CN109837292A - A kind of Chimeric antigen receptor T cell of targeting EGFR and its preparation method and application knocking out PD1 - Google Patents

Abstract

The present invention provides a kind of preparation methods of the Chimeric antigen receptor T cell of targeting EGFR for knocking out PD1, it include: that the encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is connected with carrier, carry out the building of recombinant slow virus, and transfect CD3 positive t lymphocytes, the PD1 gene of T cell after transfection is knocked out, the Chimeric antigen receptor T cell for knocking out the targeting EGFR of PD1 is obtained.The Chimeric antigen receptor T cell of the targeting EGFR for knocking out PD1 has knocked out PD1 gene, be conducive to T cell in the amplification of patient's body, avoid neoplastic cells escape immunosurveillance, performance with efficient and specific killing tumor cell, and damage is not will cause to normal cell, while the Chimeric antigen receptor T cell of the Humanized single chain antibody targeting EGFR obtained for knocking out PD1 can preferably maintain the vigor and lethality of Chimeric antigen receptor T cell.

Description

A kind of Chimeric antigen receptor T cell of targeting EGFR and preparation method thereof knocking out PD1 And application

Technical field

The present invention relates to field of medical biotechnology, in particular to a kind of Chimeric antigen receptor T for the targeting EGFR for knocking out PD1 is thin Born of the same parents and its preparation method and application.

Background technique

People's epithelial growth factor receptor (EGFR) is the major receptors of people's epidermal growth factor.Upon activation, EGFR can be sent out Raw homologous dimerization, or heterodimeric occurs with the member of other people epithelial growth factor receptor families, to induce EGFR cell The autophosphorylation of interior structure, and activate downstream passages such as Mitogen-actived protein kinase (MAPK) access etc., and mediate thin Born of the same parents' amplification.Research shows that EGFR is expression or a unconventionality expression in a variety of solid tumor cells such as lung cancer, and in ordinary cells The very faint cell surface antigen of middle expression.The proliferation of EGFR and tumour cell, tumor invasion, transfer and the inhibition of Apoptosis It is related.In cancer, the EGFR of variation obtains the activation of duration, so as to cause the immoderate amplification of cell and eventually leads to cancer Disease.Therefore, EGFR is a cancer specific target spot.

Death protein (PD1) is the one kind for being expressed in T cell and the receptor protein on precursor B cells film, is belonged to Immunoglobulin superfamily.PD1 is the representativeness in immunologic test point inhibition (immune checkpoint in hibition) Molecule, by preventing t cell activation come negative regulation immune response in immunity of organism, to prevent autoimmune disease and guarantee Self-tolerance.In tumor microenvironment, PD1 can inhibit the activity of killer T cell, to make the immune prison of neoplastic cells escape Depending on.

Immune cell therapy is that the method for thoroughly removing cancer cell is uniquely possible in existing science and technology, and treatment tumour has High specificity, almost non-toxic side effect huge advantage the drawbacks of compensating for traditional remedies, at home and abroad have been used to clinic and control Malignant tumour is treated, Chimeric antigen receptor T cell technology (CAR-T) is that current adoptive cell adoptive therapy technology is newest immune One of cell technology, because it can activate self immune system in vivo, routinely targets neoplastic cells are killed, most Achieve the purpose that remove malignant cell and widely paid close attention to and studied eventually.There is not the inosculating antibody of targeting EGFR also at present Original receptor T cell and the research that can avoid neoplastic cells escape immunosurveillance.

Summary of the invention

In view of this, the present invention provides a kind of Chimeric antigen receptor T cell of targeting EGFR for knocking out PD1, it is described to strike Except the Chimeric antigen receptor T cell of the targeting EGFR of PD1 has knocked out PD1 gene, be conducive to T cell in the amplification of patient's body, Neoplastic cells escape immunosurveillance is avoided, the performance with efficient and specific killing tumor cell preferably maintains cell Vigor and lethality, and not will cause damage to normal cell, while the Humanized single chain antibody target obtained for knocking out PD1 The vigor and lethality of Chimeric antigen receptor T cell can be preferably maintained to the Chimeric antigen receptor T cell of EGFR.

In a first aspect, the present invention provides a kind of preparation sides of the Chimeric antigen receptor T cell of targeting EGFR for knocking out PD1 Method, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is provided, including sequentially from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of connection, the encoding gene of the single-chain antibody of targeting EGFR, the encoding gene of extracellular hinge area, cross-film The encoding gene in area, intracellular signal area encoding gene, wherein the single-chain antibody of the targeting EGFR is anti-for Humanized single chain Body, the encoding gene of the single-chain antibody of the targeting EGFR include the core for encoding the amino acid sequence as shown in SEQ ID NO:1 Nucleotide sequence；

(2) encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, obtains pWPX LD-CAR-EGFR weight Group plasmid；

(3) the pWPXLD-CAR-EGFR recombinant plasmid is packed, recombinant slow virus is obtained；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, obtains Chimeric antigen receptor T cell；

(5) the PD1 gene for knocking out the Chimeric antigen receptor T cell, obtains the chimeric antigen for knocking out the targeting EGFR of PD1 Recipient T cells.

