CN107828730B

CN107828730B - Universal CART/TCRT cell and its construction method with antibody drug resistance

Info

Publication number: CN107828730B
Application number: CN201711082916.6A
Authority: CN
Inventors: 贺小宏; 王延宾; 袁鹏
Original assignee: Nanjing North Heng Biological Technology Co Ltd
Current assignee: Nanjing North Heng Biological Technology Co Ltd
Priority date: 2017-11-07
Filing date: 2017-11-07
Publication date: 2019-04-30
Anticipated expiration: 2037-11-07
Also published as: CN107828730A

Abstract

The invention belongs to genetic engineerings and synthetic biology field, more particularly to a kind of universal CART/TCRT cell and its construction method with antibody drug resistance, the universal CART/TCRT cell is the allogeneic T cells with Chimeric antigen receptor and T cell receptor, and T cell receptor α, β chain and CD52 molecule are knocked；The knockout technology of T cell receptor α, β chain and CD52 molecule is CRISPR technology；The universal CART/TCRT cell had both remained the targeting of tumour specific antigen, the problem of eliminating GvHD again and repelling, and weakening to the susceptibility of antibody drug Alemtuzumab can be used as a kind of universal simple, low cost, high activity CART/TCRT cell preparation use.

Description

Universal CART/TCRT cell and its construction method with antibody drug resistance

Technical field

The invention belongs to genetic engineerings and synthetic biology field, and in particular to a kind of with the general of antibody drug resistance Type CART/TCRT cell and its construction method.

Background technique

Chimeric antigen receptor T cell (CART) is one of tumour immunotherapy most promising at present, substantially former Reason mainly extract patient itself T cell, and by gene and cell engineering means make its expression specificity chimeric antigen by Body enables to identify and combines tumor cell surface antigen, to play the role of target killing tumor cell.CAR-T exists Significant curative effect, on August 30th, 2017, U.S. food hygiene and safety committee FDA a surname are obtained in leukaemia and lymphoma treating Cloth ratifies Novartis CAR-T cell therapy official listing, for treating recurrent or the leaching of intractable children and adolescents B- cell acute Bar chronic myeloid leukemia (rALL), trade name Kymriah (tisagenlecleucel).Acute lymphoblastic leukemia is 15 Year old or less account for about 25% in childhood cancer confirmed cases, be the most common children with cancer in the U.S., in China, acute lymphocytic is white Blood disease accounts for the 80% of acute leukemia, and disease incidence is ten a ten thousandths.Adult can also be betided, Zhan Suoyou adult is white The 20% of blood disease disease incidence.

Estimated according to National Cancer Institute, there are about 3100 ages every year at 20 years old, and patient below is diagnosed as This disease has about 10000 children and youth to suffer from acute leukemia every year due to the population base that China is huge.Effectively Therapeutic choice it is extremely limited, in multiple relapse or intractable B- cell Acute Lymphoblastic Leukemia children and adolescent patient In, disease-free survival rate is lower than 10%-30% within 5 years.The safety of Kymriah and validity obtain in multi-center clinical trial It confirms, the overall relief rate in treatment three months is 83%, it is shown that its brilliant curative effect.

In addition to B cell acute lymphoblastic leukemia, CAR-T is in relapsed or stubborn chronic lymphocytic leukemia (rCLL), it is also achieved in the treatment of recurrent or Refractory Multiple Myeloma (rNHL) and non-Hodgkin lymphoma (NHL) Significant curative effect.In a treatment diffusivity large B cell lymphoid tumor (DLBCL), conversion follicular lymphoma (TFL), Yi Jiyuan Hair property mediastinal B-cell lymphoma (PMBCL) these three non-Hodgkin lymphoma, in 2 clinical trial phases of entitled ZUMA-1, Kite CAR-T therapy reached Major Clinical terminal on objective remission rate (objective response rate).Receiving list After the infusion of secondary axicabtagene ciloleucel, there is up to 82% patient to occur alleviating.It is 8.7 in median In the follow-up of the moon, there is 44% patient to be still in the paracmasis, and there is 39% patient to be in complete incidence graph.In Bluebird The key name that Bio company carries out is 100% patient in two dosage groups in the experiment of the treatment Huppert's disease of b2121 (n=6) objective reaction is realized；Two patients are negative in MRD；General reaction rate (ORR) is 78%.6 and when follow-up in 4 months, Two patients reach the complete reaction (CR) of stricti jurise.

Other than CART, T cell receptor (TCR) T cell therapy also achieves considerable progress.TCRT cell technology is earliest From tumor-infiltrated T cell (TIL), this kind of T cell " would generally invade " cancerous tissue, this illustrates that they have centainly cancer cell Recognition capability.Fact-finding TIL has cancer cell related antigen (Tumor Associated Antigen, TAA) special Property recognition capability.This kind of antigen includes CEA, Her-2, CD19, gp100, MART-1, MAGA-A3, NY-ESO-1, etc..These TAA has the expression of opposite particularity in different cancers, thus also becomes the object of attack of immune system.Nevertheless, The recognition capability of these natural TIL is usually weaker, therefore cannot form the advantageous attack to cancer cell.In this case, may be used Modification is oriented to T cell to achieve the purpose that attack tumour to import the specific TCR of TIL by external source.2016 " from So-medicine " the exciting TCR of magazine ran numerical example treats case, and this clinical test cured by Univ Maryland-Coll Park USA A entitled " TCR of gene modification " of institute, medical college, University of Pennsylvania and immunization therapy company Adaptimmune cooperative research and development Immunotherapeutic.After having modified several key amino acids, the TCR of these genes modification substantially increases normal with one kind The affinity of the cancer TAA, NY-ESO-1 that see, and then the cancer to NY-ESO-1 overexpression is significantly improved, such as multiple The therapeutic effect of property myeloma (Multiple Myeloma).In current clinical test, 80% multiple myeloma patients Good clinical response is produced, wherein 70% patient reaches completely or nearly complete response, average progression free survival phase reaches By 19 months.The treatment of other cancers of NY-ESO-1 overexpression is being carried out successively at present.

