CN109837302A - A kind of Chimeric antigen receptor T cell and its preparation method and application for the targeting CD20 knocking out PD1 - Google Patents

Abstract

The present invention provides a kind of preparation methods of the Chimeric antigen receptor T cell of targeting CD20 for knocking out PD1, it include: that the encoding gene for the Chimeric antigen receptor CAR-CD20 for targeting CD20 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 CD20 for knocking out PD1 is obtained.The T cell has knocked out PD1 gene, is conducive to T cell in the amplification of patient's body, avoids neoplastic cells escape immunosurveillance, has the performance for efficiently and specifically killing CD20 positive tumor cell, enduringly maintains the vigor and lethality of cell.

Description

A kind of Chimeric antigen receptor T cell and preparation method thereof for the targeting CD20 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 CD20 for knocking out PD1 is thin Born of the same parents and its preparation method and application.

Background technique

CAR-T (Chimeric antigen receptor T cell) technology is a kind of novel cell therapy, it is will be by the T of CA R transformation Cell is fed back to human body, activates self immune system, kills to tumour cell, in acute leukemia and non-Hodgkin's lymph There is significant curative effect in the treatment of tumor, it is considered to be one of the therapeutic modality of current most effective malignant tumour.

CAR-T technology is in CD19 positive blood tumor (such as B cell lymphoma, acute and chronic bone-marrow-derived lymphocyte leukaemia) at present Achieve the effect to attract people's attention.But since similar tumour often expresses different immunophenotype, for example, work as tumour Cell occur CD19 feminine gender escape, that is, there is the tumour cell for not expressing CD19, then CD19 target spot fail, and CART-CD19 without Method plays antitumous effect again.

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.Therefore, it is necessary to provide the CAR-T cell of other wide expression target spots of energy targeted malignant tumour and can avoid tumour Cells escape immunosurveillance.

Summary of the invention

In view of this, the present invention provides the Chimeric antigen receptor T cell of targeting CD20 for knocking out PD1 a kind of, the target PD1 gene has been knocked out to CD20 Chimeric antigen receptor T cell, has been conducive to T cell in the amplification of patient's body, avoids tumour cell Immunosurveillance is escaped, the performance with efficient and specific killing tumor cell enduringly maintains vigor and the killing of cell Power.

In a first aspect, the present invention provides a kind of preparation sides of the Chimeric antigen receptor T cell of targeting CD20 for knocking out PD1 Method, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-CD20 of targeting CD20 is provided, including sequentially from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of connection, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, cross-film for targeting CD20 The encoding gene in area, intracellular signal area encoding gene, wherein the encoding gene of the single-chain antibody of the targeting CD20 includes compiling The nucleotide sequence of code amino acid sequence as shown in SEQ ID NO:1；

(2) encoding gene of the CAR-CD20 is inserted into pWPXLD carrier, obtains pWPX LD-CAR-CD20 weight Group plasmid；

(3) the pWPXLD-CAR-CD20 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 CD20 of PD1 Recipient T cells.

Optionally, the amino acid sequence of the single-chain antibody of the targeting CD20 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 CD20 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 CD20 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-CD20 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 CD20 knot on the single-chain antibody and tumour of the targeting CD20 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 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-CD20.

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: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-CD20 of the targeting CD20 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-CD20 is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and position After the extension factor 1 α (EF1 α) of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-CD20 is inserted into When to pWPXLD carrier, initiation codon (such as ATG) can be added for 5 ' ends of the encoding gene of the CAR-CD20 and pWPXLD is carried BamH1 restriction enzyme site is connected in body, and EcoR1 restriction enzyme site in terminator codon (such as TAA) and pWPXLD carrier can be added in 3 ' ends It is connected.

Optionally, the amino acid sequence of the targeting Chimeric antigen receptor CAR-CD20 includes as shown in SEQ ID N O:4 Amino acid sequence.

Optionally, the packaging pWPXLD-CAR-CD20 recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-CD20 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 Cas9mRNA；

The sgRNA sequence of targeting PD1 gene is provided；By the corresponding gene order insertion of the sgRNA of the targeting PD1 gene Into pcDNA3.1 carrier, pcDNA3.1-PD1-sgRNA recombinant plasmid is obtained, with pc DNA3.1-PD1-sgRNA recombination Plasmid is that template is transcribed in vitro to obtain sgRNA；

The sgRNA sequence and the Chimeric antigen receptor T cell of the Cas9mRNA and the targeting PD1 gene are carried out Mixing is placed in electroporation and carries out electricity turn, completes the knockout of PD1 gene in 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 Cas9mRNA and the PD1 gene is 1:1-1:5.

