CN109837246A

CN109837246A - A kind of Chimeric antigen receptor T cell and its preparation method and application for the targeting ROR1 knocking out PD1

Info

Publication number: CN109837246A
Application number: CN201711197296.0A
Authority: CN
Inventors: 张宏玲; 龙丽梅; 钟春颖
Original assignee: Shenzhen Benta Biological Technology Co Ltd
Current assignee: Shenzhen Benta Biological Technology Co Ltd
Priority date: 2017-11-25
Filing date: 2017-11-25
Publication date: 2019-06-04

Abstract

The present invention provides a kind of preparation methods of the Chimeric antigen receptor T cell of targeting ROR1 for knocking out PD1, it include: that the encoding gene of Chimeric antigen receptor CAR-ROR1 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, targeting ROR1 Chimeric antigen receptor T cell is obtained.The Chimeric antigen receptor T cell of the targeting ROR1 for knocking out PD1 has knocked out PD1 gene, be conducive to T cell in the amplification of patient's body, avoid neoplastic cells escape immunosurveillance, performance with efficient and specific killing tumor cell, the vigor and lethality of cell are enduringly maintained, and not will cause damage to normal cell.

Description

A kind of Chimeric antigen receptor T cell and preparation method thereof for the targeting ROR1 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 ROR1 for knocking out PD1 is thin Born of the same parents and its preparation method and application.

Background technique

Malignant tumour is to seriously endanger the disease of human health the most, and about three million peoples of annual China are diagnosed as newly sending out Tumor patient, two million peoples die of cancer.Nearest studies have shown that tyrosine protein kinase transmembrane receptor (Receptor Tyrosine kinase like orphan receptor 1, ROR1) be expressed in a variety of B cell malignant tumours, and And then including lymphoma mantle cell (MCL), acute lymphoblastic leukemia (ALL) and lung cancer, breast cancer, colon ROR1 is identified on the surface of many cancers including cancer, cancer of pancreas, kidney and oophoroma subgroup.In addition, ROR1 can be in cancer High expression on stem cell such as ovary cancer stem cell, and it is mediated to migrate and invade.Therefore, ROR1 is that a kind of tumour-specific is high, And the ideal tumour target antigen with wide spectrum expression in all kinds of tumours.

Chimeric antigen receptor T cell technology (Chimeric Antigen Receptor-Modified T Cells, CAR- T) as one of current newest immunocyte technology, because it can activate self immune system in vivo, routinely target It is killed to tumour cell, is finally reached fully erased malignant cell, is widely paid close attention to and studied.But The current application of CART technology is also limited to blood tumor, does not there is the correlative study to most solid tumors such as lung cancer also.

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 ROR1 for knocking out PD1 also at present and can avoid neoplastic cells escape and is immunized The research of monitoring.

Summary of the invention

In view of this, the present invention provides the Chimeric antigen receptor T cell of targeting ROR1 for knocking out PD1 a kind of, it is described to strike Except the Chimeric antigen receptor T cell of the targeting ROR1 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, there is the tumour cell of ROR1 albumen with efficient and specific recognition surface, and is swashed The immunization of T cell living kills ROR1 positive tumor cell, has more lasting cellular vigor and lethality, and Damage is not will cause to normal cell.

In a first aspect, the present invention provides a kind of preparation sides of the Chimeric antigen receptor T cell of targeting ROR1 for knocking out PD1 Method, comprising:

(1) encoding gene of Chimeric antigen receptor CAR-ROR1 is provided, including holds sequentially connected signals from 5 ' ends to 3 ' The encoding gene of peptide, the encoding gene of single-chain antibody for targeting ROR1, the encoding gene of extracellular hinge area, transmembrane region coding base Because of the encoding gene in, intracellular signal area, wherein the encoding gene of the single-chain antibody of the targeting ROR1 includes coding such as SEQ ID The nucleotide sequence of amino acid sequence shown in NO:1；

(2) encoding gene of the CAR-ROR1 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-ROR1 recombination Plasmid；

(3) the pWPXLD-CAR-ROR1 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 ROR1 of PD1 Recipient T cells.