But since CART/TCRT is a kind of method of height personalization, everyone cell and the treatment received Difference, many patients receive CART/TCRT treatment before received various treatment methods, as bone marrow transplant, chemotherapy, Targeted therapy etc., the preparation and programming of T cell obtained also can be different.This individualized treatment mode consumes a large amount of people Power material resources, so at high price.The CART cell therapy product that the Kymriah of Novartis ratifies as first FDA, is priced at 47.5 ten thousand U.S. dollars, the medical expense of such great number can make some patient families be difficult to bear, while also greatly limit its city Field application.

What the CART/TCRT first echelon including Novartis, Kite pharmacy, Juno was all made of at present is autotransplantation side Method prepares CART/TCRT cell, carries out treatment using allogeneic T cells preparation CART/TCRT cell and provides another thinking, but It is the row due to donor T-cells to the graft-versus-host reaction (GvHD) of host and host immune system to allogeneic T cells Reprimand, significantly limits its application.

The TCR that CART cell surface is knocked out by way of gene editing can be eliminated since allogeneic T cells are to host's Identification and caused by GvHD, be used to prepare universal CART cell to a certain extent.Currently used gene editing tool Mainly there are TALEN and CRISPR.

TAL effector (TAL effector, TALE) is most early in phytopathogen Xanthomonas campestris (Xanthomonas Sp.) to being found in the invasion infection research of plant.Since TALE has the specific binding capacity of DNA sequence dna, follow-up study FokI DNA nuclease and artificial T ALE are connected by engineering science means, formd a kind of with specific gene group volume Collect the strong tools of function, i.e. TALEN.

Typical TALEN by nuclear localization sequence (Nuclear localization signal, NLS) N-terminal structural domain, It can recognize the central domain of the series connection TALE repetitive sequence of specific dna sequence, the C-terminal knot with FokI endonuclease function Structure domain composition.In recent years, TALEN is widely used to yeast, animal and plant cells and arabidopsis, drosophila, zebra fish and mouse Etc. the research of all kinds of model organisms.

A large amount of molecular cloning and sequencing procedures are needed since single TALEN module is carried out assembling, it is very complicated, and And because the building of TALEN needs to carry out cumbersome and complicated protein engineering according to target dna sequence and designs and optimize, in this way Just substantially prolong the experimental period of building TALEN element.Simultaneously and since the knockout efficiency of TALEN is influenced by DNA sequence dna It is larger, and by these many protein deliveries to being used in the cell that multiple genetic operates simultaneously right and wrong often with challenging, Therefore so very big that limit its application.

In addition to above-mentioned technological difficulties, since TALEN needs two FokI cutting domains to form dimerization the shearing of DNA Body, and at least one identification structural domain is needed to combine targeting DNA.Although DNA identifies that structural domain has stronger sequence specific Property, but since the shear history of TALEN not fully relies on the formation of homodimer, so heterodimeric once being formed Body is just likely to cause undershooting-effect, and the mispairing of DNA and sequence may finally be caused to change, and generates stronger cytotoxicity. When these adverse effects accumulation is excessive, when the range born more than cellular repair mechanisms, the apoptosis of cell will be caused.It is above all More limitations cause TALEN to be difficult to promote and apply.

CRISPR/Cas system as a kind of newest genome edit tool, know by the specific DNA that can complete RNA guiding Not and edit.CRISPR/Cas technology uses the guide RNA molecule (sequence-specific of one section of sequence-specific Guide RNA) guidance endonuclease is at target spot, to complete the editor of genome.The exploitation of CRISPR/Cas system is It constructs more efficient gene site-directed modification technique and provides completely new platform.

CRISPR/Cas technology gets rid of synthesis and assembles the cumbersome behaviour with specific DNA recognition capability module Make, the design and synthesis workload of gRNA is far smaller than the building process of the DNA identification module of TALEN and ZFN technology, and poison Property is well below ZFN technology." science " magazine in 2013 has delivered two being of great significance respectively from MIT and Harvard CRISPR technical papers, independently completed for the first time using CRISPR technology to human genome editor.Due to CRISPR The simplicity of system and efficient characteristic, application have obtained great popularization.In addition to gene knockout, CRISPR system is engineered It is applied to more extensive field, including gene inhibits, gene activation, gene simple point modification, epigenetic modification, light science of heredity Field, CRISPR's uses very big the research range for having expanded biology.

Due to the huge applications prospect and possible commercial interest of CRISPR gene editing technology, foreign countries are at present with the technology Based on obtain huge risk investment biotech firm mainly have Editas Medicine, CRISPR Therapeutics, Intellia Therapeutics etc., master plan carry out related human genetic disease such as retina degenerative disease, genetic muscle The pilot study of the prevention and control fields such as disease and hematologic disease.