Optionally, the amino acid sequence of the single-chain antibody of the targeting EGFR includes the amino as shown in SEQ ID NO:1 Acid sequence.

Optionally, the encoding gene of the amino acid sequence of the single-chain antibody of the targeting EGFR includes such as SEQ I D NO:5 Shown in nucleotide sequence.

Optionally, the encoding gene of the amino acid sequence of the single-chain antibody of the targeting EGFR should consider degeneracy base, I.e. the encoding gene of the amino acid sequence as shown in SEQ ID NO:1 includes the nucleotide sequence as shown in SEQ ID NO:5, is protected Shield range should also protect the nucleotide sequence for having base degeneracy matter with SEQ ID NO:5, these nucleotide sequences are corresponding Amino acid sequence remain as SEQ ID NO:1.

In the present invention, the signal peptide is for instructing the Chimeric antigen receptor CAR-EGFR expression to cell surface, institute Signal peptide is stated to be cut in protein translation maturation by signal peptidase.

Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:7.

Optionally, the encoding gene of the amino acid sequence of the signal peptide includes the nucleotide as shown in SEQ ID NO:8 Sequence.

Optionally, the encoding gene of the amino acid sequence of the signal peptide should consider degeneracy base, i.e., such as SE Q ID The encoding gene of amino acid sequence shown in NO:7 includes the nucleotide sequence as shown in SEQ ID NO:8, and protection scope is also answered The protection and SEQ ID NO:8 have the nucleotide sequence of base degeneracy matter, the corresponding amino acid sequence of these nucleotide sequences Column remain as SEQ ID NO:7.

In the present invention, the extracellular hinge area is used to promote the EGFR knot on the single-chain antibody and tumour of the targeting EGFR It closes.

Optionally, the extracellular hinge area include CD8 α hinge area, CD28 hinge area, CD4 hinge area, C D5 hinge area, One of CD134 hinge area, CD137 hinge area, ICOS hinge area or a variety of combinations.

Further alternative, the extracellular hinge area includes CD8 α hinge area.

Optionally, the amino acid sequence of the CD8 α hinge area includes the amino acid sequence as shown in SEQ ID NO:9.

Optionally, the encoding gene of the amino acid sequence of the CD8 α hinge area includes as shown in SEQ ID NO:1 0 Nucleotide sequence.

Optionally, the encoding gene of the amino acid sequence of the CD8 α hinge area should consider degeneracy base, i.e., such as SEQ The encoding gene of amino acid sequence shown in ID NO:9 includes the nucleotide sequence as shown in SEQ ID NO:10, protection scope The nucleotide sequence that there is base degeneracy matter with SEQ ID NO:10, the corresponding amino of these nucleotide sequences should also be protected Acid sequence remains as SEQ ID NO:9.

In the present invention, the transmembrane region is for fixing the Chimeric antigen receptor CAR-EGFR.

Optionally, the transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region Or a variety of combination.

Further alternative, the transmembrane region includes CD8 transmembrane region.

Optionally, the amino acid sequence of the CD8 transmembrane region includes the amino acid sequence as shown in SEQ ID NO:11.

Optionally, the encoding gene of the amino acid sequence of the CD8 transmembrane region includes the core as shown in SEQ ID NO:12 Nucleotide sequence.

Optionally, the encoding gene of the amino acid sequence of the CD8 transmembrane region should consider degeneracy base, i.e., such as SEQ ID The encoding gene of amino acid sequence shown in NO:11 includes the nucleotide sequence as shown in SEQ ID NO:12, and protection scope is also The nucleotide sequence that there is base degeneracy matter with SEQ ID NO:12, the corresponding amino acid of these nucleotide sequences should be protected Sequence remains as SEQ ID NO:11.

In the present invention, the intracellular signal area for providing the signal of T cell activation, maintain T cell life span and Activate T cell proliferation signal access.

Optionally, the intracellular signal area includes 4-1BB signaling zone, CD3 ζ signaling zone, ICOS signaling zone, C D27 signal Area, OX40 signaling zone, CD27 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, in TNFRSF19L signaling zone One or more combinations.

Optionally, the intracellular signal area includes 4-1BB signaling zone and CD3 ζ signaling zone.

Optionally, the amino acid sequence of the 4-1BB signaling zone includes the amino acid sequence as shown in SEQ ID NO:13.

Optionally, the encoding gene of the amino acid sequence of the 4-1BB signaling zone includes as shown in SEQ ID NO:1 4 Nucleotide sequence.

