrier, pLEX-MCS carrier, pSico carrier and pCgpV carrier.
The recombinant viral vector that third aspect present invention provides is a safe and reliable carrier tool, can efficiently be turned Move the encoding gene of the CAR-EGFR；The recombinant viral vector can be used for preparing the coding base for carrying the CAR-EGFR The virus of cause.The recombinant viral vector can be also used for the preparation of the Chimeric antigen receptor T cell of targeting EGFR, make the T The expression of cell realization height tissue specificity.
Fourth aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in the third aspect Group viral vectors.
Optionally, the host cell may include HEK293T cell, 293 cells, 293T cell, 293FT cell, SW480 cell, u87MG cell, HOS cell, COS1 cell or COS7 cell etc., but not limited to this.
Still optionally further, the host cell is HEK293T cell.
The host cell that fourth aspect present invention provides is used to provide and carries the recombinant virus as described in the third aspect The assembling of body simultaneously prepares the place for generating corresponding virus, and the heredity of the CAR-EGFR is carried by virus prepared by host cell Information has strong infectivity.
5th aspect, the present invention provides a kind of preparation methods of the Chimeric antigen receptor T cell of targeting EGFR, comprising:
(1) encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR, the coding base of the CAR-EGFR are provided Because including nucleotide sequence corresponding to the amino acid sequence as shown in SEQ ID NO:1；
(2) encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, obtains pWPXLD-CAR-EGFR recombination Plasmid；
(3) it by the pWPXLD-CAR-EGFR recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains To recombinant slow virus；
(4) recombinant slow virus is infected into CD3 positive t lymphocytes；
(5) separate and obtain the Chimeric antigen receptor T cell of targeting EGFR.
Optionally, the encoding gene of the CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:3.Accordingly Ground, the amino acid sequence of the CAR-EGFR is as shown in SEQ ID NO:4.
It include the encoding gene of a signal peptide in the encoding gene of CAR-EGFR of the present invention；The signal peptide is for referring to The Chimeric antigen receptor CAR-EGFR expression is led to cell surface, the signal peptide is in protein translation maturation by signal Peptase cutting.
Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:5.
Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:6.
Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. amino as shown in SEQ ID NO:5 The encoding gene of acid sequence includes the nucleotide sequence as shown in SEQ ID NO:6, and protection scope should also be protected and SEQ ID NO:6 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:5。
Optionally, the encoding gene of the CAR-EGFR be inserted into pWPXLD carrier BamH I and I restriction enzyme site of EcoR it Between, and be located at after the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-EGFR is inserted into When pWPXLD carrier, initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-EGFR Middle I restriction enzyme site of BamH is connected, and terminator codon (such as TAA) and I restriction enzyme site phase of EcoR in pWPXLD carrier can be added in 3 ' ends Even.
Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T Cell.
The envelope plasmid PMD2G encodes vesicular stomatitis virus glycoprotein capsid, the vesicular stomatitis virus sugar egg White capsid assists recombinant slow virus to adhere to cell membrane, and keeps the infectivity of recombinant slow virus.
Recombinant slow virus of the present invention can further contain the envelope protein from other viruses.For example, as this Kind protein, is preferably the virus enveloped protein for carrying out self-infection human cell.To this protein, there is no particular limitation, can example The amphophilic virus hand epithelium albumen etc. for enumerating retrovirus, can be used for example from mouse leukemia virus (MuMLV) 4070A plants of envelope protein.Alternatively, it is also possible to use the envelope protein from MuMLV 10Al.In addition, as herpetoviridae Albumen, it can be cited for example that, gB, gD, gH, gp85 albumen of herpes simplex virus, gp350, gp220 albumen of Epstein-Barr virus Deng.As the albumen of Hepadna Virus section, the S protein etc. of hepatitis B virus can be included.
The packaging of recombinant slow virus is generallyd use transient transfection or is packed using cell line.It may be used as wrapping when transient transfection Human cell's strain that dress cell uses, for example including 293 cells, 293T cell, 293FT cell, 293LTV cell, 293EBNA Cell and other clones separated from 293 cells；SW480 cell, u87MG cell, HOS cell, C8166 cell, MT-4 are thin Born of the same parents, Molt-4 cell, Hela cell, HT1080 cell, TE671 cell etc..It can also be using the cell strain from monkey, example Such as, COS1 cell, COS7 cell, CV-1 cell, BMT10 cell etc..Moreover, the calcium phosphate and PEI transfection reagent that generally use, It is also well used there are also some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin.