Optionally, the amino acid sequence of the single-chain antibody of the targeting ROR1 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 ROR1 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 ROR1 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-ROR1 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 ROR1 knot on the single-chain antibody and tumour of the targeting ROR1 It closes.

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-ROR1.

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-ROR1 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 is in albumen It is cut during translation is mature by signal peptidase.

The encoding gene of the CAR-ROR1 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-ROR1 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-ROR1 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-ROR1 includes as shown in SEQ ID NO:4 Amino acid sequence.

Optionally, the packaging pWPXLD-CAR-ROR1 recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-ROR1 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 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 Cas9mRNA and the targeting PD1 gene are mixed with the Chimeric antigen receptor T cell It closes, is placed in electroporation and carries out electricity turn, 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 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 ROR1 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 ROR1, but it is possible that the phenomenon that neoplastic cells escape immunosurveillance.It knocks out The Chimeric antigen receptor T cell of the targeting ROR1 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 ROR1 for the knockout PD1 that first aspect present invention provides, leads to The Chimeric antigen receptor for crossing preparation targeting ROR1 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 ROR1 of PD1 is knocked out, which is conducive to T cell in the amplification of patient's body, keeps away Exempt from neoplastic cells escape immunosurveillance, makes it have the performance of efficient and specific killing tumor cell, be particularly suitable for ROR1 positive tumor cell or tissue.

In another embodiment of the present invention, it can also prepare the targeting ROR1'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-ROR1 of targeting ROR1 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 ROR1 The encoding gene in area, intracellular signal area encoding gene, wherein the encoding gene of the single-chain antibody of the targeting ROR1 includes compiling The gene order of code amino acid sequence as shown in SEQ ID NO:1；

(3) encoding gene of the CAR-ROR1 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-ROR1 recombination Plasmid；

(4) the pWPXLD-CAR-ROR1 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 ROR1.

In the present invention, in the preparation process of the Chimeric antigen receptor T cell of the targeting ROR1 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 for obtaining the targeting ROR1 for knocking out PD1 It is carried out after recipient T cells.Knockout sequence is not construed as limiting in the present invention, as long as can achieve the targeting for obtaining and knocking out PD1 The purpose of the Chimeric antigen receptor T cell of ROR1.

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 ROR1 is targeted, the Chimeric antigen receptor T cell of the targeting ROR1 for knocking out PD1 does not include PD1 Gene, the Chimeric antigen receptor T cell of the targeting ROR1 for knocking out PD1 include the Chimeric antigen receptor CAR- for targeting ROR1 The Chimeric antigen receptor CAR-ROR1 of ROR1, the targeting ROR1 include the sequentially connected targeting ROR1 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 ROR1 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 ROR1 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 ROR1, 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 encoding gene of the amino acid sequence of the targeting Chimeric antigen receptor CAR-ROR1 includes such as SEQ ID Nucleotide sequence shown in NO:6.

Optionally, the encoding gene of the amino acid sequence of the CAR-ROR1 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-ROR1 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-ROR1 expression to cell surface, but signal peptide in protein translation maturation by signal peptidase Cutting.

The Chimeric antigen receptor T cell of the targeting ROR1 for the knockout PD1 that second aspect of the present invention provides, can be single-minded Property targeting ROR1, especially express ROR1 solid tumor cell.After CAR-ROR1 is in conjunction with ROR1, the born of the same parents of the T cell Interior signaling zone is activated, amplification of the promotion T cell in patient's body, efficient and specific killing tumor cell, and to normal Cell hardly causes to damage；In addition, the single-chain antibody based on targeting ROR1 is Humanized single chain antibody, this makes the T thin Born of the same parents avoid the immune response for causing human organism, enduringly maintain the vigor and lethality of cell；The T cell has knocked out PD1 base Cause can make it have the performance of efficient and specific killing tumor cell to avoid neoplastic cells escape immunosurveillance.This hair The Chimeric antigen receptor T cell of the bright targeting HER2 can include acute lymphoblastic leukemia with efficient identification and killing There is the cancer cell of ROR1 with expression such as breast cancer, colon cancer, cancer of pancreas, kidney and oophoromas, is particularly suitable for the ROR1 positive Lung carcinoma cell or tissue.