CAR-T cell (Chimeric antigen receptor-T) is current as immune cell therapy treatment tumour One of tumour immunotherapy hot research field.PD1 (Programmed death 1) is used as a kind of immunosuppression molecule, has become For effective target molecule of important oncotherapy, 2016 studies have reported that knock out the PD1 on CART cell using CRISPR technology Molecule can significantly improve CART cell to the therapeutic effect of solid tumor.The report of research in 2017, using CRISPR technology, at Function constructs the CAR-T cell of the gene site-directed insertion CAR gene of mouse TRAC, can significantly extend the modification mouse survival time.

Cellectis company knocks out the intracellular TCRa and CD52 molecule of CART using TALEN technology, has been applied to face Bed research, but since TALEN that they the use T cell edited all produces serious chromosomal shift phenomenon, this is to it Clinical use causes great tumor formation risk, but the high degree of specificity due to CRISPR in T cell, is compiled using CRISPR The universal CART cell collected carries out oncotherapy, has very big advantage.Simultaneously because TALEN is to the inefficient of CART cell Rate editor, can the CART cell to gene editing carry out purifying and cause difficulty, while can because TCR positive cell separate it is unclean And increase the risk of GvHD.Our invention is so that CRISPR efficiently edits CART cell, largely favorably In the production of universal CART cell.

Individually knockout TCR α, β chain can not generate universal CART cell, this is because this CART cell still has HLA molecule is expressed in cell surface, to be identified and repelled by recipient immune system T cell, it is difficult to achieve the purpose that treatment.Together When editor TCR α, β chain and inhibit molecule, such as PD1, TIM3, CTLA4 can not generate universal CART cell.

Summary of the invention

The present invention solves the above-mentioned technical problems in the prior art, provides a kind of with the general of antibody drug resistance Type CART/TCRT cell and its construction method.

To solve the above problems, technical scheme is as follows:

Universal CART/TCRT cell with antibody drug resistance, the universal CART/TCRT cell be have it is embedding The allogeneic T cells of antigen receptor and T cell receptor are closed, T cell receptor α, β chain and CD52 molecule are knocked.

Preferably, the Chimeric antigen receptor is targeting CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, Alpha folate receptor, CEA, PSCA, PSMA, Her2, EGFR, IL13R α 2, The Chimeric antigen receptor of GD2, NKG2D, EGFRvIII, CS1, BCMA, Mesothelin.

Preferably, the T cell receptor is targeting HBV, HPV E6, NY-ESO, mNY-ESO, WT1, MART-1, MAGE- The T cell receptor of A3, MAGE-A4, P53, Thyroglobulin, Tyrosinase.

The construction method of universal CART/TCRT cell with antibody drug resistance, using CRISPR technology in allosome T T cell receptor α, β chain and CD52 molecule on intracellular specific knockdown T cell surface.

Preferably, using sgRNA and crRNA sequence used in CRISPR technology are as follows:

SgRNA-TCR α is selected from following sequence:

sgRNA-TCRα-1:GAGAATCAAAATCGGTGAAT(SEQ ID NO.1)

sgRNA-TCRα-2:GCTGGTACACGGCAGGGTCA(SEQ ID NO.2)

sgRNA-TCRα-3:GTCAGGGTTCTGGATATCTG(SEQ ID NO.3)

sgRNA-TCRα-4:ACAAAACTGTGCTAGACATG(SEQ ID NO.4)

sgRNA-TCRα-5:CTTCAAGAGCAACAGTGCTG(SEQ ID NO.5)

sgRNA-TCRα-6:TGGAATAATGCTGTTGTTGA(SEQ ID NO.6)

sgRNA-TCRα-7:TAGGCAGACAGACTTGTCAC(SEQ ID NO.7)

sgRNA-TCRα-8:TGGATTTAGAGTCTCTCAGC(SEQ ID NO.8)

sgRNA-TCRα-9:TCTCTCAGCTGGTACACGGC(SEQ ID NO.9)

sgRNA-TCRα-10:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

SgRNA-TCR β is selected from following sequence:

sgRNA-TCRβ-1:GAGCAGCCGCCTGAGGGTCT(SEQ ID NO.11)

sgRNA-TCRβ-2:GAGCTGGTGGGTGAATGGGA(SEQ ID NO.12)

sgRNA-TCRβ-3:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-TCRβ-4:GGCTCTCGGAGAATGACGAG(SEQ ID NO.14)

sgRNA-TCRβ-5:GGAGAATGACGAGTGGACCC(SEQ ID NO.15)

sgRNA-TCRβ-6:GGCGCTGACGATCTGGGTGA(SEQ ID NO.16)

sgRNA-TCRβ-7:GTTGCGGGGGTTCTGCCAGA(SEQ ID NO.17)

sgRNA-TCRβ-8:GCAGTATCTGGAGTCATTGA(SEQ ID NO.18)

sgRNA-TCRβ-9:GGCACACCAGTGTGGCCTTT(SEQ ID NO.19)

sgRNA-TCRβ-10:GGCTCAAACACAGCGACCTC(SEQ ID NO.20)

SgRNA-CD52 is selected from following sequence:

sgRNA-CD52-1:CCTACTCACCATCAGCCTCC(SEQ ID NO.21)

sgRNA-CD52-2:GCATCCAGCAACATAAGCGG(SEQ ID NO.22)

sgRNA-CD52-3:GTTATGGTACAGATACAAAC(SEQ ID NO.23)

sgRNA-CD52-4:CACCATCAGCCTCCTGGTTA(SEQ ID NO.24)

sgRNA-CD52-5:GTTATGGTACAGATACAAAC(SEQ ID NO.25)