The packaging of recombinant slow virus also uses some slow virus package cell lines, such as most common Env glycoprotein of use, Stable cell lines caused by VSVG albumen or HIV-1gag-pol albumen.
For the sake of security, the slow virus carrier system of large-scale use is all the method using split gene group, i.e., will The assignment of genes gene mapping of different miscellaneous functions is played in different plasmids.There are four pUC pUCs (encoding gag-pol gene, Rev base at present Cause, VSVG gene, SIN metastatic gene be located at four different plasmids), three pUC pUCs (eliminate coding Rev gene Plasmid, gag-pol gene uses the codon of the preferences in people's cell in gag-pol plasmid) and two pUC pUCs are (slowly Auxiliary gene necessary to viral vectors is packed is located on the same plasmid, these auxiliary genes are single gene orders；Separately One is then transgenosis plasmid).Also the slow virus packaging system for having more than four pUC pUCs is using.
Optionally, in step (4), the CD3 positive t lymphocytes are to separate to obtain from source of people peripheral blood mononuclear cells .
Optionally, the source of people peripheral blood mononuclear cells derives from autologous vein blood, autologous bone marrow, Cord blood and placenta Blood etc..
Still optionally further, the fresh peripheral acquired after cancer patient's operation one month, after chemicotherapy one month Blood or marrow.
Specifically, the acquisition process of the CD3 positive t lymphocytes is as follows: into peripheral blood mononuclear cells by certain CD3/CD28 immunomagnetic beads are added in ratio, after being incubated for a period of time, are put into magnet and are screened, it is coated to obtain immunomagnetic beads CD3 positive t lymphocytes after removing magnetic bead, obtain CD3 positive t lymphocytes.
6th aspect, the present invention provides a kind of CAR-EGFR as described in relation to the first aspect, as described in second aspect or The Chimeric antigen receptor T cell of targeting EGFR made from preparation method as described in terms of the 5th, the weight as described in the third aspect Group viral vectors or host cell the answering in the drug for preparing prevention, diagnosing and treating malignant tumour as described in fourth aspect With.Specifically, can be used for preventing, the tables such as diagnosing and treating kinds of tumors or cancer, including glioma, cancer of pancreas and lung cancer Up to the cancer cell for having EGFR；It is suitable for the solid tumor cell or tissue of the EGFR positive simultaneously.
The application specifically: provide a kind of kit, the kit includes targeting EGFR described in first aspect Chimeric antigen receptor, the Chimeric antigen receptor T cell of targeting EGFR as described in second aspect, the weight as described in the third aspect One of group viral vectors, host cell as described in fourth aspect are a variety of.
Beneficial effects of the present invention:
The Chimeric antigen receptor T cell of targeting EGFR provided by the invention can promote T cell with the targeting EGFR of specificity In the amplification of patient's body, killing tumor cell that can be efficient and specific, while EGFR wide expression in tumour cell, And expressed in ordinary cells it is very faint, therefore the Chimeric antigen receptor T cell of targeting EGFR can specificity combination EGFR Positive tumor cell generates fragmentation effect to EGFR positive tumor cell, not will cause damage to normal cell.
Detailed description of the invention
Fig. 1 is the plasmid map of pWPXLd-CAR-EGFR recombinant plasmid provided in an embodiment of the present invention.
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as Protection scope of the present invention.
Embodiment one
A kind of preparation method of the Chimeric antigen receptor T cell of targeting EGFR, comprising the following steps:
(1) gene order of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is prepared
The coding gene sequence of the Chimeric antigen receptor CAR-EGFR of targeting EGFR, institute are prepared by the method for PCR The encoding gene for stating CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:3；
(2) pWPXLd-CAR-EGFR recombinant plasmid is constructed
The encoding gene of CAR-EGFR is inserted between I restriction enzyme site of BamH I and EcoR of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.When the encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, institute 5 ' end additions initiation codon (such as ATG) for stating the encoding gene of CAR-EGFR and I restriction enzyme site phase of BamH in pWPXLD carrier Even, 3 ' ends are also connected added with terminator codon (such as TAA) with I restriction enzyme site of EcoR in pWPXLD carrier.Then it is transferred to large intestine Bacillus competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and survey Sequence identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-EGFR recombinant plasmid, is as shown in Figure 1 PWPXLd-CAR-EGFR recombinant plasmid.