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 ROR1 of knockout PD1 described in face a kind of is in preparation prevention, diagnosing and treating malignant tumour Drug in application.Specifically, can be used for preventing, diagnosing and treating kinds of tumors, including the white blood of acute lymphoblastic The expression such as disease and breast cancer, colon cancer, cancer of pancreas, kidney and oophoroma have the cancer cell of ROR1, are particularly suitable for the ROR1 positive Lung carcinoma cell or tissue.

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 ROR1 of knockout PD1 that method is prepared or as described in second aspect a kind of.

Beneficial effects of the present invention:

It is provided by the invention knock out PD1 targeting ROR1 Chimeric antigen receptor T cell can with the targeting ROR1 of specificity, Promote T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific, while ROR1 is in tumour cell Wide expression, and expressed in ordinary cells very faint, therefore the Chimeric antigen receptor T cell of targeting ROR1 for knocking out PD1 can There is the tumour cell of ROR1 antigen with the mating surface of specificity, fragmentation effect is generated to ROR1 positive tumor cell, to just Normal cell not will cause damage, while knock out the PD1 gene of T cell, is conducive to T cell in the amplification of patient's body, avoids Neoplastic cells escape immunosurveillance makes it have the performance of efficient and specific killing tumor cell, promotes T cell to have and holds Long vigor and lethality.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-ROR1 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 ROR1 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 ROR1 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 ROR1 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 ROR1 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 ROR1 knocking out PD1, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-ROR1 of preparation targeting ROR1

Prepare respectively signal peptide, target the single-chain antibody of ROR1, 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 ROR1'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 ROR1 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the chimeric of targeting ROR1 The encoding gene of antigen receptor CAR-ROR1, the encoding gene of the CAR-ROR1 is as shown in SEQ ID NO:2.

(2) pWPXLd-CAR-ROR1 recombinant plasmid is constructed

The encoding gene of CAR-ROR1 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-ROR1 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-ROR1 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 pWPXLd-CAR-ROR1 recombinant plasmid as shown in Figure 1.

(3) recombinant slow virus constructs

PWPXLd-CAR-ROR1 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 pcDNA3.1-PD1-sgRNA recombination 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 ROR1 for knocking out PD1.

The PD1 that the Chimeric antigen receptor T cell of the targeting ROR1 of above-mentioned knockout PD1 is measured using flow cytometer is expressed Amount calculates knockout rate, as a result, it has been found that the knockout rate for knocking out PD1 gene in the Chimeric antigen receptor T cell of the targeting ROR1 of PD1 reaches 79%.

In order to assess the Chimeric antigen receptor T for targeting ROR1 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 ROR1 of knockout PD1 prepared by the assessment present invention Positive rate

Will by the method for the present invention preparation knock out PD1 targeting ROR1 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 ROR1 Chimeric antigen receptor T cell.It can be obtained compared with (B) by (A) in Fig. 2, knockout PD1 prepared by the present invention Targeting ROR1 Chimeric antigen receptor T cell positive rate be 49.9%.

Effect example two: the tumor cell in vitro that assessment knocks out the Chimeric antigen receptor T cell of the targeting ROR1 of PD1 kills Condition of the injury condition

Will by the targeting ROR1 of knockout PD1 made from the method for the present invention Chimeric antigen receptor T cell (experimental group) with The Vitro Tumor fragmentation effect of T lymphocyte (negative control group) without preparation is compared, specific: in vitro by effect Cell (knocking out the Chimeric antigen receptor T cell of the targeting ROR1 of PD1 or the T lymphocyte without preparation) and target cell (H526 Cell) it in quantity ratio is 1:10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, after incubation 15-18 hours, collect cell, carry out streaming dyeing, detect cell killing situation, as a result as shown in Figure 3.It can from Fig. 3 Find out, the Chimeric antigen receptor T cell of the targeting ROR1 of the knockout PD1 of addition is more, they get over the lethality of tumour cell By force.The tumor-killing power of the Chimeric antigen receptor T cell of the targeting ROR1 of knockout PD1 by method of the present invention preparation 20% or more, even up to 45%, significantly larger than negative control group, this illustrates the knockout PD1 prepared through the method for the present invention Targeting ROR1 Chimeric antigen receptor T cell have strong tumor-killing ability.