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

In the present invention, in step (5), the Chimeric antigen receptor T cell of the targeting CD20 of the knockout PD1 of acquisition, is in step Suddenly the PD1 gene of T cell has been knocked out on the basis of the Chimeric antigen receptor T cell in (4).Chimeric antigen in step (4) by Body T cell also has the function of certain targeting CD20, but it is possible that the phenomenon that neoplastic cells escape immunosurveillance.It knocks out The Chimeric antigen receptor T cell of the targeting CD20 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 CD20 for the knockout PD1 that first aspect present invention provides, leads to The Chimeric antigen receptor for crossing preparation targeting CD20 obtains Chimeric antigen receptor T cell, and knocks out the PD1 gene in T cell and be made The Chimeric antigen receptor T cell of the targeting CD20 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, make it have the performance of efficient and specific killing tumor cell, is suitable for CD20 sun Property tumour cell killing, be particularly suitable for killing and do not express the tumour cell of CD19, tumour cell CD19 feminine gender is avoided to escape When CART-CD19 can not play antitumous effect again.

In other embodiments of the invention, it can also prepare the targeting C D20'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-CD20 of targeting CD20 is provided, including sequentially from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of connection, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, cross-film for targeting CD20 The encoding gene in area, intracellular signal area encoding gene, wherein the encoding gene of the single-chain antibody of the targeting CD20 includes compiling The nucleotide sequence of code amino acid sequence as shown in SEQ ID NO:1；

(3) encoding gene of the CAR-CD20 is inserted into pWPXLD carrier, obtains pWPX LD-CAR-CD20 weight Group plasmid；

(4) the pWPXLD-CAR-CD20 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 CD20.

In the present invention, in the preparation process of the Chimeric antigen receptor T cell of the targeting CD20 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 CD20 After carry out.Knockout sequence is not construed as limiting in the present invention, as long as can achieve the inosculating antibody for obtaining the targeting CD20 for knocking out PD1 The purpose of original receptor T 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 CD20 is targeted, the Chimeric antigen receptor T cell of the targeting CD20 for knocking out PD1 is free of PD1 base Cause, the Chimeric antigen receptor T cell of the targeting CD20 for knocking out PD1 include the Chimeric antigen receptor CAR- for targeting CD20 The Chimeric antigen receptor CAR-CD20 of CD20, the targeting CD20 include the sequentially connected targeting CD20 from aminoterminal to c-terminus Single-chain antibody, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein it is described targeting CD20 it is single-stranded Antibody includes 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 sequence of the single-chain antibody of the targeting CD20 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 targeting single-chain antibody of CD20, 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-CD20 includes as shown in SEQ ID N O:4 Amino acid sequence.

Optionally, the encoding gene of the amino acid sequence of the targeting Chimeric antigen receptor CAR-CD20 includes such as SEQ ID Nucleotide sequence shown in NO:6.

Optionally, the encoding gene of the amino acid sequence of the CAR-CD20 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-CD20 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-CD20 expression to cell surface, but signal peptide in protein translation maturation by signal peptidase Cutting.

The Chimeric antigen receptor T cell of the targeting CD20 for the knockout PD1 that second aspect of the present invention provides, can be single-minded Property targeting CD20, after CAR-CD20 receptor is in conjunction with CD20, the intracellular signal area of the T cell is activated, promote T cell In the amplification of patient's body, the malignant tumour of efficient and specific killing tumor cell, especially killing expression CD20 (including B cell malignant tumour etc.), especially suitable for killing the tumour cell for not expressing CD19, tumour cell CD19 feminine gender is avoided to escape When CART-CD19 can not play antitumous effect again.In addition, the single-chain antibody based on targeting CD20 is Humanized single chain antibody, This makes the T cell be avoided the immune response for causing human organism, enduringly maintains the vigor and lethality of cell；The T cell PD1 gene has been knocked out, efficient and specific killing tumor cell can have been made it have to avoid neoplastic cells escape immunosurveillance Performance.

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 The Chimeric antigen receptor T cell of the targeting CD20 of knockout PD1 described in face a kind of is in preparation prevention, diagnosing and treating malignant tumour Drug in application.It is particularly suitable for the malignant tumour of expression CD20.Especially suitable for B cell malignant tumour, (such as B cell is drenched Bar tumor, bone-marrow-derived lymphocyte leukaemia etc.) prevention, diagnosing and treating, it is especially relevant to infantilism lymphocyte pernicious swollen Tumor.

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 CD20 of knockout PD1 that method is prepared or as described in second aspect a kind of.