(3) recombinant slow virus constructs
PWPXLd-CAR-EGFR recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into training The HEK293T cell supported.48h harvest is protected in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration It deposits；Supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration merge with the viral supernatants of 48h harvest and are added together It in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature are controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added Liquid is saved, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence method after being centrifuged 5min Measuring titre, virus is according to 100 μ l, and 2 × 108A/ml packing, is stored in -80 DEG C of ultra low temperature freezers, obtains recombinant slow virus.
(4) preparation of the Chimeric antigen receptor T cell of targeting EGFR
A) separation of PBMC (peripheral blood mononuclear cells)
PBMC is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Preferably derive from cancer patient's hand The fresh peripheral blood or marrow acquired after art one month, after chemicotherapy one month.
Extract patient blood, sample presentation to blood separating chamber；Peripheral blood mononuclear cells is acquired, is taken after Ficoll centrifuge separation Intermediate layer cell；After PBS is washed, PBMC is obtained.
B) immunomagnetic beads antigenspecific T lymphocyte
Above-mentioned PBMC is taken, the basal medium for being free of serum is added, is made into cell suspension；Ratio in magnetic bead and cell is 3:1, is added CD3/CD28 immunomagnetic beads, and room temperature incubates 1-2h；The cell for being incubated for magnetic bead is screened using magnet；PBS is washed It washs, after removing immunomagnetic beads, obtains CD3 positive t lymphocytes.
C) virus transfection method prepares antigenspecific T lymphocyte
The above-mentioned CD3 positive t lymphocytes obtained by magnetic activated cell seperation are taken, are added and CD3 positive cell number phase The recombinant slow virus for the virus titer answered is cultivated.
The 3rd day of culture carries out cell count and changes liquid, and adjustment cell concentration is 1 × 106A/ml is inoculated with, culture；Training Cell state is observed in feeding the 5th day, if cell density increases, diluting cells concentration is 1 × 106A/ml detects cell Activity continues to cultivate.Amplification cultivation collected cell, and obtained the Chimeric antigen receptor T cell of targeting EGFR by the 9-11 days, and It is stored in and feeds back in dedicated cells frozen storing liquid.
Effect example
Effect example one assesses the tumor cell in vitro killing situation of the Chimeric antigen receptor T cell of targeting EGFR
By by the Chimeric antigen receptor T cell (being abbreviated as CAR-T-EGFR) of targeting EGFR made from the method for the present invention with The Vitro Tumor fragmentation effect of T lymphocyte (negative control group) without preparation is compared, specific: in vitro by 105 A effector cell's (CAR-T-EGFR or T lymphocyte without preparation) and target cell (U251 neuroglia cell of human oncocyte) Quantity ratio is 1:10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO2Under co-cultured, after incubation 15-18 hours, cell is collected, carries out streaming dyeing, detects cell killing situation, the results showed that method preparation of the present invention The Chimeric antigen receptor T cell tumor-killing effect of targeting EGFR be significantly larger than negative control group, therefore through the method for the present invention The Chimeric antigen receptor T cell of the targeting EGFR of preparation has strong tumor-killing ability.
