CN109837303A - A kind of Chimeric antigen receptor T cell and its preparation method and application for the targeting CD317 knocking out PD1 - Google Patents

Abstract

The present invention provides a kind of preparation methods of the CD317 Chimeric antigen receptor T cell of targeting for knocking out PD1, it include: that the encoding gene for the Chimeric antigen receptor CAR-CD317 for targeting CD317 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 the targeting CD317 for knocking out PD1 is obtained.The targeting CD317 Chimeric antigen receptor T cell has knocked out PD1 gene, be conducive to T cell in the amplification of patient's body, avoid neoplastic cells escape immunosurveillance, it can efficiently and specifically kill CD317 positive tumor cell, vigor and lethality are enduringly maintained, and not will cause damage to normal cell.

Description

It is a kind of knock out PD1 targeting CD317 Chimeric antigen receptor T cell and its preparation side Method 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 CD317 for knocking out PD1 Cell and its preparation method and application.

Background technique

Malignant tumour (cancer) has become the arch-criminal for threatening human life, and disease incidence is at ascendant trend.CD317 is Lipid Rafts phase Albumen is closed, main expression is on a variety of solid tumor cells such as liver cancer, colon cancer, and the almost seldom expression in ordinary cells. The expression of CD317 is often that cell shows the response of gamma- interferon access, therefore has substantial connection with tumour.

CAR-T (Chimeric antigen receptor T cell) technology is a kind of novel cell therapy, it is that the T by CAR transformation is thin Born of the same parents are fed back to human body, activate self immune system, kill to tumour cell, it is considered to be current most effective malignant tumour One of therapeutic modality.But the application of current CAR-T technology is also limited to blood tumor, to a variety of solid tumors such as liver cancer do not have also into Exhibition.

Death protein (PD1) is that immunologic test point inhibits in (immune checkpoint inhibition) Representative 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, so that neoplastic cells escape be made to exempt from Epidemic disease monitoring.There is not the Chimeric antigen receptor T cell for the targeting CD317 for knocking out PD1 also at present and can avoid neoplastic cells escape and exempts from The research of epidemic disease monitoring.

Summary of the invention

In view of this, the present invention provides the Chimeric antigen receptor T cell of targeting CD317 for knocking out PD1 a kind of, it is described to strike Except the Chimeric antigen receptor T cell of the targeting CD317 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 CD317 positive tumor cell, preferably The vigor and lethality of cell are maintained, and not will cause damage to normal cell.

In a first aspect, the present invention provides a kind of preparations of the Chimeric antigen receptor T cell of targeting CD317 for knocking out PD1 Method, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-CD317 of targeting CD317 is provided, including suitable from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of secondary connection, the encoding gene of single-chain antibody for targeting CD317, extracellular hinge area encoding gene, The encoding gene of transmembrane region, intracellular signal area encoding gene, wherein the encoding gene of the single-chain antibody of the targeting CD317 Nucleotide sequence including encoding the amino acid sequence as shown in SEQ ID NO:1；

(2) encoding gene of the CAR-CD317 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD317 weight Group plasmid；

(3) the pWPXLD-CAR-CD317 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 inosculating antibody for knocking out the targeting CD317 of PD1 Original receptor T cell.

Optionally, the amino acid sequence of the single-chain antibody of the targeting CD317 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 CD317 includes such as SEQ ID NO:5 Shown in nucleotide sequence.

Optionally, the encoding gene of the amino acid sequence of the single-chain antibody of the targeting CD317 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-CD317 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 SEQ 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 CD317 on the single-chain antibody and tumour of the targeting CD317 In conjunction with.

Optionally, the extracellular hinge area include CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 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 the core as shown in SEQ ID NO:10 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-CD317.

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, CD27 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:14 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 the core as shown in SEQ ID NO:16 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-CD317 of the targeting CD317 includes such as SEQ ID NO: Nucleotide sequence shown in 2.Nucleotide sequence shown in SEQ ID NO:2 contains the encoding gene of the signal peptide, described Signal peptide is cut in protein translation maturation by signal peptidase.

The encoding gene of the CAR-CD317 is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and After the extension factor 1 α (EF1 α) of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-CD317 is inserted When entering to pWPXLD carrier, initiation codon (such as ATG) and pWPXLD can be added in 5 ' ends of the encoding gene of the CAR-CD317 BamH1 restriction enzyme site is connected in carrier, and EcoR1 digestion position in terminator codon (such as TAA) and pWPXLD carrier can be added in 3 ' ends Point is connected.