CrRNA-TCR α is selected from following sequence:

crRNA-TCRα-1:GAGTCTCTCAGCTGGTACACGGC(SEQ ID NO.26)

crRNA-TCRα-2:CATGTGCAAACGCCTTCAACAAC(SEQ ID NO.27)

crRNA-TCRα-3:CACATGCAAAGTCAGATTTGTTG(SEQ ID NO.28)

crRNA-TCRα-4:TTGCTCCAGGCCACAGCACTGTT(SEQ ID NO.29)

crRNA-TCRα-5:TCTGTGATATACACATCAGAATC(SEQ ID NO.30)

crRNA-TCRα-6:TGACACATTTGTTTGAGAATCAA(SEQ ID NO.31)

crRNA-TCRα-7:TTTGAGAATCAAAATCGGTGAAT(SEQ ID NO.32)

crRNA-TCRα-8:AGAATCAAAATCGGTGAATAGGC(SEQ ID NO.33)

crRNA-TCRα-9:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.34)

CrRNA-TCR β is selected from following sequence:

crRNA-TCRβ-1:GGTGTGGGAGATCTCTGCTTCTG(SEQ ID NO.35)

crRNA-TCRβ-2:CCTCGGTGTCCTACCAGCAAGGG(SEQ ID NO.36)

crRNA-TCRβ-3:GCCCTATCCTGGGTCCACTCGTC(SEQ ID NO.37)

crRNA-TCRβ-4:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.38)

CrRNA-CD52 is selected from following sequence:

crRNA-CD52-1:GCTGGTGTCGTTTTGTCCTGAGA(SEQ ID NO.39)

crRNA-CD52-2:TCCTGAGAGTCCAGTTTGTATCT(SEQ ID NO.40)

crRNA-CD52-3:TATCTGTACCATAACCAGGAGGC(SEQ ID NO.41)

crRNA-CD52-4:CTTTTCTTCGTGGCCAATGCCAT(SEQ ID NO.42)

crRNA-CD52-5:TTCGTGGCCAATGCCATAATCCA(SEQ ID NO.43)

sgRNA-TCRα:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

sgRNA-TCRβ:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-CD52:CACCATCAGCCTCCTGGTTA(SEQ ID NO.24)

crRNA-TCRα:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.34)

crRNA-TCRβ:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.38)

crRNA-CD52:TATCTGTACCATAACCAGGAGGC(SEQ ID NO.41)。

Compared with the existing technology, advantages of the present invention is as follows,

Universal CART/TCRT cell with antibody drug resistance of the invention realizes the progress of TCR and CD52 molecule Double knockouts, had both remained the targeting of tumour specific antigen, and the knockout of TCR eliminates GvHD；The antibody drug of CD52 simultaneously The use of Alemtuzumab removes the intracorporal T cell of host, so that allogeneic T cells is prevented to be ostracised, and CD52 molecule strikes The allogeneic T cells removed are because can over a long time survive resistant to Alemtuzumab plays anti-tumor activity；It can be used as A kind of universal simple, low cost, the use of high activity CART/TCRT cell preparation.

The construction method of universal CART/TCRT cell with antibody drug resistance of the invention, using Cas9 and TCR and CD52 gene in Cpf1 Technique on T cell carries out double knockouts, and the double negative cells group of generation respectively reaches 40.1% He 48.3%, this cell mass loses GvHD activity simultaneously and weakens the susceptibility to antibody drug Alemtuzumab；Pass through single The sorting of TCR/CD3 feminine gender, TCR negative cells group reach 99%.

The present invention edits the intracellular TCR and CD52 molecule of CART using CRISPR, can produce without GvHD, but simultaneously With the CART cell to CD52 antibody drug Alemtuzumab resistance；When being treated to patient, use The T cell that Alemtuzumab removes patient's body prevents rejection, and CD52 molecule is knocked out with Alemtuzumab resistance Universal CART cell do not cause GvHD while treating tumour, thus greatly reduce at produce and treatment cost.

Detailed description of the invention

The primary T cells surface TCR/CD3 compound that Fig. 1 .Cas9 and Cpf1 are mediated knocks out；1a is CRISPR/Cas9 Jie The TCR/CD3 compound led knocks out, and 1b is that the TCR/CD3 compound that CRISPR/Cpf1 is mediated knocks out.

The primary T cells surface C D52 molecule that Fig. 2 .Cas9 and Cpf1 are mediated knocks out.

The primary T cells surface TCR/CD52 molecule that Fig. 3 .Cas9 and Cpf1 are mediated is double to be knocked out；3a is CRISPR/Cas9 Jie The TCR/CD52 molecule led is double to be knocked out, and 3b is that the TCR/CD52 molecule pair that CRISPR/Cpf1 is mediated knocks out.

The CART cell surface TCR/CD52 molecule that Fig. 4 .Cas9 and Cpf1 are mediated is double to be knocked out.

The bis- knockout CART/TCRT gene expression detections of Fig. 5 .TCR/CD52；The bis- knockout CART gene expressions of 5aTCR/CD52, 5b is the bis- knockout TCRT gene expressions of TCR/CD52.