Advantages of the present invention will be illustrated partially in the following description, and a part is apparent according to specification , or can implementation through the embodiment of the present invention and know.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-CD20 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 CD20 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 C D20 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 CD20 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 CD20 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 CD20 knocking out PD1, comprising the following steps:

(1) gene order of Chimeric antigen receptor CAR-CD20 is prepared

Prepare respectively signal peptide, target the single-chain antibody of CD20, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and The encoding gene of CD3 ζ signaling zone, for the encoding gene of the signal peptide as shown in SEQ ID NO:8, the targeting CD20's is single-stranded The encoding gene of antibody is as shown in SEQ ID NO:5, and the encoding gene of the CD8 α hinge area is as shown in SEQ ID NO:10, institute The encoding gene of CD8 transmembrane region is stated as shown in SEQ ID NO:12, the encoding gene of the 4-1BB signaling zone such as SEQ ID NO: Shown in 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 CD20 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-CD20, the encoding gene of the CAR-CD20 is as shown in SEQ I D NO:2.

(2) pWPXLd-CAR-CD20 recombinant plasmid is constructed

The encoding gene of CAR-CD20 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.When the encoding gene of the CAR-CD20 is inserted into pWPXLD carrier, BamH1 restriction enzyme site in 5 ' end additions initiation codon (such as ATG) of the encoding gene of the CAR-CD20 and pWPXLD carrier It is connected, 3 ' ends are also connected added with terminator codon (such as TAA) with EcoR1 restriction enzyme site in pWPXLD carrier.Then it is transferred to big Enterobacteria competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and Sequencing identification meets target fragment size and sequence, successfully constructs p WPXLd-CAR-CD20 recombinant plasmid as shown in Figure 1.

(3) recombinant slow virus constructs

PWPXLd-CAR-CD20 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 Cas9mRNA；

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 pc DNA3.1-PD1-sgRNA weight Group plasmid, is transcribed in vitro to obtain sgRNA as template using the pcDNA3.1-PD1-sgRNA recombinant plasmid；

The sgRNA sequence of the Cas9mRNA and the targeting PD1 gene are obtained into Chimeric antigen receptor T cell with above-mentioned It is mixed, is placed in electroporation and carries out electricity turn, knock out the PD1 gene of T cell；T cell after electricity is turned is cultivated, and is obtained To the Chimeric antigen receptor T cell for the targeting CD20 for knocking out PD1.

The PD1 that the Chimeric antigen receptor T cell of the targeting CD20 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 P D1 gene in the Chimeric antigen receptor T cell of the targeting CD20 of PD1 Up to 72%.

In order to assess the Chimeric antigen receptor T for targeting CD20 for knocking out PD1 of above method preparation described in the invention The effect of cell carries out following effect example.

Effect example one: the Chimeric antigen receptor T cell of the targeting CD20 of knockout PD1 prepared by the assessment present invention Positive rate

Will by the method for the present invention preparation knock out PD1 targeting CD20 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 CD20 Chimeric antigen receptor T cell.(a) can be obtained compared with (b) in Fig. 2, knockout PD1 prepared by the present invention Targeting CD20 Chimeric antigen receptor T cell positive rate be 34.7%.

Effect example two: the tumor cell in vitro that assessment knocks out the Chimeric antigen receptor T cell of the targeting CD20 of PD1 kills Condition of the injury condition

(CAR- will be abbreviated as by the Chimeric antigen receptor T cell of the targeting CD20 of knockout PD1 made from the method for the present invention T-CD20 (PD1KO) group), do not knock out PD1 Chimeric antigen receptor T cell (being abbreviated as CAR-T-CD20) and the T without preparation The Vitro Tumor fragmentation effect of lymphocyte (negative control group) is compared, specific: in vitro (knocking out effector cell The Chimeric antigen receptor T cell or T lymphocyte without preparation of the targeting CD20 of PD1) with target cell (Nalm-6 cell, it is anxious Property people B _ Lymphoid Leukemic Cells) in quantity ratio be 1:10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Lower progress It co-cultures, after incubation 15-18 hours, collects cell, carry out streaming dyeing, detect cell killing situation, as a result such as Fig. 3 It is shown.

As can be seen from Figure 3, CAR-T-CD20 (PD1KO) cell of addition is more, their lethality to tumour cell It is stronger.The tumor-killing power of CAR-T-CD20 (PD1KO) cell of the targeting CD20 by method of the present invention preparation is equal 20% or more, even up to 80%, significantly larger than negative control group, in addition, being more than CAR-T-CD20 after 1:1 in effect target ratio (PD1KO) group starts to be apparently higher than the CAR-T-CD20 group for not knocking out PD1 to the killing ability of tumour.These results suggest that warp The Chimeric antigen receptor T cell for the targeting CD20 that having knocked out for the method for the present invention preparation is after PD1 gene, knocks out PD1 has height The tumor-killing ability of effect and specificity can avoid neoplastic cells escape immunosurveillance.

