CN107828730A - Universal CART/TCRT cells and its construction method with antibody drug resistance - Google Patents
Universal CART/TCRT cells and its construction method with antibody drug resistance Download PDFInfo
- Publication number
- CN107828730A CN107828730A CN201711082916.6A CN201711082916A CN107828730A CN 107828730 A CN107828730 A CN 107828730A CN 201711082916 A CN201711082916 A CN 201711082916A CN 107828730 A CN107828730 A CN 107828730A
- Authority
- CN
- China
- Prior art keywords
- seq
- sgrna
- crrna
- tcrα
- tcrβ
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70592—CD52
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Abstract
The invention belongs to genetic engineering and synthetic biology field, more particularly to a kind of universal CART/TCRT cells and its construction method with antibody drug resistance, the universal CART/TCRT cells are the allogeneic T cells for having Chimeric antigen receptor and φt cell receptor, and φt cell receptor α, β chain and CD52 molecules are knocked；The knockout technology of φt cell receptor α, β chain and CD52 molecules is CRISPR technologies；The universal CART/TCRT cells had both remained the targeting of tumour specific antigen, the problem of eliminating GvHD again and repelling, and weakening to antibody drug Alemtuzumab susceptibility, can be as a kind of universal simple, low cost, high activity CART/TCRT cell preparations use.
Description
Technical field
The invention belongs to genetic engineering and synthetic biology field, and in particular to a kind of general with antibody drug resistance
Type CART/TCRT cells and its construction method.
Background technology
Chimeric antigen receptor T cell (CART) is one of tumour immunotherapy most promising at present, and it is substantially former
Reason mainly extract patient itself T cell, and by gene and cell engineering means make its expression specificity chimeric antigen by
Body, enable to identify and with reference to tumor cell surface antigen, so as to play a part of target killing tumor cell.CAR-T exists
The effect of notable, on August 30th, 2017, the food sanitation safe committee of U.S. FDA a surnames are obtained in leukaemia and lymphoma treating
Cloth ratifies Novartis CAR-T cell therapy official listings, for treating recurrent or the leaching of intractable children and adolescents B- cell acutes
Bar chronic myeloid leukemia (rALL), its trade name Kymriah (tisagenlecleucel).ALL is 15
25% is accounted in year following childhood cancer confirmed cases, is the most common children with cancer in the U.S., acute lymphocytic is white in China
Blood disease accounts for the 80% of acute leukemia, and the incidence of disease is ten a ten thousandths.Also adult can be betided, it is white to account for all adults
The 20% of the blood disease incidence of disease.
Estimated according to National Cancer Institute, there are about patient of 3100 ages below 20 years old every year and be diagnosed as
This disease, due to the population base that China is huge, there are about 10000 children and youth to suffer from acute leukemia every year.Effectively
Therapeutic choice it is extremely limited, in multiple relapse or intractable B- cell Acute Lymphoblastic Leukemias children and adolescent patient
In, disease-free survival rate is less than 10%-30% within 5 years.Kymriah security and validity obtains in multi-center clinical trial
Confirm, it is 83% to treat the overall relief rate in three months, it is shown that the effect of its is remarkable.
Except B cell ALL, CAR-T is in relapsed or stubborn chronic lymphocytic leukemia
(rCLL), also achieved in the treatment of recurrent or Refractory Multiple Myeloma (rNHL) and NHL (NHL)
The effect of notable.Diffusivity large B cell lymphoid tumor (DLBCL), conversion follicular lymphoma (TFL), Yi Jiyuan are treated at one
Hair property mediastinal B-cell lymphoma (PMBCL) these three NHLs, in entitled ZUMA-1 2 clinical trial phases, Kite
CAR-T therapies reached Major Clinical terminal on objective remission rate (objective response rate).Receiving list
After secondary axicabtagene ciloleucel infusion, there is up to 82% patient to occur alleviating.It it is 8.7 in median
In the follow-up of the moon, the patient for having 44% is still in the paracmasis, and the patient for having 39% is in complete incidence graph.In Bluebird
The key name that Bio companies are carried out is 100% patient in two dosage groups in the experiment of b2121 treatment Huppert's disease
(n=6) objective reaction is realized；Two patients are negative in MRD；General reaction rate (ORR) is 78%.6 and during follow-up in 4 months,
Two patients reach the complete reaction (CR) of stricti jurise.