The bis- knockout CART/TCRT cell cytokine secretions of Fig. 6 .TCR/CD52；6a is that the bis- knockout CART of TCR/CD52 are thin Born of the same parents' cytokine secretion, 6b are the bis- knockout TCRT cell cytokine secretions of TCR/CD52.

The bis- knockout CART/TCRT cell tumour killing activity detections of Fig. 7 .TCR/CD52；7a, which is that TCR/CD52 is bis-, to be knocked out The anti-tumor activity of CART cell detects, and 7b is the bis- anti-tumor activity detections for knocking out TCRT cell of TCR/CD52.

Fig. 8 is using different sgRNA and crRNA to the editorial efficiency of T cell TCR α, TCR β and CD52 molecule.

Specific embodiment

Embodiment 1:

Primary T cells amplification.Primary human T-Cells are being contained into 10%FBS, 100U/ml penicillin, 100 μ g/ml sulfate chains Mycin is cultivated in the RPMI 1640 of 10mM Hepes, and using the magnetic bead of coupling CD3/CD28 antibody according to 1:3 ratio into Line activating stimulation.Cell is counted, every 2 days addition culture mediums, collects T on appropriate opportunity by cell size and growth parameter(s) Cell carries out functional examination or freezen protective.

The design and implementation of CRISPR

Sequence in sgRNA and crRNA targeting TCR α constant region exon 1, TCR β constant region 1 and 2 exons 1s share Sequence, the exons 1 sequence of CD52, and be cloned into the carrier containing T7 promoter.These plasmids restriction enzyme line Property.It is transcribed using MEGAscript T7Transcription kit (Life Technologies, Carlsbad, CA) RNA is stored in -80 DEG C by sgRNA/crRNA.

SgRNA the and crRNA sequence of Select to use of the present invention is as follows:

SgRNA-TCR α is selected from following sequence:

sgRNA-TCRα-1:GAGAATCAAAATCGGTGAAT(SEQ ID NO.1)

sgRNA-TCRα-2:GCTGGTACACGGCAGGGTCA(SEQ ID NO.2)

sgRNA-TCRα-3:GTCAGGGTTCTGGATATCTG(SEQ ID NO.3)

sgRNA-TCRα-4:ACAAAACTGTGCTAGACATG(SEQ ID NO.4)

sgRNA-TCRα-5:CTTCAAGAGCAACAGTGCTG(SEQ ID NO.5)

sgRNA-TCRα-6:TGGAATAATGCTGTTGTTGA(SEQ ID NO.6)

sgRNA-TCRα-7:TAGGCAGACAGACTTGTCAC(SEQ ID NO.7)

sgRNA-TCRα-8:TGGATTTAGAGTCTCTCAGC(SEQ ID NO.8)

sgRNA-TCRα-9:TCTCTCAGCTGGTACACGGC(SEQ ID NO.9)

sgRNA-TCRα-10:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

SgRNA-TCR β is selected from following sequence:

sgRNA-TCRβ-1:GAGCAGCCGCCTGAGGGTCT(SEQ ID NO.11)

sgRNA-TCRβ-2:GAGCTGGTGGGTGAATGGGA(SEQ ID NO.12)

sgRNA-TCRβ-3:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-TCRβ-4:GGCTCTCGGAGAATGACGAG(SEQ ID NO.14)

sgRNA-TCRβ-5:GGAGAATGACGAGTGGACCC(SEQ ID NO.15)

sgRNA-TCRβ-6:GGCGCTGACGATCTGGGTGA(SEQ ID NO.16)

sgRNA-TCRβ-7:GTTGCGGGGGTTCTGCCAGA(SEQ ID NO.17)

sgRNA-TCRβ-8:GCAGTATCTGGAGTCATTGA(SEQ ID NO.18)

sgRNA-TCRβ-9:GGCACACCAGTGTGGCCTTT(SEQ ID NO.19)

sgRNA-TCRβ-10:GGCTCAAACACAGCGACCTC(SEQ ID NO.20)

SgRNA-CD52 is selected from following sequence:

sgRNA-CD52-1:CCTACTCACCATCAGCCTCC(SEQ ID NO.21)

sgRNA-CD52-2:GCATCCAGCAACATAAGCGG(SEQ ID NO.22)

sgRNA-CD52-3:GTTATGGTACAGATACAAAC(SEQ ID NO.23)

sgRNA-CD52-4:CACCATCAGCCTCCTGGTTA(SEQ ID NO.24)

sgRNA-CD52-5:GTTATGGTACAGATACAAAC(SEQ ID NO.25)

CrRNA-TCR α is selected from following sequence:

crRNA-TCRα-1:GAGTCTCTCAGCTGGTACACGGC(SEQ ID NO.26)

crRNA-TCRα-2:CATGTGCAAACGCCTTCAACAAC(SEQ ID NO.27)

crRNA-TCRα-3:CACATGCAAAGTCAGATTTGTTG(SEQ ID NO.28)

crRNA-TCRα-4:TTGCTCCAGGCCACAGCACTGTT(SEQ ID NO.29)

crRNA-TCRα-5:TCTGTGATATACACATCAGAATC(SEQ ID NO.30)

crRNA-TCRα-6:TGACACATTTGTTTGAGAATCAA(SEQ ID NO.31)

crRNA-TCRα-7:TTTGAGAATCAAAATCGGTGAAT(SEQ ID NO.32)

crRNA-TCRα-8:AGAATCAAAATCGGTGAATAGGC(SEQ ID NO.33)

crRNA-TCRα-9:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.34)