Optionally, the amino acid sequence of the targeting Chimeric antigen receptor CAR-CD317 includes as shown in SEQ ID NO:4 Amino acid sequence.

Optionally, the packaging pWPXLD-CAR-CD317 recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-CD317 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, 293FT cell, 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 Cas9 mRNA；

The corresponding gene order of sgRNA of targeting PD1 gene is provided；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 Cas9 mRNA 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, complete the knockout of the PD1 gene of the Chimeric antigen receptor T cell.

Optionally, the corresponding gene order of sgRNA of the targeting PD1 gene includes the core as shown in SEQ ID NO:3 Nucleotide sequence.

Optionally, the mass ratio of the sgRNA of the Cas9 mRNA and the PD1 gene is 1:1-1:5.

Further alternative, the mass ratio of the sgRNA of the Cas9 mRNA and the PD1 gene is 1:3.

In the present invention, in step (5), the Chimeric antigen receptor T cell of the targeting CD317 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 also has the function of certain targeting CD317, but it is possible that the phenomenon that neoplastic cells escape immunosurveillance.It knocks out The Chimeric antigen receptor T cell of the targeting CD317 for the knockout PD1 that PD1 gene obtains is stronger to the targeting of tumour cell, and keeps away The case where having exempted from neoplastic cells escape immunosurveillance.

The preparation method of the Chimeric antigen receptor T cell of the targeting CD317 for the knockout PD1 that first aspect present invention provides, Chimeric antigen receptor T cell is obtained by the Chimeric antigen receptor of preparation targeting CD317, and knocks out the PD1 gene system in T cell The Chimeric antigen receptor T cell of the targeting CD317 of PD1 must be knocked out, which is conducive to T cell in the expansion of patient's body Increase, avoids neoplastic cells escape immunosurveillance, make it have the performance of efficient and specific killing tumor cell, it is especially suitable In CD317 positive tumor cell.

In other embodiments of the invention, it can also prepare the targeting CD317's for knocking out PD1 using following methods Chimeric antigen receptor T cell, comprising the following steps:

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

(2) encoding gene of the Chimeric antigen receptor CAR-CD317 of targeting CD317 is provided, including suitable from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of secondary connection, the encoding gene of single-chain antibody for targeting CD317, extracellular hinge area encoding gene, The encoding gene of transmembrane region, intracellular signal area encoding gene, wherein the encoding gene of the single-chain antibody of the targeting CD317 Nucleotide sequence including encoding the amino acid sequence as shown in SEQ ID NO:1；

(3) encoding gene of the CAR-CD317 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD317 weight Group plasmid；

(4) the pWPXLD-CAR-CD317 recombinant plasmid is packed, recombinant slow virus is obtained；

(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 CD317.

In the present invention, in the preparation process of the Chimeric antigen receptor T cell of the targeting CD317 for knocking out PD1, knocked out Journey carries out when can be directed to the CD3 positive t lymphocytes, can also be in the Chimeric antigen receptor T cell for obtaining targeting CD317 After carry out.Knockout sequence is not construed as limiting in the present invention, obtains the chimeric of the targeting CD317 for knocking out PD1 as long as can achieve The purpose of antigen receptor T cell.

Second aspect, the present invention provides the knockout PD1's being prepared using preparation method as described in relation to the first aspect The Chimeric antigen receptor T cell of CD317 is targeted, the Chimeric antigen receptor T cell of the targeting CD317 for knocking out PD1 is free of PD1 Gene, the Chimeric antigen receptor T cell of the targeting CD317 for knocking out PD1 include the Chimeric antigen receptor CAR- for targeting CD317 The Chimeric antigen receptor CAR-CD317 of CD317, the targeting CD317 include the sequentially connected targeting from aminoterminal to c-terminus The single-chain antibody of CD317, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein the targeting CD317 Single-chain antibody include the amino acid sequence as shown in SEQ ID NO:1.

Above-mentioned " being sequentially connected with from aminoterminal to c-terminus " specifically: the amino acid of the single-chain antibody of the targeting CD317 The c-terminus of sequence is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, the amino acid sequence of the extracellular hinge area The c-terminus of column is 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 It is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.