In addition to CART, φt cell receptor (TCR) T cell therapy also achieves considerable progress.TCRT cell technologies are earliest
From tumor-infiltrated T cell (TIL), this kind of T cell " would generally invade " cancerous tissue, and this illustrates that they have necessarily to cancer cell
Recognition capability.Fact-finding TIL has special to cancer cell related antigen (Tumor Associated Antigen, TAA)
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 relative particularity in different cancers, thus also turns into the object of attack of immune system.Nevertheless,
These natural TIL recognition capability is generally weaker, therefore can not form the favourable attack to cancer cell.In this case, may be used
Modification is oriented to reach the purpose of attack tumour to import TIL specific TCR by external source to T cell.2016《From
So-medical science》The exciting TCR treatment cases of magazine ran numerical example, this clinical test cured by Univ Maryland-Coll Park USA
A entitled " the TCR " of gene modification of institute, medical college of University of Pennsylvania and immunization therapy company Adaptimmune cooperative research and development
Immunotherapeutic.After it have modified several key amino acids, these genes modification TCR substantially increase it is normal with one kind
The cancer TAA, NY-ESO-1 that see affinity, and then the cancer to NY-ESO-1 overexpressions is significantly improved, such as it is multiple
The therapeutic effect of property myeloma (Multiple Myeloma).In current clinical test, 80% multiple myeloma patients
Good clinical response is generated, wherein 70% patient reaches completely or nearly complete response, average progression free survival phase reaches
By 19 months.The treatment for other cancers of NY-ESO-1 overexpressions is being carried out successively at present.
But because CART/TCRT is a kind of personalized method of height, 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 the T cell obtained also can be different.This individualized treatment mode consumes substantial amounts of people
Power material resources, so price is high.The CART cell therapy products that the Kymriah of Novartis ratifies as first FDA, it is priced at
47.5 ten thousand U.S. dollars, the medical expense of such great number, some patient families can be caused to be difficult to bear, while also greatly limit its city
Field application.
The CART/TCRT first echelons including Novartis, Kite pharmacy, Juno are using autotransplantation side at present
Method prepares CART/TCRT cells, and carrying out treatment using allogeneic T cells preparation CART/TCRT cells provides another thinking, but
The row for being due to donor T-cells to the graft-versus-host reaction (GvHD) of host and host immune system to allogeneic T cells
Reprimand, significantly limit its application.
The TCR of CART cell surfaces is knocked out by way of gene editing can eliminate because allogeneic T cells are to host's
Identification and caused by GvHD, be used to prepare universal CART cells to a certain extent.Currently used gene editing instrument
Mainly there are TALEN and CRISPR.
TAL effectors (TAL effector, TALE) are most early in phytopathogen Xanthomonas campestris (Xanthomonas
Sp.) it is found in the invasion and attack infection research to plant.Because TALE has the specific binding capacity of DNA sequence dna, follow-up study
FokI DNA nucleases 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 domain,
The central domain of the series connection TALE repetitive sequences of recognizable specific dna sequence, there is the C-terminal knot of FokI endonuclease functions
Structure domain forms.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.
Substantial amounts of molecular cloning and sequencing procedures are needed due to single TALEN modules are carried out into assembling, it is very cumbersome, and
And because TALEN structure needs to carry out cumbersome and complicated protein engineering design and optimization according to target dna sequence, so
It just substantially prolongs the experimental period of structure TALEN elements.Simultaneously and because TALEN knockout efficiency is influenceed by DNA sequence dna
It is larger, and by these many protein deliveries into the cell for multiple genetic operation simultaneously right and wrong often with challenging,
Therefore it is very big to limit its application.