Effect example two assesses the mouse interior tumor cell killing feelings of the Chimeric antigen receptor T cell of targeting EGFR Condition
By the Chimeric antigen receptor T cell (CAR-T-EGFR) of the targeting EGFR by the method for the present invention preparation, without system Standby T lymphocyte (negative control group) and physiological saline (blank control group) gives every mouse in mouse tumor model Tail vein injection 1 × 106A cell (n=9), obtains the survivorship curve of mouse, the results showed that the targeting prepared by this method The Chimeric antigen receptor T cell of EGFR is dead caused by capable of preferably protecting mice against because of tumour, and effect is right better than negative According to group and blank group.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
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Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln
50 55 60
Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr
65 70 75 80
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Thr Arg Leu Lys His Gln Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Ser Ala Leu Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val
130 135 140
Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg
145 150 155 160
Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val
165 170 175
Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg
180 185 190
Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly
195 200 205
Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser
210 215 220
Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ala
225 230 235 240
Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
245 250 255
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
260 265 270
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
275 280 285
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
290 295 300
Leu Val Ile Thr Leu Tyr Cys Arg Lys Tyr Arg Ser Asn Lys Gly Glu
305 310 315 320
Ser Pro Val Glu Pro Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu
325 330 335
Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu
340 345 350
Pro Ala Cys Ser Pro Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
355 360 365
Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
370 375 380
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
385 390 395 400
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
405 410 415
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
Ala Leu Pro Pro Arg
465
<210> 2
<211> 1407
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggcccagg tgcagctggt gcagtctggg gctgaggtga agaagcctgg gtcctcggtg 60
aaggtctcct gcaaggcttc tggaggcacc ttcagcagct atgctatcag ctgggtgcga 120
caggcccctg gacaagggct tgagtggatg ggagggatca tccctatctt tggtacagca 180
aactacgcac agaagttcca gggcagagtc acgattaccg cggacgaatc cacgagcaca 240
gcctacatgg agctgagcag cctgagatct gaggacacgg ccgtgtatta ctgtgcaaga 300
actcggctta agcatcagtg gggccaaggt accctggtca ccgtctcgag tggtggaggc 360
ggttcaggcg gaggtggctc tggcggtagt gcactttctt ctgagctgac tcaggaccct 420
gctgtgtctg tggccttggg acagacagtc aggatcacat gccaaggaga cagcctcaga 480
agctattatg caagctggta ccagcagaag ccaggacagg cccctgtact tgtcatctat 540
ggtaaaaaca accggccctc agggatccca gaccgattct ctggctccag ctcaggaaac 600
acagcttcct tgaccatcac tggggctcag gcggaagatg aggctgacta ttactgtaac 660
tcccgggaca gcagtggtcc ggtattcggc ggagggacca agctgaccgt cctaggtgcg 720
gccgcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900
cttctcctgt cactggttat caccctttac tgcaggaaat atagatcaaa caaaggagaa 960
agtcctgtgg agcctgcaga gccttgtcgt tacagctgcc ccagggagga ggagggcagc 1020
accatcccca tccaggagga ttaccgaaaa ccggagcctg cctgctcccc cagagtgaag 1080
ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1140
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1200
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1260
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1320
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1380
cttcacatgc aggccctgcc ccctcgc 1407
<210> 3
<211> 1467
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
atggcccagg tgcagctggt gcagtctggg gctgaggtga agaagcctgg gtcctcggtg 120
aaggtctcct gcaaggcttc tggaggcacc ttcagcagct atgctatcag ctgggtgcga 180
caggcccctg gacaagggct tgagtggatg ggagggatca tccctatctt tggtacagca 240
aactacgcac agaagttcca gggcagagtc acgattaccg cggacgaatc cacgagcaca 300
gcctacatgg agctgagcag cctgagatct gaggacacgg ccgtgtatta ctgtgcaaga 360
actcggctta agcatcagtg gggccaaggt accctggtca ccgtctcgag tggtggaggc 420
ggttcaggcg gaggtggctc tggcggtagt gcactttctt ctgagctgac tcaggaccct 480
gctgtgtctg tggccttggg acagacagtc aggatcacat gccaaggaga cagcctcaga 540
agctattatg caagctggta ccagcagaag ccaggacagg cccctgtact tgtcatctat 600
ggtaaaaaca accggccctc agggatccca gaccgattct ctggctccag ctcaggaaac 660
acagcttcct tgaccatcac tggggctcag gcggaagatg aggctgacta ttactgtaac 720
tcccgggaca gcagtggtcc ggtattcggc ggagggacca agctgaccgt cctaggtgcg 780
gccgcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960
cttctcctgt cactggttat caccctttac tgcaggaaat atagatcaaa caaaggagaa 1020
agtcctgtgg agcctgcaga gccttgtcgt tacagctgcc ccagggagga ggagggcagc 1080
accatcccca tccaggagga ttaccgaaaa ccggagcctg cctgctcccc cagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc ccctcgc 1467
<210> 4
<211> 489
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu
20 25 30
Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly
35 40 45
Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala
65 70 75 80
Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
85 90 95
Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Arg Thr Arg Leu Lys His Gln Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Ser Ala Leu Ser Ser Glu Leu Thr Gln Asp Pro
145 150 155 160
Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly
165 170 175
Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly
195 200 205
Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu
210 215 220
Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn
225 230 235 240
Ser Arg Asp Ser Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr
245 250 255
Val Leu Gly Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
260 265 270
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
275 280 285
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
290 295 300
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
305 310 315 320
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Lys Tyr Arg Ser
325 330 335
Asn Lys Gly Glu Ser Pro Val Glu Pro Ala Glu Pro Cys Arg Tyr Ser
340 345 350
Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr
355 360 365
Arg Lys Pro Glu Pro Ala Cys Ser Pro Arg Val Lys Phe Ser Arg Ser
370 375 380
Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
385 390 395 400
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
405 410 415
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
420 425 430
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
435 440 445
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
465 470 475 480
Leu His Met Gln Ala Leu Pro Pro Arg
485
<210> 5
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro
20
<210> 6
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

Claims (10)

1. a kind of Chimeric antigen receptor of targeting EGFR, which is characterized in that the Chimeric antigen receptor CAR- of the targeting EGFR EGFR includes the amino acid sequence as shown in SEQ ID NO:1.