Effect example three: the mouse interior tumor that assessment knocks out the Chimeric antigen receptor T cell of the targeting ROR1 of PD1 is thin Born of the same parents kill situation

By the Chimeric antigen receptor T cell (experimental group) of the targeting ROR1 of the knockout PD1 by the method for the present invention preparation, not T lymphocyte (negative control group) and physiological saline (blank control group) through preparing give every in mouse tumor model Mouse tail vein injection 1 × 10⁶A H526 cell (n=9), draws the survivorship curve of mouse, as a result as shown in Figure 4.It can from Fig. 4 To find out, the Chimeric antigen receptor T cell of the targeting ROR1 of the knockout PD1 by this method preparation makes mouse in culture 50 days When survival rate be also higher than 50%, cultivating 80 days and survival rate when the longer time is more than 20%, considerably beyond negative control group And blank control group.Fig. 4's the result shows that, by this method preparation knockout PD1 targeting ROR1 Chimeric antigen receptor T it is thin Born of the same parents are 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 ROR1 for knocking out PD1

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 332

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser

1 5 10 15

Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe

20 25 30

Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro

35 40 45

Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe

50 55 60

Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys

65 70 75 80

Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile

85 90 95

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

100 105 110

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

115 120 125

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330

<210> 2

<211> 575

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg

20 25 30

Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn

35 40 45

Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val

50 55 60

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

65 70 75 80

Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys

85 90 95

Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro

100 105 110

Trp Ser Asp Ile Glu Cys Val His Lys Glu Glu Pro Lys Ser Cys Asp

115 120 125

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

130 135 140

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

275 280 285

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

290 295 300

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

305 310 315 320

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

325 330 335

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

<210> 3

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cacgaagctc tccgatgtgt tgg 23

<210> 4

<211> 555

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser

1 5 10 15

Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe

20 25 30

Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro

35 40 45

Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe

50 55 60

Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys

65 70 75 80

Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile

85 90 95

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

100 105 110

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

115 120 125

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

355 360 365

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

485 490 495

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

500 505 510

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

515 520 525

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

530 535 540

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

545 550 555

<210> 5

<211> 996

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tgtccacctg gacaccatat ctcagaagac ggtagagatt gcatctcctg caaatatgga 60

caggactata gcactcactg gaatgacctc cttttctgct tgcgctgcac caggtgtgat 120

tcaggtgaag tggagctaag tccctgcacc acgaccagaa acacagtgtg tcagtgcgaa 180

gaaggcacct tccgggaaga agattctcct gagatgtgcc ggaagtgccg cacagggtgt 240

cccagaggga tggtcaaggt cggtgattgt acaccctgga gtgacatcga atgtgtccac 300

aaagaagagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 360

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 420

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 480

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 540

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 600

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 660

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 720

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 780

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 840

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 900

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 960

aaccactaca cgcagaagag cctctccctg tctccg 996

<210> 4

<211> 1665

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tgtccacctg gacaccatat ctcagaagac ggtagagatt gcatctcctg caaatatgga 60