Optionally, the encoding gene of the amino acid sequence of the 4-1BB signaling zone should consider degeneracy base, i.e., such as SEQ The encoding gene of amino acid sequence shown in ID NO:13 includes the nucleotide sequence as shown in SEQ ID NO:14, protection scope The nucleotide sequence that there is base degeneracy matter with SEQ ID NO:14, the corresponding amino of these nucleotide sequences should also be protected Acid sequence remains as SEQ ID NO:13.

Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:15.

Optionally, the encoding gene of the amino acid sequence of the CD3 ζ signaling zone includes as shown in SEQ ID NO:1 6 Nucleotide sequence.

Optionally, the encoding gene of the amino acid sequence of the CD3 ζ signaling zone should consider degeneracy base, i.e., such as SEQ The encoding gene of amino acid sequence shown in ID NO:15 includes the nucleotide sequence as shown in SEQ ID NO:16, protection scope The nucleotide sequence that there is base degeneracy matter with SEQ ID NO:16, the corresponding amino of these nucleotide sequences should also be protected Acid sequence remains as SEQ ID NO:15.

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.Nucleotide sequence shown in SEQ ID NO:2 contains the encoding gene of the signal peptide, the letter Number peptide is cut in protein translation maturation by signal peptidase.

The encoding gene of the CAR-EGFR is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and After the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CA R-EGFR is inserted into pWPXLD load When body, BamH1 enzyme in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-EGFR Enzyme site is connected, and 3 ' ends can be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site in pWPXLD carrier.

Optionally, the amino acid sequence of the Chimeric antigen receptor CAR-EGFR of the targeting EGFR includes such as SE Q ID Amino acid sequence shown in NO:4.

Optionally, the packaging pWPXLD-CAR-EGFR recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-EGFR recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, obtain To the recombinant slow virus.

Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2.

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.

Optionally, the host cell may include HEK293T cell, 293 cells, 293T cell, 293 FT cells, SW480 cell, u87MG cell, HOS cell, COS1 cell or COS7 cell.

Further alternative, the host cell is HEK293T cell.

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 envelope protein can also be morbilli It is formed after viral glycoprotein and other single chain antibody fusions.

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..

Fresh peripheral that is further alternative, being 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.

Optionally, the PD1 gene for knocking out the Chimeric antigen receptor T cell uses electrotransfection and Crispr/Cas9 Technology knocks out the PD1 gene of the Chimeric antigen receptor T cell.

Further, the PD1 gene for knocking out the Chimeric antigen receptor T cell, comprising the following steps:

The encoding gene of the Cas9 is inserted into pcDNA3.1 carrier, pcDNA3.1-Cas9 recombinant plasmid is obtained, with The pcDNA3.1-Cas9 recombinant plasmid is that template is transcribed in vitro to obtain Cas9mRNA；

Synthesis targets the corresponding gene order of sgRNA of the PD1 gene, by the sg RNA couple of the targeting PD1 gene The gene order answered is inserted into pcDNA3.1 carrier, pcDNA3.1-PD1-sgRNA recombinant plasmid is obtained, with described PcDNA3.1-PD1-sgRNA recombinant plasmid is that template is transcribed in vitro to obtain sgRNA；

The sgRNA of the Cas9mRNA and the targeting PD1 gene are mixed with the Chimeric antigen receptor T cell It closes, is placed in electroporation and carries out electricity turn, knock out the PD1 gene of the Chimeric antigen receptor T cell.

Optionally, the corresponding gene order of sgRNA of the targeting PD1 gene includes as shown in SEQ ID N O:3 Nucleotide sequence.

Optionally, the mass ratio of the sgRNA of the Cas9mRNA and the targeting PD1 gene is 1:1-1:5.

Further alternative, the mass ratio of the sgRNA of the Cas9mRNA and the targeting PD1 gene is 1:3.

In the present invention, in step (5), the Chimeric antigen receptor T cell of the targeting EGFR of the knockout PD1 of acquisition, is in step Suddenly the PD1 gene of cell has been knocked out on the basis of the Chimeric antigen receptor T cell in (4).Chimeric antigen receptor in step (4) T cell can also be with targeting EGFR, but it is possible that the phenomenon that neoplastic cells escape immunosurveillance.Knock out what PD1 gene obtained The Chimeric antigen receptor T cell for knocking out the targeting EGFR of PD1 is stronger to the targeting of tumour cell, avoids neoplastic cells escape The case where immunosurveillance.