CrRNA-TCR β is selected from following sequence:

crRNA-TCRβ-1:GGTGTGGGAGATCTCTGCTTCTG(SEQ ID NO.35)

crRNA-TCRβ-2:CCTCGGTGTCCTACCAGCAAGGG(SEQ ID NO.36)

crRNA-TCRβ-3:GCCCTATCCTGGGTCCACTCGTC(SEQ ID NO.37)

crRNA-TCRβ-4:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.38)

CrRNA-CD52 is selected from following sequence:

crRNA-CD52-1:GCTGGTGTCGTTTTGTCCTGAGA(SEQ ID NO.39)

crRNA-CD52-2:TCCTGAGAGTCCAGTTTGTATCT(SEQ ID NO.40)

crRNA-CD52-3:TATCTGTACCATAACCAGGAGGC(SEQ ID NO.41)

crRNA-CD52-4:CTTTTCTTCGTGGCCAATGCCAT(SEQ ID NO.42)

crRNA-CD52-5:TTCGTGGCCAATGCCATAATCCA(SEQ ID NO.43)

By the comparison of gene editing efficiency, it is preferable to use sgRNA and crRNA sequence it is as follows:

sgRNA-TCRα:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

sgRNA-TCRβ:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-CD52:CACCATCAGCCTCCTGGTTA(SEQ ID NO.24)

crRNA-TCRα:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.34)

crRNA-TCRβ:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.38)

crRNA-CD52:TATCTGTACCATAACCAGGAGGC(SEQ ID NO.41)。

The gene editing in T cell is realized using the method for electroporation

Gene editing in T cell is realized using the means of electroporation: firstly, T cell at the 0th day, passes through coupling CD3/ The magnetic bead of CD28 antibody is activated, and the transfection of CRISPR was carried out at the 2nd~5 day, collects gene editing at the 9th to the 11st day T cell；

For the gene editing of CART/TCRT cell, except the 1st day plus in addition to virus carries out CAR/TCR transfection to T cell, He is same as above step.

The operating procedure of electroporation method approximately as:

1. cleaning T cell 3 times, each 300g using Opti-MEM, it is centrifuged 10 minutes removal supernatants；

2. T cell is resuspended in Opti-MEM, make its final concentration of 10^6~10^8 cell/mL；

3. 20~100ug Cas9/Cpf1 albumen and 20~100ug sgRNA/crRNA mixed at room temperature are incubated for 10 minutes；

4. albumen is mixed with sgRNA/crRNA mixture with 100ul cell suspension

5. carrying out electricity using electroporation to turn.The parameter of BTX-ECM830 is 200~500V, 100us~3ms.(electroporation packet Include BTX, Biorad, Lonza etc.)

6. after electroporation, cell is immediately placed in 2mL pre-temperature culture medium, and at 37 DEG C, 5%CO₂Culture or 32 DEG C, 5%CO₂Lower culture 1 day, then puts back to 37 DEG C, 5%CO₂Culture.

The gene knockout in primary T cells is realized by CRISPR

Since the TCR on allogeneic T cells can cause GvHD to react, while allogeneic T cells can be clear by host T cell identification It removes, present invention employs knock out TCR the and CD52 molecule on T cell surface simultaneously to prepare universal allogeneic T cells.TCR's strikes Except eliminating GvHD；The use of the antibody drug Alemtuzumab of CD52 removes the intracorporal T cell of host simultaneously, thus anti- Stop allogeneic T cells to be ostracised, and the allogeneic T cells that CD52 molecule knocks out are because resistant to Alemtuzumab can grow Period survival plays anti-tumor activity.

We carry out gene knockout to TCR α, the β chain on allogeneic T cells using CRISPR/Cas9 first, find TCR α, β The knockout of chain destroys the formation of the TCR/CD3 complex on T cell surface, and the editorial efficiency of TCR α, β chain has respectively reached 90% With 85% (Fig. 1 a).CRISPR/Cpf1 is a kind of novel gene editing system, and identification module (PAM) is different from Cas9's NGG, and it is TTTN, the analysis tool case of gene editing is expanded, provides possibility for operation AT enriched DNA fragments.To realize Gene editing in primary T cells, using Cpf1 to TCR α, β chain is cut for we, is knocked out efficiency and is respectively reached 82% and 80% (Fig. 1 b).

In order to produce the T cell resistant to antibody drug Alemtuzumab, the present invention simultaneously to allogeneic T cells into Row gene editing destroys the response molecule of its antibody drug to generate resistance T cell, and the production of universal T cell is realized with this. The CD52 gene knockout that Cas9 and Cpf1 is mediated has respectively reached 83% and 73.5% (Fig. 2).

Universal allogeneic T cells are constructed by CRISPR gene knockout

By the present invention in that being operated simultaneously to TCR the and CD52 gene in T cell with Cas9 and Cpf1, structure is realized Build universal allogeneic T cells purpose.

Double knockouts, the double negative cells group point of generation are carried out to TCR the and CD52 gene in T cell using Cas9 and Cpf1 Do not reach 40.1% and 48.3% (Fig. 3), this cell mass loses GvHD activity simultaneously and weakens to antibody drug Alemtuzumab Susceptibility.It is sorted by single TCR/CD3 feminine gender, TCR negative cells group reaches 99%.