caggactata gcactcactg gaatgacctc cttttctgct tgcgctgcac caggtgtgat 120

tcaggtgaag tggagctaag tccctgcacc acgaccagaa acacagtgtg tcagtgcgaa 180

gaaggcacct tccgggaaga agattctcct gagatgtgcc ggaagtgccg cacagggtgt 240

cccagaggga tggtcaaggt cggtgattgt acaccctgga gtgacatcga atgtgtccac 300

aaagaagagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 360

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 420

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 480

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 540

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 600

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 660

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 720

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 780

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 840

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 900

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 960

aaccactaca cgcagaagag cctctccctg tctccgacca ctaccccagc accgaggcca 1020

cccaccccgg ctcctaccat cgcctcccag cctctgtccc tgcgtccgga ggcatgtaga 1080

cccgcagctg gtggggccgt gcatacccgg ggtcttgact tcgcctgcga tatctacatt 1140

tgggcccctc tggctggtac ttgcggggtc ctgctgcttt cactcgtgat cactctttac 1200

tgtaagcgcg gtcggaagaa gctgctgtac atctttaagc aacccttcat gaggcctgtg 1260

cagactactc aagaggagga cggctgttca tgccggttcc cagaggagga ggaaggcggc 1320

tgcgaactgc gcgtgaaatt cagccgcagc gcagatgctc cagcctacaa gcaggggcag 1380

aaccagctct acaacgaact caatcttggt cggagagagg agtacgacgt gctggacaag 1440

cggagaggac gggacccaga aatgggcggg aagccgcgca gaaagaatcc ccaagagggc 1500

ctgtacaacg agctccaaaa ggataagatg gcagaagcct atagcgagat tggtatgaaa 1560

ggggaacgca gaagaggcaa aggccacgac ggactgtacc agggactcag caccgccacc 1620

aaggacacct atgacgctct tcacatgcag gccctgccgc ctcgg 1665

<210> 7

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro

20

<210> 8

<211> 60

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

gccctccctg tcaccgccct gctgcttccg ctggctcttc tgctccacgc cgctcggccc 60

<210> 9

<211> 46

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

<210> 10

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatc 138

<210> 11

<211> 26

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

20 25

<210> 12

<211> 78

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

tacatttggg cccctctggc tggtacttgc ggggtcctgc tgctttcact cgtgatcact 60

ctttactgta agcgcggt 78

<210> 13

<211> 39

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

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

20 25 30

Glu Glu Gly Gly Cys Glu Leu

35

<210> 14

<211> 117

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 60

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactg 117

<210> 15

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 16

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

Claims

1. a kind of preparation method of the Chimeric antigen receptor T cell for the targeting ROR1 for knocking out PD1 characterized by comprising

(1) encoding gene of Chimeric antigen receptor CAR-ROR1 is provided, including holding sequentially connected signal peptides from 5 ' ends to 3 ' Encoding gene, target the encoding gene of single-chain antibody of ROR1, the encoding gene of extracellular hinge area, transmembrane region encoding gene, The encoding gene in intracellular signal area, wherein the encoding gene of the single-chain antibody of the targeting ROR1 includes coding such as SEQ ID The nucleotide sequence of amino acid sequence shown in NO:1；

(2) encoding gene of the CAR-ROR1 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-ROR1 recombination matter Grain；

(5) the PD1 gene for knocking out the Chimeric antigen receptor T cell obtains the Chimeric antigen receptor for knocking out the targeting ROR1 of PD1 T cell.

2. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting ROR1 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 ROR1 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-ROR1 includes the nucleotide sequence as shown in SEQ IDNO:2.

4. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting ROR1 of PD1, feature as described in claim 1 It is, the packaging pWPXLD-CAR-ROR1 recombinant plasmid, obtaining recombinant slow virus includes:

By the pWPXLD-CAR-ROR1 recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, institute is obtained State recombinant slow virus.

5. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting ROR1 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.

6. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting ROR1 of PD1, feature as claimed in claim 5 It is, the corresponding gene order of sgRNA of the targeting PD1 gene includes the nucleotide sequence as shown in SEQ ID NO:3.

7. knocking out the preparation method of the Chimeric antigen receptor T cell of the targeting ROR1 of PD1, feature as claimed in claim 5 It is, the mass ratio of the sgRNA of the Cas9mRNA and the PD1 gene is 1:1-1:5.

8. the Chimeric antigen receptor T of the targeting ROR1 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 ROR1 for knocking out PD1 does not include PD1 gene, described to strike Except the Chimeric antigen receptor T cell of the targeting ROR1 of PD1 includes the Chimeric antigen receptor CAR-ROR1 for targeting ROR1, the targeting The Chimeric antigen receptor CAR-ROR1 of ROR1 includes the single-chain antibody of sequentially connected targeting ROR1, 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 ROR1 includes such as SEQ Amino acid sequence shown in ID NO:1.

9. knocking out the Chimeric antigen receptor T cell of the targeting ROR1 of PD1 as claimed in claim 8, which is characterized in that the target Amino acid sequence to the Chimeric antigen receptor CAR-ROR1 of ROR1 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 ROR1 of PD1 With.