2. the Chimeric antigen receptor of targeting EGFR as described in claim 1, which is characterized in that the inosculating antibody of the targeting EGFR The encoding gene of original receptor CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:2.
3. a kind of Chimeric antigen receptor T cell of targeting EGFR, which is characterized in that the Chimeric antigen receptor including targeting EGFR CAR-EGFR, the CAR-EGFR include the amino acid sequence as shown in SEQ ID NO:1.
4. the Chimeric antigen receptor T cell of targeting EGFR as claimed in claim 3, which is characterized in that the CAR-EGFR's Amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:2.
5. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes as described in claim any one of 3-4 Targeting EGFR Chimeric antigen receptor T cell CAR-EGFR encoding gene.
6. recombinant viral vector as claimed in claim 5, which is characterized in that the encoding gene of the CAR-EGFR includes such as Nucleotide sequence shown in SEQ ID NO:3.
7. a kind of host cell, which is characterized in that the host cell includes such as the described in any item recombination diseases of claim 5-6 Poisonous carrier.
8. a kind of preparation method of the Chimeric antigen receptor T cell of targeting EGFR characterized by comprising
(1) encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR, the encoding gene packet of the CAR-EGFR are provided Include nucleotide sequence corresponding to the amino acid sequence as shown in SEQ ID NO:1；
(2) encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, obtains pWPXLD-CAR-EGFR recombination matter Grain；
(3) by the pWPXLD-CAR-EGFR recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, weight is obtained Group slow virus；
(4) recombinant slow virus is infected into CD3 positive t lymphocytes, obtains the Chimeric antigen receptor T cell of targeting EGFR.
9. the preparation method of the Chimeric antigen receptor T cell of targeting EGFR as claimed in claim 8, which is characterized in that described The encoding gene of CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:3.
10. a kind of Chimeric antigen receptor CAR-EGFR such as the described in any item targeting EGFRs of claim 1-2, such as claim 3-4 is described in any item or preparation method as described in claim 8-9 made from targeting EGFR Chimeric antigen receptor T it is thin It is prepared by born of the same parents or such as the described in any item recombinant viral vectors of claim 5-6 or host cell as claimed in claim 7 Prevent, the application in the drug of diagnosing and treating malignant tumour.
CN201810560790.7A 2018-05-25 2018-05-25 A kind of Chimeric antigen receptor of targeting EGFR, Chimeric antigen receptor T cell and its preparation method and application Withdrawn CN110526992A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014055771A1 (en) * 2012-10-05 2014-04-10 The Trustees Of The University Of Pennsylvania Human alpha-folate receptor chimeric antigen receptor
CN105920592A (en) * 2015-09-11 2016-09-07 中国人民解放军总医院 Application of CARHER1-NKT cells in preparation of preparations for treatment of HER1 positive lung cancer in progressive stage

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014055771A1 (en) * 2012-10-05 2014-04-10 The Trustees Of The University Of Pennsylvania Human alpha-folate receptor chimeric antigen receptor
CN105920592A (en) * 2015-09-11 2016-09-07 中国人民解放军总医院 Application of CARHER1-NKT cells in preparation of preparations for treatment of HER1 positive lung cancer in progressive stage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GENBANK: "anti-epidermal growth factor receptor scFv, partial [synthetic construct]", 《GENBANK》 *
孙梦梅: "抗EGFR人源化抗体的构建、表达、纯化和功能分析", 《中国学位论文全文数据库》 *

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Application publication date: 20191203