Wherein, the targeting single-chain antibody of CD317, 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 targeting Chimeric antigen receptor CAR-CD317 includes as shown in SEQ ID NO:4 Amino acid sequence.

Optionally, the encoding gene of the amino acid sequence of the targeting Chimeric antigen receptor CAR-CD317 includes such as SEQ Nucleotide sequence shown in ID NO:6.

Optionally, the encoding gene of the amino acid sequence of the CAR-CD317 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 Chimeric antigen receptor CAR-CD317 includes the nucleosides as shown in SEQ ID NO:2 Acid sequence.Nucleotide sequence shown in SEQ ID NO:2 contains the encoding gene of the signal peptide, and the signal peptide can instruct Chimeric antigen receptor CAR-CD317 expression to cell surface, but signal peptide in protein translation maturation by signal peptide Digestion is cut.

The Chimeric antigen receptor T cell of the targeting CD317 for the knockout PD1 that second aspect of the present invention provides, Ke Yizhuan The targeting CD317 of one property especially expresses the solid tumor cell of CD317.After CAR-CD317 is in conjunction with CD317, the T is thin The intracellular signal area of born of the same parents is activated, amplification of the promotion T cell in patient's body, efficient and specific killing tumor cell, and Normal cell is hardly caused to damage；In addition, the single-chain antibody based on targeting CD317 is Humanized single chain antibody, this makes It obtains the T cell and avoids the immune response for causing human organism, enduringly maintain the vigor and lethality of cell；The T cell knocks out PD1 gene, can make it have the property of efficient and specific killing tumor cell to avoid neoplastic cells escape immunosurveillance Energy.

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 CD317 of knockout PD1 described in face is pernicious swollen in preparation prevention, diagnosing and treating Application in the drug of tumor.Specifically, suitable for expressing prevention, the diagnosing and treating of the solid tumor of CD317, such as liver cancer, knot Intestinal cancer, lung cancer, cervical carcinoma etc..

The application specifically: provide a kind of kit, the kit includes preparation side as described in relation to the first aspect The Chimeric antigen receptor T cell of the targeting CD317 of knockout PD1 that method is prepared or as described in second aspect a kind of.

Beneficial effects of the present invention:

The Chimeric antigen receptor T cell of the targeting CD317 provided by the invention for knocking out PD1 can be with the targeting of specificity CD317 promotes T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific, while CD317 swollen Wide expression in oncocyte, and expressed in ordinary cells it is very faint, therefore knock out PD1 targeting CD317 chimeric antigen by Body T cell can specificity combination tumour cell, to tumour cell generate fragmentation effect, not will cause damage to normal cell Wound, while the PD1 gene of T cell has been knocked out, be conducive to T cell in the amplification of patient's body, avoid neoplastic cells escape immune Monitoring, 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-CD317 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 CD317 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 CD317 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 external swollen of the Chimeric antigen receptor T cell of the targeting CD317 provided in an embodiment of the present invention for knocking out PD1 Cytotoxic effect effect picture.

Fig. 4 is that the Chimeric antigen receptor T cell of the targeting CD317 provided in an embodiment of the present invention for knocking out PD1 treats tumour 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 CD317 knocking out PD1, comprising the following steps:

(1) gene order of Chimeric antigen receptor CAR-CD317 is prepared

Prepare respectively signal peptide, target the single-chain antibody of CD317, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and The encoding gene of CD3 ζ signaling zone, the encoding gene of the signal peptide is as shown in SEQ ID NO:8, the list of the targeting CD317 The encoding gene of chain 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.

By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of CD317 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-CD317, the encoding gene of the CAR-CD317 is as shown in SEQ ID NO:2.

(2) pWPXLd-CAR-CD317 recombinant plasmid is constructed

The encoding gene of CAR-CD317 is inserted between BamH1 the and EcoR1 restriction enzyme site of pWPXLD carrier, and After the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-CD317 is inserted into pWPXLD carrier When, 5 ' end additions initiation codon (such as ATG) and the BamH1 digestion in pWPXLD carrier of the encoding gene of the CAR-CD317 Site is connected, and 3 ' ends are also connected added with terminator codon (such as TAA) with EcoR1 restriction enzyme site in pWPXLD carrier.Then turn Enter competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.It is examined by PCR product gel electrophoresis It surveys and sequencing identification meets target fragment size and sequence, successfully construct pWPXLd-CAR-CD317 recombination matter as shown in Figure 1 Grain.