Optionally, the Chimeric antigen receptor T cell preparation process of the targeting EGFR for knocking out PD1 may be: (1) mentioning For the encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR, including holding sequentially connected signal peptides from 5 ' ends to 3 ' Encoding gene, the encoding gene of single-chain antibody of targeting EGFR, the encoding gene of extracellular hinge area, transmembrane region encoding gene, The encoding gene in intracellular signal area, wherein the encoding gene of the single-chain antibody of the targeting EGFR includes coding such as SEQ ID The nucleotide sequence of amino acid sequence shown in NO:1；

(2) encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, obtains pWPX LD-CAR-EGFR weight Group plasmid；

(3) the pWPXLD-CAR-EGFR recombinant plasmid is packed, recombinant slow virus is obtained；

(4) CD3 positive t lymphocytes are prepared, the PD1 gene of the CD3 positive t lymphocytes is knocked out, obtain knocking out PD1 CD3 positive t lymphocytes；

(5) recombinant slow virus is transfected into the CD3 positive t lymphocytes for knocking out PD1, obtains the target for knocking out PD1 To the Chimeric antigen receptor T cell of EGFR.

Optionally, the knockout process can be carried out when obtaining the CD3 positive t lymphocytes, can also obtained It is carried out when the Chimeric antigen receptor T cell of targeting EGFR, finally can obtain the chimeric antigen of the targeting EGFR for knocking out PD1 Recipient T cells, in the present invention to this knockout sequence and be not construed as limiting, can achieve obtain knock out PD1 targeting EGFR it is embedding Close the purpose of antigen receptor T cell.

The preparation method of the Chimeric antigen receptor T cell of the targeting EGFR for the knockout PD1 that first aspect present invention provides, leads to It crosses and prepares the Chimeric antigen receptor of targeting EGFR and obtain Chimeric antigen receptor T cell, and knock out the PD1 gene in T cell and be made The Chimeric antigen receptor T cell of the targeting EGFR of PD1 is knocked out, which is conducive to T cell in the amplification of patient's body, keeps away Exempt from neoplastic cells escape immunosurveillance, makes it have the performance of efficient and specific killing tumor cell, while humanization list The Chimeric antigen receptor T cell of the chain antibody targeting EGF R obtained for knocking out PD1 can preferably maintain Chimeric antigen receptor T The vigor and lethality of cell.

Second aspect, the present invention provides the knockout P D1's being prepared using preparation method as described in relation to the first aspect The Chimeric antigen receptor T cell of the Chimeric antigen receptor T cell of targeting EGFR, the targeting EGFR for knocking out PD1 is free of PD1 base The Chimeric antigen receptor T cell of cause, the targeting EGFR for knocking out PD1 includes the Chimeric antigen receptor CAR- of targeting EGFR EGFR, the Chimeric antigen receptor CAR-EGF R of the targeting EGFR include the sequentially connected targeting from aminoterminal to c-terminus The single-chain antibody of EGFR, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein the targeting EGFR Single-chain antibody is Humanized single chain antibody, and the single-chain antibody of the targeting EGFR includes the amino acid as shown in SEQ ID NO:1 Sequence.

Above-mentioned " being sequentially connected with from aminoterminal to c-terminus " specifically: the amino acid sequence of the single-chain antibody of the targeting EGFR The c-terminus of column is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, the amino acid sequence of the extracellular hinge area C-terminus be connected with the aminoterminal of the amino acid sequence of the transmembrane region, the c-terminus of the amino acid sequence of the transmembrane region with The aminoterminal of the amino acid sequence in the intracellular signal area is connected.

Wherein, the single-chain antibody of the targeting EGFR, extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and Corresponding amino acid sequence is as described in first aspect present invention part, and details are not described herein.

Optionally, the amino acid sequence of the Chimeric antigen receptor CAR-EGFR of the targeting EGFR includes such as SE Q ID Amino acid sequence shown in NO:4.

Optionally, the encoding gene of the amino acid sequence of the Chimeric antigen receptor CAR-EGFR of the targeting EGFR includes such as Nucleotide sequence shown in SEQ ID NO:6.

Optionally, the encoding gene of the amino acid sequence of the CAR-EGFR should consider degeneracy base, i.e., such as SEQ ID The encoding gene of amino acid sequence shown in NO:4 includes the nucleotide sequence as shown in SEQ ID NO:6, and protection scope is also answered The protection and SEQ ID NO:6 have the nucleotide sequence of base degeneracy matter, the corresponding amino acid sequence of these nucleotide sequences Column remain as SEQ ID NO:4.

Preferably, the encoding gene of the CAR-EGFR includes the nucleotide sequence as shown in SEQ ID NO:2.SEQ ID Nucleotide sequence shown in NO:2 contains the encoding gene of the signal peptide, the signal peptide can instruct the chimeric antigen by Body CAR-EGFR is expressed to cell surface, but signal peptide is cut in protein translation maturation by signal peptidase.