Effect example three: the mouse interior tumor that assessment knocks out the Chimeric antigen receptor T cell of the targeting CD20 of PD1 is thin Born of the same parents kill situation

The Chimeric antigen receptor T cell of the targeting CD20 of knockout PD1 by the method for the present invention preparation (is abbreviated as CAR- T-CD20 (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 Nalm-6 cell (n=9), draws the survivorship curve of mouse, As a result as shown in Figure 4.From fig. 4, it can be seen that the targeting CD20's of the knockout PD1 of the knockout PD1 by this method preparation is chimeric Antigen receptor T cell stablize mouse survival rate after culture 40 days can 65%, and can maintain 70 days or more, much super Cross negative control group and blank control group.Fig. 4's the result shows that, by this method preparation knockout PD1 targeting CD20 it is embedding Conjunction antigen receptor T cell is dead caused by capable of preferably protecting mice against 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 CD20 for knocking out PD1

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 191

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

1 5 10 15

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

20 25 30

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

35 40 45

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

50 55 60

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

65 70 75 80

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

85 90 95

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

100 105 110

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

115 120 125

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

130 135 140

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

145 150 155 160

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

165 170 175

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

180 185 190

<210> 2

<211> 1302

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 120

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 180

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 240

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 300

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 360

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 420

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 480

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 540

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 600

acgcagaaga gcctctccct gtctccgggt aaaaccacga cgccagcgcc gcgaccacca 660

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 720

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 780

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 840

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 900

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 960

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 1020

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1080

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1140

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1200

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1260

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 1302

<210> 3

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cacgaagctc tccgatgtgt tgg 23

<210> 4

<211> 414

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

1 5 10 15

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

20 25 30

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

35 40 45

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

50 55 60

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

65 70 75 80

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

85 90 95

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

100 105 110

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

115 120 125

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

130 135 140

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

145 150 155 160

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

165 170 175

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Thr

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

260 265 270

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

275 280 285

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

290 295 300

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn

305 310 315 320

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410

<210> 5

<211> 573

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 60

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 120

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 180

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 240

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 300

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 360

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 420

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 480

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 540

acgcagaaga gcctctccct gtctccgggt aaa 573

<210> 6

<211> 1242

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 60

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 120

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 180

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 240

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 300

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 360

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 420

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 480

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 540

acgcagaaga gcctctccct gtctccgggt aaaaccacga cgccagcgcc gcgaccacca 600

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 660

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 720

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 780

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 840

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 900

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 960

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1020

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1080

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1140

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1200

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 1242

<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 CD20 for knocking out PD1 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-CD20 of targeting CD20 is provided, including is sequentially connected with from 5 ' ends to 3 ' ends Signal peptide encoding gene, target the encoding gene of single-chain antibody of CD20, the encoding gene of extracellular hinge area, transmembrane region Encoding gene, intracellular signal area encoding gene, wherein it is described targeting CD20 single-chain antibody encoding gene include coding such as The nucleotide sequence of amino acid sequence shown in SEQ ID NO:1；

(2) encoding gene of the CAR-CD20 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD20 recombination matter Grain；

(3) the pWPXLD-CAR-CD20 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 CD20 of PD1 T cell.

2. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting CD20 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 CD20 of PD1 is knocked out as claimed in claim 1 or 2, it is special Sign is that the encoding gene of the Chimeric antigen receptor CAR-CD20 of the targeting CD20 includes the core 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 CD20 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 CD20 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 CD20 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 CD20 of PD1, feature as described in claim 1 It is, the packaging pWPXLD-CAR-CD20 recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-CD20 recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, institute is obtained State recombinant slow virus.

8. the Chimeric antigen receptor T of the targeting CD20 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 CD20 for knocking out PD1 is free of PD1 gene, the knockout The Chimeric antigen receptor T cell of the targeting CD20 of PD1 includes the Chimeric antigen receptor CAR-CD20 for targeting CD20, the targeting The Chimeric antigen receptor CAR-CD20 of CD20 includes the single-chain antibody of sequentially connected targeting CD20, 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 CD20 includes such as SEQ Amino acid sequence shown in ID NO:1.

9. knocking out the Chimeric antigen receptor T cell of the targeting CD20 of PD1 as claimed in claim 8, which is characterized in that the target Amino acid sequence to the Chimeric antigen receptor CAR-CD20 of CD20 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 CD20 of PD1 With.