(3) recombinant slow virus constructs

PWPXLd-CAR-CD317 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 mMESSAGET7 kit carries out external Transcription obtains Cas9 mRNA；

The corresponding gene order of sgRNA of targeting PD1 gene is provided, sequence is as shown in SEQ ID NO:3；By the target It is inserted into pcDNA3.1 carrier to the corresponding gene order of sgRNA of PD1 gene, obtains pcDNA3.1-PD1-sgRNA recombination Plasmid is transcribed in vitro to obtain sgRNA as template using the pcDNA3.1-PD1-sgRNA recombinant plasmid；

By the sgRNA sequence of the Cas9 mRNA and the targeting PD1 gene with above-mentioned to obtain Chimeric antigen receptor T thin Born of the same parents mix, and are placed in electroporation and carry out electricity turn, knock out the PD1 gene of T cell；T cell after electricity is turned is cultivated, Obtain knocking out the Chimeric antigen receptor T cell of the targeting CD317 of PD1.

The PD1 that the Chimeric antigen receptor T cell of the targeting CD317 of above-mentioned knockout PD1 is measured using flow cytometer is expressed Amount calculates 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 CD317 of PD1 Up to 75%.

In order to assess the Chimeric antigen receptor T for targeting CD317 for knocking out PD1 of above method preparation described in the invention Cell effect carries out following effect example.

Effect example one: the Chimeric antigen receptor T cell of the targeting CD317 of knockout PD1 prepared by the assessment present invention Positive rate

Will by the method for the present invention preparation knock out PD1 targeting CD317 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 CD317 Chimeric antigen receptor T cell.(a) can be obtained compared with (b) in Fig. 2, knockout prepared by the present invention The positive rate of the Chimeric antigen receptor T cell of the targeting CD317 of PD1 is 37.1%.

Effect example two: assessment knocks out the tumor cell in vitro of the Chimeric antigen receptor T cell of the targeting CD317 of PD1 Kill situation

It (will be abbreviated as by the Chimeric antigen receptor T cell of the targeting CD317 of knockout PD1 made from the method for the present invention CAR-T-CD317 (PD1KO) group) it is carried out with the Vitro Tumor fragmentation effect of the T lymphocyte (negative control group) without preparation Compare, it is specific: effector cell (CAR-T-CD317 (PD1KO) cell) and target cell (HeLa cell) being pressed into quantity in vitro Than for 1:10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, 15-18 after incubation is small When, cell is collected, streaming dyeing is carried out, detects cell killing situation, as a result as shown in Figure 3.As can be seen from Figure 3, addition CAR-T-CD317 (PD1KO) cell is more, they are stronger to the lethality of tumour cell.By method system of the present invention The tumor-killing power of standby CAR-T-CD317 (PD1KO) cell is in 20% or more, even up to 60%, significantly larger than feminine gender The Chimeric antigen receptor T cell of control group, the targeting CD317 for the knockout PD1 that this explanation is prepared through the method for the present invention has strong Tumor-killing ability.

Effect example three: assessment knocks out the mouse interior tumor of the Chimeric antigen receptor T cell of the targeting CD317 of PD1 Cell killing situation

By the Chimeric antigen receptor T cell (CAR-T- of the targeting CD317 of the knockout PD1 by the method for the present invention preparation CD317 (PD1KO) group), the T lymphocyte (negative control group) without preparation and physiological saline (blank control group), small In mouse tumor model, every mouse tail vein injection 1 × 10 is given⁶A HeLa cell (n=9), draws the survivorship curve of mouse, knot Fruit is as shown in Figure 4.From fig. 4, it can be seen that training mouse by CAR-T-CD317 (PD1KO) cell prepared by this method Survival rate is also higher than 50% when supporting 80 days, 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 CD317 of the knockout PD1 by this method preparation preferably can protect mouse to exempt from It is dead caused by because of 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 and its preparation method and application for the targeting CD317 for knocking out PD1

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 243

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Leu Ile Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu His Ser Ala

50 55 60

Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Ala Arg Asp Tyr Arg Ser Met Asp Tyr Trp Gly Ala Gly Thr