Universal allosome CART/TCRT cell is constructed by CRISPR gene knockout

The present invention is by combining virus transfection external source CAR/TCR gene and using Cas9/Cpf1 simultaneously in CART cell TCR and CD52 gene operated, realize the universal allosome CART/TCRT cell purpose of building.

CAR/TCR transfection is carried out to primary T cells by integration virus, then using Cas9 and Cpf1 in T cell TCR and CD52 gene carries out double knockouts, and the double negative cells group of generation respectively reaches 35.3% and 35.2% (Fig. 4), this cell Group loses GvHD activity simultaneously and weakens the susceptibility to antibody drug Alemtuzumab.The T cell of the gene editing of generation CAR/TCR expression rate is respectively 86.5% and 90.1% (Fig. 5), can be used as universal CART/TCRT cell.

Use flow cytomery T cell surface gene expression

The monoclonal antibody and reagent used.BD Biosciences (San Jose, CA): APC-anti-CD3 (555335), (555366) FITC-anti-CD8, PE-anti-CD8 (555635), PE-anti-CD107 (555801), PE- Anti-B2m (551337), FITC-anti-HLA (555552)；Beckman Coulter (Pasadena, CA): PE-anti- Vb13.1(IM2021U).Data are obtained using BD LSR2, and by FlowJo version 7.6.1 (Tree Star, Inc.Ashland, OR) analysis.

The function assessment of universal CAR/TCRT cell is studied

In order to verify the function of universal CAR/TCRT cell, the present invention respectively to its targets neoplastic cells toxicity, and It is tested in terms of cytokine secretion.

The CAR/TCRT cell of the bis- knockouts of TCR and CD52 has the anti-tumor activity same with wild type CAR/TCRT； And secrete the cell factor IL2 and IFNr (Fig. 6,7) of peer-level.

CD107a staining procedure approximately as: in the RPMI culture medium of 96 orifice plates, with effector cell: T cell ratio is The plating cells of 1:1 (1 × 10^5 effector cell: 1 × 10^5 target).CD107 antibody is added, and plate is incubated at 37 DEG C It educates 1 hour, Golgi stop is then added, and in addition plate is incubated for 2.5~3 hours.Then addition CD8 and CD3 antibody, and 37 DEG C incubate 30 minutes.After incubation, cell is washed with FACS buffer solution, passes through flow cytometer showed.

Cell factor ELISA measurement approximately as: in the complete medium of the RPMI 1640 containing 10% fetal calf serum Middle washing target tumour cell, and suspended with 1 × 10^6 cell/ml.100 every kind of target cell types of μ l are added in duplicate In 96 hole round bottom plates.Washing effect T cell, and suspended in complete medium with 1 × 10^6 cell/ml, then will 100ul T cell is combined with the target cell in designation hole.Plate is incubated for 18 to 20 hours at 37 DEG C.After incubation, collect Supernatant simultaneously carries out ELISA measurement.

Cytotoxic T cell killing activity detection based on luciferase.By the tumour cell of targeted expression luciferase It washs in complete medium, and is resuspended with 1 × 10^5 cell/ml, then by the T of 100ul tumour cell and different proportion Cell (such as 30:1,15:1 etc.) is incubated with, and is cultivated 18 to 20 hours at 37 DEG C.After incubation, substrate is added to carefully In born of the same parents and measurement immediately shines, and calculated result.

It should be noted that above-described embodiment is only presently preferred embodiments of the present invention, there is no for the purpose of limiting the invention Protection scope, the equivalent substitution or substitution made on the basis of the above all belong to the scope of protection of the present invention.

Sequence table

<110>Nanjing Bei Heng Biotechnology Co., Ltd

<120>with the universal CART/TCRT cell and its construction method of antibody drug resistance

<160> 43

<170> SIPOSequenceListing 1.0

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<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

gagaatcaaa atcggtgaat 20

<210> 2

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gctggtacac ggcagggtca 20

<210> 3

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gtcagggttc tggatatctg 20

<210> 4

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

acaaaactgt gctagacatg 20

<210> 5

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<400> 5

cttcaagagc aacagtgctg 20

<210> 6

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<400> 6

tggaataatg ctgttgttga 20

<210> 7

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<400> 7

taggcagaca gacttgtcac 20

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<400> 8

tggatttaga gtctctcagc 20

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<400> 9

tctctcagct ggtacacggc 20

<210> 10

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<400> 10

agagtctctc agctggtaca 20

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gagcagccgc ctgagggtct 20

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gagctggtgg gtgaatggga 20

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gctgtcaagt ccagttctac 20

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ggctctcgga gaatgacgag 20

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<400> 15

ggagaatgac gagtggaccc 20

<210> 16

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<400> 16

ggcgctgacg atctgggtga 20

<210> 17

<211> 20

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<400> 17

gttgcggggg ttctgccaga 20

<210> 18

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<213>artificial sequence (Artificial Sequence)

<400> 18

gcagtatctg gagtcattga 20

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<211> 20

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<213>artificial sequence (Artificial Sequence)

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ggcacaccag tgtggccttt 20

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<400> 20

ggctcaaaca cagcgacctc 20

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<400> 21

cctactcacc atcagcctcc 20

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<400> 22

gcatccagca acataagcgg 20

<210> 23

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<400> 23

gttatggtac agatacaaac 20

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<400> 24

caccatcagc ctcctggtta 20

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gttatggtac agatacaaac 20

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gagtctctca gctggtacac ggc 23