The Chimeric antigen receptor T cell of the targeting EGFR for the knockout PD1 that second aspect of the present invention provides can be single-minded Property targeting EGFR, especially express EGFR solid tumor cell, to tumour cell generate fragmentation effect, will not to normal cell It causes to damage, the Chimeric antigen receptor T cell of the Humanized single chain antibody targeting EGFR obtained for knocking out PD1 can be tieed up preferably The vigor and lethality of Chimeric antigen receptor T cell are held, and knocks out the PD1 gene of T cell, is conducive to T cell in patient's body Interior amplification avoids neoplastic cells escape immunosurveillance, makes it have the performance of efficient and specific killing tumor cell.

The third aspect, a kind of be prepared the present invention provides preparation method as described in relation to the first aspect or such as second party A kind of Chimeric antigen receptor T cell of the targeting EGFR of knockout PD1 described in face is in preparation prevention, diagnosing and treating malignant tumour Drug in application.

The application specifically: provide a kind of kit, the kit includes preparation side as described in relation to the first aspect A kind of Chimeric antigen receptor T cell of the targeting EGFR of knockout PD1 that method is prepared or as described in second aspect.

Beneficial effects of the present invention:

(1) EGFR wide expression in tumour cell, and very faint increasing with tumour cell is expressed in ordinary cells Grow, tumor invasion, transfer and the inhibition of Apoptosis it is related, therefore the targeting EGFR provided by the invention for knocking out P D1 is chimeric Antigen receptor T cell can specificity combination tumour cell, the tumour cell of the targeted expression EGFR of specificity, promote T it is thin Born of the same parents generate fragmentation effect to tumour cell, not will cause damage to normal cell in the amplification of patient's body；

(2) single-chain antibody of targeting EGFR is Humanized single chain antibody, and the targeting E GFR's obtained for knocking out PD1 is chimeric Antigen receptor T cell preferably maintains the vigor and lethality of cell；

(3) the Chimeric antigen receptor T cell for knocking out the targeting EGFR of PD1 is conducive to T cell in the amplification of patient's body, keeps away Exempt from neoplastic cells escape immunosurveillance, makes it have the performance of efficient and specific killing tumor 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.

Fig. 2 is the positive rate of the Chimeric antigen receptor T cell of the targeting EGFR provided in an embodiment of the present invention for knocking out PD1； (a) is negative control group in Fig. 2, and (b) is the chimeric antigen of the targeting EGFR provided in an embodiment of the present invention for knocking out PD1 in Fig. 2 The experimental group of recipient T cells.

Fig. 3 is the Vitro Tumor of the Chimeric antigen receptor T cell of the targeting EGFR provided in an embodiment of the present invention for knocking out PD1 Cell killing efficacy figure.

Fig. 4 is that the Chimeric antigen receptor T cell treatment tumour of the targeting EGFR provided in an embodiment of the present invention for knocking out PD1 is small The effect picture of mouse.

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 for the targeting EGFR knocking out PD1, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is prepared

Prepare respectively signal peptide, the single-chain antibody of targeting EGFR, CD8 α hinge area, CD8 transmembrane region, 4-1 BB signaling zone and The encoding gene of CD3 ζ signaling zone, the encoding gene of the signal peptide as shown in SEQ ID NO:8, the targeting EGFR it is single-stranded The encoding gene of antibody as shown in SEQ ID NO:5, the encoding gene of the CD8 α hinge area as shown in SEQ ID NO:10, The encoding gene of the CD8 transmembrane region is as shown in SEQ ID NO:12, the encoding gene of the 4-1BB signaling zone such as SEQ ID Shown in NO:14, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:16, wherein the targeting EGFR it is single-stranded Antibody is Humanized single chain antibody.

By the method for PCR by above-mentioned signal peptide, the single-chain antibody of targeting EGFR, CD8 α hinge area, CD8 transmembrane region, 4- 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains Chimeric antigen receptor The encoding gene of CAR-EGFR, the encoding gene of the CAR-EGFR is as shown in SEQ I D NO:2.

(2) pWPXLd-CAR-EGFR recombinant plasmid is constructed

The encoding gene of CAR-EGFR is inserted between BamH1 the and EcoR1 restriction enzyme site 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 BamH1 restriction enzyme site in initiation codon (such as ATG) and pWPXLD carrier can be added in the 5 ' ends for stating the encoding gene of CAR-EGFR It is connected, 3 ' ends can be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site 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 p WPXLd-CAR-EGFR recombinant plasmid as shown in Figure 1.

(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 Chimeric antigen receptor T cell

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 Chimeric antigen receptor T cell by the 9-11 days.