100 105 110

Ile Val Thr Val Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Thr Pro Ser Leu Ala

130 135 140

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

145 150 155 160

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Phe Gln Gln Lys Pro

165 170 175

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

180 185 190

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

195 200 205

Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

225 230 235 240

Glu Ile Lys

<210> 2

<211> 1458

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gaggtgaagc tggtggagtc tggaggaggc ttggtacagc ctgggggttc tctgagactc 120

tcctgtgcaa cttctgggtt cacgttctct gattactaca tggcctgggt ccgccagcct 180

ccaggaaagg cacttgagtg gttgggttta attagaaaca aaggtaatgg ttacacaaca 240

gagcacagtg catctgtgag gggtcggttc accatctcca gagataattc ccaaagcatc 300

ctctatcttc aaatgaacac cctgagacct gaggacagtg ccacttatta ctgtgcaaga 360

gattaccggt ctatggacta ctggggcgca ggaactatag tcacagtcaa tggtggcggt 420

ggctcgggcg gtggtgggtc gggtggcggc ggatctgaca ttgtgctcac acaatctaca 480

ccttctttgg ctgtgtctct agggcagagg gccaccatat cctgcagagc cagtgaaagt 540

gttgatagtt atggcaacag ttttatgcac tggttccagc agaaaccagg acagccaccc 600

aaactcctca tctatcgtgc atccaaccta gaatctggga tccctgccag gttcagtggc 660

agtgggtcta ggacagactt caccctcacc attaatcctg tggaggctga tgatgttgca 720

acctattact gtcagcaaag taatgaggat ccgtacacgt tcggaggggg gaccaagctg 780

gaaataaaaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgc 1458

<210> 3

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cacgaagctc tccgatgtgt tgg 23

<210> 4

<211> 466

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Leu Ile Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu His Ser Ala

50 55 60

Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Ala Arg Asp Tyr Arg Ser Met Asp Tyr Trp Gly Ala Gly Thr

100 105 110

Ile Val Thr Val Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Thr Pro Ser Leu Ala

130 135 140

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

145 150 155 160

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Phe Gln Gln Lys Pro

165 170 175

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

180 185 190

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

195 200 205

Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

355 360 365

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

370 375 380

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

385 390 395 400

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

405 410 415

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

420 425 430

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

435 440 445

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

450 455 460

Pro Arg

465

<210> 5

<211> 729

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gaggtgaagc tggtggagtc tggaggaggc ttggtacagc ctgggggttc tctgagactc 60

tcctgtgcaa cttctgggtt cacgttctct gattactaca tggcctgggt ccgccagcct 120

ccaggaaagg cacttgagtg gttgggttta attagaaaca aaggtaatgg ttacacaaca 180

gagcacagtg catctgtgag gggtcggttc accatctcca gagataattc ccaaagcatc 240

ctctatcttc aaatgaacac cctgagacct gaggacagtg ccacttatta ctgtgcaaga 300

gattaccggt ctatggacta ctggggcgca ggaactatag tcacagtcaa tggtggcggt 360

ggctcgggcg gtggtgggtc gggtggcggc ggatctgaca ttgtgctcac acaatctaca 420

ccttctttgg ctgtgtctct agggcagagg gccaccatat cctgcagagc cagtgaaagt 480

gttgatagtt atggcaacag ttttatgcac tggttccagc agaaaccagg acagccaccc 540

aaactcctca tctatcgtgc atccaaccta gaatctggga tccctgccag gttcagtggc 600

agtgggtcta ggacagactt caccctcacc attaatcctg tggaggctga tgatgttgca 660

acctattact gtcagcaaag taatgaggat ccgtacacgt tcggaggggg gaccaagctg 720

gaaataaaa 729

<210> 6

<211> 1398

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gaggtgaagc tggtggagtc tggaggaggc ttggtacagc ctgggggttc tctgagactc 60