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<400> 27

catgtgcaaa cgccttcaac aac 23

<210> 28

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<400> 28

cacatgcaaa gtcagatttg ttg 23

<210> 29

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<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 29

ttgctccagg ccacagcact gtt 23

<210> 30

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<400> 30

tctgtgatat acacatcaga atc 23

<210> 31

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<212> DNA

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<400> 31

tgacacattt gtttgagaat caa 23

<210> 32

<211> 23

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<400> 32

tttgagaatc aaaatcggtg aat 23

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<400> 33

agaatcaaaa tcggtgaata ggc 23

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<213>artificial sequence (Artificial Sequence)

<400> 34

attctcaaac aaatgtgtca caa 23

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<400> 35

ggtgtgggag atctctgctt ctg 23

<210> 36

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<400> 36

cctcggtgtc ctaccagcaa ggg 23

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<211> 23

<212> DNA

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<400> 37

gccctatcct gggtccactc gtc 23

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<400> 38

agccatcaga agcagagatc tcc 23

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gctggtgtcg ttttgtcctg aga 23

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<400> 40

tcctgagagt ccagtttgta tct 23

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tatctgtacc ataaccagga ggc 23

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<400> 42

cttttcttcg tggccaatgc cat 23

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ttcgtggcca atgccataat cca 23

Claims

1. the construction method of the universal CART or TCRT cell with antibody drug resistance, which is characterized in that described universal CART or TCRT cell is the allogeneic T cells with Chimeric antigen receptor or T cell receptor, and the universal CART or TCRT is thin T cell receptor α, β chain and CD52 molecule of born of the same parents is knocked；

Described knock out uses CRISPR technology；

SgRNA and crRNA sequence used in the CRISPR technology are as follows:

SgRNA-TCR α is selected from following sequence:

SgRNA-TCR α -2:GCTGGTACACGGCAGGGTCA(SEQ ID NO.2)

SgRNA-TCR α -3:GTCAGGGTTCTGGATATCTG(SEQ ID NO.3)

SgRNA-TCR α -8:TGGATTTAGAGTCTCTCAGC(SEQ ID NO.8)

SgRNA-TCR α -9:TCTCTCAGCTGGTACACGGC(SEQ ID NO.9)

SgRNA-TCR α -10:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)；

SgRNA-TCR β is selected from following sequence:

SgRNA-TCR β -1:GAGCAGCCGCCTGAGGGTCT(SEQ ID NO.11)

SgRNA-TCR β -2:GAGCTGGTGGGTGAATGGGA(SEQ ID NO.12)

SgRNA-TCR β -3:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

SgRNA-TCR β -4:GGCTCTCGGAGAATGACGAG(SEQ ID NO.14)

SgRNA-TCR β -5:GGAGAATGACGAGTGGACCC(SEQ ID NO.15)

SgRNA-TCR β -6:GGCGCTGACGATCTGGGTGA(SEQ ID NO.16)

SgRNA-TCR β -7:GTTGCGGGGGTTCTGCCAGA(SEQ ID NO.17)

SgRNA-TCR β -8:GCAGTATCTGGAGTCATTGA(SEQ ID NO.18)

SgRNA-TCR β -10:GGCTCAAACACAGCGACCTC(SEQ ID NO.20)；

SgRNA-CD52 is selected from following sequence:

SgRNA-CD52-1:CCTACTCACCATCAGCCTCC(SEQ ID NO.21)

SgRNA-CD52-2:GCATCCAGCAACATAAGCGG(SEQ ID NO.22)

SgRNA-CD52-3:GTTATGGTACAGATACAAAC(SEQ ID NO.23)

SgRNA-CD52-4:CACCATCAGCCTCCTGGTTA(SEQ ID NO.24)

SgRNA-CD52-5:GTTATGGTACAGATACAAAC(SEQ ID NO.25)；

CrRNA-TCR α is following sequence:

CrRNA-TCR α -9:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.34)；

CrRNA-TCR β is selected from following sequence:

CrRNA-TCR β -3:GCCCTATCCTGGGTCCACTCGTC(SEQ ID NO.37)

CrRNA-TCR β -4:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.38)；

CrRNA-CD52 is selected from following sequence:

CrRNA-CD52-2:TCCTGAGAGTCCAGTTTGTATCT(SEQ ID NO.40)

CrRNA-CD52-3:TATCTGTACCATAACCAGGAGGC(SEQ ID NO.41).

2. construction method as described in claim 1, which is characterized in that the Chimeric antigen receptor be targeting CD19, CD20, CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、AFP、Alpha folate receptor、CEA、PSCA , PSMA, Her2, EGFR, IL13R α 2, GD2, NKG2D, EGFRvIII, CS1, BCMA or Mesothelin chimeric antigen by Body.

3. construction method as described in claim 1, which is characterized in that the T cell receptor is targeting HBV, HPV E6, NY- The T cell of ESO, mNY-ESO, WT1, MART-1, MAGE-A3, MAGE-A4, P53, Thyroglobulin or Tyrosinase by Body.

4. construction method as described in claim 1, which is characterized in that using sgRNA and crRNA used in CRISPR technology Sequence are as follows:

α: AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10 of sgRNA-TCR)

β: GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13 of sgRNA-TCR)

SgRNA-CD52:CACCATCAGCCTCCTGGTTA(SEQ ID NO.24)

α: ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.34 of crRNA-TCR)

β: AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.38 of crRNA-TCR)

CrRNA-CD52: TATCTGTACCATAACCAGGAGGC(SEQ ID NO.41).