D) PD1 gene is knocked out

The encoding gene of the Cas9 is inserted into pcDNA3.1 carrier, pcDNA3.1-Cas9 recombinant plasmid is obtained, with The pcDNA3.1-Cas9 recombinant plasmid is template, utilizes mMESSAGE T7 kit is turned in vitro Record obtains Cas9mRNA；Synthesis targets the corresponding gene order of sgRNA of the PD1 gene, the targeting PD1 gene The corresponding gene order of sgRNA nucleotide sequence as shown in SEQ ID NO:3；The sgRNA of the targeting PD1 gene is corresponding Gene order be inserted into pcDN A3.1 carrier, pcDNA3.1-PD1-sgRNA recombinant plasmid is obtained, with described PcDNA3.1-PD1-sgR NA recombinant plasmid is template, utilizes mMESSAGET7 kit carries out external Transcription obtains sgRNA；By the Cas9mRNA and it is described targeting PD1 gene sgRNA and the Chimeric antigen receptor T cell into Row mixing, is placed in electroporation and carries out electricity turn, knocks out the P D1 gene of the Chimeric antigen receptor T cell, obtains and knocks out PD1 Targeting EGFR Chimeric antigen receptor T cell.

The PD 1 that the Chimeric antigen receptor T cell of the targeting EGFR of above-mentioned knockout PD1 is measured using flow cytometer is expressed Amount, measures its knockout rate, as a result, it has been found that knocking out the knockout rate of PD1 gene in the Chimeric antigen receptor T cell of the targeting EGFR of PD1 It is 65%.

Effect example

In order to assess the Chimeric antigen receptor T of the targeting EGFR for knocking out PD1 prepared by the above method described in the invention Cell effect carries out following effect example.

Effect example one: the Chimeric antigen receptor T cell of the targeting EGFR of knockout PD1 prepared by the assessment present invention Positive rate

By by the method for the present invention preparation knock out PD1 targeting EGFR Chimeric antigen receptor T cell (experimental group) with not T lymphocyte (negative control group) through preparing, using its positive rate of flow cytomery, as a result as shown in Fig. 2, wherein scheming (a) is negative control group in 2, i.e., without the T cell of preparation, (b) is experimental group, knockout PD1 as produced by the present invention in Fig. 2 Targeting EGFR Chimeric antigen receptor T cell.(a) can be obtained compared with (b) in Fig. 2, knockout PD1 prepared by the present invention Targeting EGFR Chimeric antigen receptor T cell positive rate be 50.5%.

Effect example two: the tumor cell in vitro that assessment knocks out the Chimeric antigen receptor T cell of the targeting EGFR of PD1 kills Condition of the injury condition

Will by the Chimeric antigen receptor T cell (experimental group) of the targeting EGFR of knockout PD1 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 effect Cell (knocking out the Chimeric antigen receptor T cell of the targeting EGFR of PD1 or the T lymphocyte without preparation) and target cell (HT1080 cell) is 1:10,1:3,1:1,3:1 and 10:1 ratio in quantity ratio, at 37 DEG C, 5%CO₂Under co-cultured, 15-18 hours after culture, cell is collected, carries out streaming dyeing, detects cell killing situation, as a result as shown in Figure 3.From figure It can be seen that in 3, the Chimeric antigen receptor T cell of the targeting EGFR of the knockout PD1 of addition is more, their killings to tumour cell Power is stronger.The tumour of the Chimeric antigen receptor T cell of the targeting EGFR of knockout PD1 by method of the present invention preparation is killed Overstrain is 20% or more, and even up to 50%, significantly larger than negative control group, this illustrates the knockout prepared through the method for the present invention The Chimeric antigen receptor T cell of the targeting EGFR of PD1 has strong tumor-killing ability.

Effect example three: the mouse interior tumor that assessment knocks out the Chimeric antigen receptor T cell of the targeting EGFR of PD1 is thin Born of the same parents kill situation

By the Chimeric antigen receptor T cell (experimental group) of the targeting EGFR of the knockout PD1 by the method for the present invention preparation, not T lymphocyte (negative control group) and physiological saline (blank control group) through preparing give every in mouse tumor model Mouse tail vein injection 1 × 10⁶A HT1080 cell (n=9), draws the survivorship curve of mouse, as a result as shown in Figure 4.From Fig. 4 As can be seen that the Chimeric antigen receptor T cell of the targeting EGFR of the knockout PD1 by this method preparation makes mouse in culture 95 It when survival rate be also higher than 50%, almost close to 60%, considerably beyond negative control group and blank control group.The result table of Fig. 4 Bright, the Chimeric antigen receptor T cell of the targeting EGFR of the knockout PD1 by this method preparation can be protected mice against preferably Because dead caused by tumour.