tcctgtgcaa cttctgggtt cacgttctct gattactaca tggcctgggt ccgccagcct 120

ccaggaaagg cacttgagtg gttgggttta attagaaaca aaggtaatgg ttacacaaca 180

gagcacagtg catctgtgag gggtcggttc accatctcca gagataattc ccaaagcatc 240

ctctatcttc aaatgaacac cctgagacct gaggacagtg ccacttatta ctgtgcaaga 300

gattaccggt ctatggacta ctggggcgca ggaactatag tcacagtcaa tggtggcggt 360

ggctcgggcg gtggtgggtc gggtggcggc ggatctgaca ttgtgctcac acaatctaca 420

ccttctttgg ctgtgtctct agggcagagg gccaccatat cctgcagagc cagtgaaagt 480

gttgatagtt atggcaacag ttttatgcac tggttccagc agaaaccagg acagccaccc 540

aaactcctca tctatcgtgc atccaaccta gaatctggga tccctgccag gttcagtggc 600

agtgggtcta ggacagactt caccctcacc attaatcctg tggaggctga tgatgttgca 660

acctattact gtcagcaaag taatgaggat ccgtacacgt tcggaggggg gaccaagctg 720

gaaataaaaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 780

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 960

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1020

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1080

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1200

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1320

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1380

caggccctgc cccctcgc 1398

<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

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 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

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatc 138

<210> 11

<211> 23

<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

20

<210> 12

<211> 69

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 60

ctttactgc 69

<210> 13

<211> 42

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 14

<211> 126

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<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

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

Claims (10)

1. a kind of preparation method of the Chimeric antigen receptor T cell for the targeting CD317 for knocking out PD1 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-CD317 of targeting CD317 is provided, including is sequentially connected from 5 ' ends to 3 ' ends The encoding gene of the signal peptide connect, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, cross-film for targeting CD317 The encoding gene in area, intracellular signal area encoding gene, wherein the encoding gene of the single-chain antibody of the targeting CD317 includes Encode the nucleotide sequence of the amino acid sequence as shown in SEQ ID NO:1；

(2) encoding gene of the CAR-CD317 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD317 recombination matter Grain；

(3) the pWPXLD-CAR-CD317 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, obtain knock out the chimeric antigen of the targeting CD317 of PD1 by Body T cell.

2. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting CD317 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 method of the Chimeric antigen receptor T cell of the targeting CD317 of PD1 is knocked out as claimed in claim 1 or 2, It is characterized in that, the encoding gene of the Chimeric antigen receptor CAR-CD317 of the targeting CD317 includes as shown in SEQ ID NO:2 Nucleotide sequence.

4. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting CD317 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；

The corresponding gene order of sgRNA of targeting PD1 gene is provided；By the corresponding gene sequence of sgRNA of the targeting PD1 gene Column are inserted into pcDNA3.1 carrier, pcDNA3.1-PD1-sgRNA recombinant plasmid are 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, complete the knockout of the PD1 gene of the Chimeric antigen receptor T cell.

5. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting CD317 of PD1, feature as claimed in claim 4 It is, the corresponding gene order of sgRNA of the targeting PD1 gene includes the nucleotide sequence as shown in SEQ ID NO:3.

6. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting CD317 of PD1, feature as claimed in claim 4 It is, the mass ratio of the sgRNA of the Cas9mRNA and the PD1 gene is 1:1-1:5.

7. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting CD317 of PD1, feature as described in claim 1 It is, the packaging pWPXLD-CAR-CD317 recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-CD317 recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, institute is obtained State recombinant slow virus.

8. the Chimeric antigen receptor of the targeting CD317 for the knockout PD1 that the method according to claim 1 to 7 is prepared T cell, which is characterized in that the Chimeric antigen receptor T cell of the targeting CD317 for knocking out PD1 is free of PD1 gene, described to strike It is described except the Chimeric antigen receptor T cell of the targeting CD317 of PD1 includes the Chimeric antigen receptor CAR-CD317 for targeting CD317 The Chimeric antigen receptor CAR-CD317 of targeting CD317 includes the single-stranded of the sequentially connected targeting CD317 from aminoterminal to c-terminus Antibody, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein it is described targeting CD317 single-chain antibody packet Include the amino acid sequence as shown in SEQ ID NO:1.

9. knocking out the Chimeric antigen receptor T cell of the targeting CD317 of PD1 as claimed in claim 8, which is characterized in that described The amino acid sequence for targeting the Chimeric antigen receptor CAR-CD317 of CD317 includes the amino acid sequence as shown in SEQ ID NO:4 Column.

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 The Chimeric antigen receptor T cell for knocking out the targeting CD317 of PD1 is prevented, in the drug of diagnosing and treating malignant tumour in preparation Using.