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

<110>Shenzhen Bin De Bioisystech Co., Ltd

<120>a kind of Chimeric antigen receptor T cell of targeting EGFR and its preparation method and application for knocking out PD1

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe Asn Ile Glu Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys Tyr Gly Pro Ile Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser Ile Asn Thr Val Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Asp Ser

130 135 140

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

145 150 155 160

Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile Ser Leu Val Ser Lys

180 185 190

Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

210 215 220

Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Val Glu Ile Lys

245

<210> 2

<211> 1467

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gccctccctg tcaccgccct gctgcttccg ctggctcttc tgctccacgc cgctcggccc 60

gagattcagc tcgtgcaatc gggagcggaa gtcaagaagc caggagagtc cttgcggatc 120

tcatgcaagg gtagcggctt taacatcgag gattactaca tccactgggt gaggcagatg 180

ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 240

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 300

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 360

ggggtctact ggggacaggg aactaccgtg actgtctcgt ccggcggagg cgggtcagga 420

ggtggcggca gcggaggagg agggtccggc ggaggtgggt ccgacgtcgt gatgacccag 480

agccctgaca gcctggcagt gagcctgggc gaaagagcta ccattaactg caaatcgtcg 540

cagagcctgc tggactcgga cggaaaaacg tacctcaatt ggctgcagca aaagcctggc 600

cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 660

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 720

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 780

acgaaagtgg agatcaagac cactacccca gcaccgaggc cacccacccc ggctcctacc 840

atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc tggtggggcc 900

gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcgg 1467

<210> 3

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cacgaagctc tccgatgtgt tgg 23

<210> 4

<211> 469

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe Asn Ile Glu Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys Tyr Gly Pro Ile Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser Ile Asn Thr Val Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Asp Ser

130 135 140

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

145 150 155 160

Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile Ser Leu Val Ser Lys

180 185 190

Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

210 215 220

Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

260 265 270

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

305 310 315 320

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

325 330 335

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

340 345 350

Gly Gly Cys Glu Leu 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> 5

<211> 738

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gagattcagc tcgtgcaatc gggagcggaa gtcaagaagc caggagagtc cttgcggatc 60

tcatgcaagg gtagcggctt taacatcgag gattactaca tccactgggt gaggcagatg 120

ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 180

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 240

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 300

ggggtctact ggggacaggg aactaccgtg actgtctcgt ccggcggagg cgggtcagga 360

ggtggcggca gcggaggagg agggtccggc ggaggtgggt ccgacgtcgt gatgacccag 420

agccctgaca gcctggcagt gagcctgggc gaaagagcta ccattaactg caaatcgtcg 480

cagagcctgc tggactcgga cggaaaaacg tacctcaatt ggctgcagca aaagcctggc 540

cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 600

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 660

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 720

acgaaagtgg agatcaag 738

<210> 6

<211> 1407

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gagattcagc tcgtgcaatc gggagcggaa gtcaagaagc caggagagtc cttgcggatc 60

tcatgcaagg gtagcggctt taacatcgag gattactaca tccactgggt gaggcagatg 120

ccggggaagg gactcgaatg gatgggacgg atcgacccag aaaacgacga aactaagtac 180

ggtccgatct tccaaggcca tgtgactatt agcgccgata cttcaatcaa taccgtgtat 240

ctgcaatggt cctcattgaa agcctcagat accgcgatgt actactgtgc tttcagagga 300

ggggtctact ggggacaggg aactaccgtg actgtctcgt ccggcggagg cgggtcagga 360

ggtggcggca gcggaggagg agggtccggc ggaggtgggt ccgacgtcgt gatgacccag 420

agccctgaca gcctggcagt gagcctgggc gaaagagcta ccattaactg caaatcgtcg 480

cagagcctgc tggactcgga cggaaaaacg tacctcaatt ggctgcagca aaagcctggc 540

cagccaccga agcgccttat ctcactggtg tcgaagctgg attcgggagt gcccgatcgc 600

ttctccggct cgggatcggg tactgacttc accctcacta tctcctcgct tcaagcagag 660

gacgtggccg tctactactg ctggcaggga acccactttc cgggaacctt cggcggaggg 720

acgaaagtgg agatcaagac cactacccca gcaccgaggc cacccacccc ggctcctacc 780

atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc tggtggggcc 840

gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc tctggctggt 900

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 960

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1020

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1080

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1140

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1200

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1260

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1320

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1380

cttcacatgc aggccctgcc gcctcgg 1407

<210> 7

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

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> 8

<211> 60

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

gccctccctg tcaccgccct gctgcttccg ctggctcttc tgctccacgc cgctcggccc 60

<210> 9

<211> 46

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 9

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

<210> 10

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatc 138

<210> 11

<211> 26

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

1 5 10 15

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

20 25

<210> 12

<211> 78

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

tacatttggg cccctctggc tggtacttgc ggggtcctgc tgctttcact cgtgatcact 60

ctttactgta agcgcggt 78

<210> 13

<211> 39

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

1 5 10 15

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

20 25 30

Glu Glu Gly Gly Cys Glu Leu

35

<210> 14

<211> 117

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 60

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactg 117

<210> 15

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 16

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

Claims (10)

1. a kind of preparation method of the Chimeric antigen receptor T cell for the targeting EGFR for knocking out PD1 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-EGFR of targeting EGFR is provided, including is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of signal peptide, the encoding gene of single-chain antibody of targeting EGFR, the encoding gene of extracellular hinge area, transmembrane region Encoding gene, intracellular signal area encoding gene, wherein the single-chain antibody of the targeting EGFR be Humanized single chain antibody, institute The encoding gene for stating the single-chain antibody of targeting EGFR includes the nucleotides sequence for encoding the amino acid sequence as shown in SEQ ID NO:1 Column；

(2) encoding gene of the CAR-EGFR is inserted into pWPXLD carrier, obtains pWPX LD-CAR-EGFR recombination matter Grain；

(3) the pWPXLD-CAR-EGFR recombinant plasmid is packed, recombinant slow virus is obtained；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, obtains Chimeric antigen receptor T cell；

(5) the PD1 gene for knocking out the Chimeric antigen receptor T cell, obtains the Chimeric antigen receptor for knocking out the targeting EGFR of PD1 T cell.

2. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting EGFR of PD1, feature as described in claim 1 It is, the extracellular hinge area includes CD8 α hinge area, and the transmembrane region includes CD8 transmembrane region, and the intracellular signal area includes 4-1BB signaling zone and CD3 ζ signaling zone.

3. the preparation side of the Chimeric antigen receptor T cell such as the targeting EGFR of the described in any item knockout PD1 of claims 1 or 2 Method, which is characterized in that the encoding gene of the Chimeric antigen receptor CAR-EGFR of the targeting EGFR includes such as SEQ ID NO:2 institute The nucleotide sequence shown.

4. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting EGFR of PD1, feature as described in claim 1 It is, the packaging pWPXLD-CAR-EGFR recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-EGFR recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, institute is obtained State recombinant slow virus.

5. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting EGFR of PD1, feature as described in claim 1 It is, the PD1 gene for knocking out the Chimeric antigen receptor T cell, comprising:

The encoding gene of the Cas9 is inserted into pcDNA3.1 carrier, pcDNA3.1-Cas9 recombinant plasmid is obtained, with described PcDNA3.1-Cas9 recombinant plasmid is that template is transcribed in vitro to obtain Cas9mRNA；

Synthesis targets the corresponding gene order of sgRNA of the PD1 gene, by the corresponding base of sgRNA of the targeting PD1 gene Because sequence is inserted into pcDNA3.1 carrier, pcDNA3.1-PD1-sgRNA recombinant plasmid is obtained, with the pcDNA3.1-PD1- SgRNA recombinant plasmid is that template is transcribed in vitro to obtain sgRNA；

The sgRNA of the Cas9mRNA and the targeting PD1 gene are mixed with the Chimeric antigen receptor T cell, and It is placed in electroporation and carries out electricity turn, knock out the PD1 gene of the Chimeric antigen receptor T cell.

6. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting EGFR of PD1, feature as claimed in claim 5 It is, the corresponding gene order of sgRNA of the targeting PD1 gene includes the nucleotides sequence as shown in SEQ ID NO:3 Column.

7. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting EGFR of PD1, feature as claimed in claim 5 It is, the mass ratio of the sgRNA of the Cas9mRNA and the targeting PD1 gene is 1:1-1:5.

8. the Chimeric antigen receptor T of the targeting EGFR for the knockout PD1 that the method according to claim 1 to 7 is prepared Cell, which is characterized in that the Chimeric antigen receptor T cell of the targeting EGFR for knocking out PD1 is free of PD1 gene, the knockout The Chimeric antigen receptor T cell of the targeting EGFR of PD1 includes the Chimeric antigen receptor CAR-EGFR of targeting EGFR, the targeting The Chimeric antigen receptor CAR-EGFR of EGFR includes the single-chain antibody of sequentially connected targeting EGFR, born of the same parents from aminoterminal to c-terminus The amino acid sequence of outer hinge area, transmembrane region and intracellular signal area, wherein the single-chain antibody of the targeting EGFR is humanization list Chain antibody, the single-chain antibody of the targeting EGFR include the amino acid sequence as shown in SEQ ID NO:1.

9. knocking out the Chimeric antigen receptor T cell of the targeting EGFR of PD1 as claimed in claim 8, which is characterized in that the target Amino acid sequence to the Chimeric antigen receptor CAR-EGFR of EGFR includes the amino acid sequence as shown in SEQ ID NO:4.

10. one kind is as made from the described in any item preparation methods of claim 1-7 or as claim 8-9 is described in any item Knock out Chimeric antigen receptor T cell the answering in the drug for preparing prevention, diagnosing and treating malignant tumour of the targeting EGFR of PD1 With.