Except above-mentioned technological difficulties, because shearings of the TALEN to DNA needs two FokI cutting domains to form dimerization
Body, and need at least one identification domain to combine targeting DNA.Although DNA identifies domain with stronger sequence specific
Property, but because TALEN shear history not fully relies on the formation of homodimer, so once forming heterodimeric
Body, just it is likely to cause effect of missing the target, and DNA mispairing and sequence may finally be caused to change, produces stronger cytotoxicity.
When these harmful effects accumulation is excessive, during the scope born more than cellular repair mechanisms, the apoptosis of cell will be caused.It is all above
More limitations cause TALEN to be difficult to popularization and application.
CRISPR/Cas systems as a kind of newest genome edit tool, know by the specific DNA that can complete RNA guiding
Not and edit.CRISPR/Cas technologies use the guide RNA molecule (sequence-specific of one section of sequence-specific
Guide RNA) guiding endonuclease is at target spot, so as to complete the editor of genome.The exploitation of CRISPR/Cas systems is
Build more efficient gene site-directed modification technique and provide brand-new platform.
CRISPR/Cas technologies, which have been broken away from, synthesizes and assembles the cumbersome behaviour with specific DNA recognition capability module
Make, its gRNA design and synthetic work amount are far smaller than the building process of the DNA identification modules of TALEN and ZFN technologies, and poison
Property is well below ZFN technologies.2013《Science》Magazine has delivered two respectively from the significant of MIT and Harvard
CRISPR technical papers, independently completed first using CRISPR technologies to human genome editor.Due to CRISPR
The easy and efficient characteristic of system, its application have obtained great popularization.Except gene knockout, CRISPR systems are engineered
More extensive field is applied to, including gene suppresses, gene activation, gene simple point modification, epigenetic modification, light science of heredity
Field, the CRISPR very big research range that must have expanded biology of use.
Huge applications prospect and possible commercial interest due to CRISPR gene editing technologies, foreign countries are with the technology at present
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 field such as disease and hematologic disease.
CAR-T cells (Chimeric antigen receptor-T) are current as immune cell therapy treatment tumour
One of tumour immunotherapy hot research field.PD1 (Programmed death 1) be used as a kind of immunosuppression molecule, into
For effective target molecule of important oncotherapy, 2016 using CRISPR technologies studies have reported that knock out the PD1 on CART cells
Molecule, therapeutic effect of the CART cells to solid tumor can be significantly improved.Research report in 2017, using CRISPR technologies, into
Work(builds the CAR-T cells of the gene site-directed insertion CAR genes of mouse TRAC, can significantly extend the modification mouse survival time.
Cellectis companies knock out the intracellular TCRa and CD52 molecules of CART using TALEN technologies, have been applied to face
Bed research, but because TALEN that they the use T cell edited all generates serious chromosomal shift phenomenon, this is to it
Clinical practice is caused greatly into knurl risk, but the high degree of specificity due to CRISPR in T cell, is compiled using CRISPR
The universal CART cells collected carry out oncotherapy, have very big advantage.Simultaneously because TALEN is to the poorly efficient of CART cells
Rate editor, purifying can be carried out to the CART cells of gene editing and causes difficulty, while can be because the separation of TCR positive cells is not clean
And increase GvHD risk.Our invention causes CRISPR efficiently to edit CART cells, largely favorably
In the production of universal CART cells.
Individually knockout TCR α, β chains can not generate universal CART cells, because this CART cells still have
HLA molecules are in cell surface expression, so as to be identified and repelled by recipient immune system T cell, it is difficult to reach the purpose for the treatment of.Together
When edit TCR α, β chains and suppress molecule, such as PD1, TIM3, CTLA4 can not produce universal CART cells.
The content of the invention
The present invention solves above-mentioned technical problem present in prior art, there is provided a kind of general with antibody drug resistance
Type CART/TCRT cells and its construction method.
To solve the above problems, technical scheme is as follows：
Universal CART/TCRT cells with antibody drug resistance, the universal CART/TCRT cells be have it is embedding
The allogeneic T cells of antigen receptor and φt cell receptor are closed, φt cell receptor α, β chain and CD52 molecules 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,
GD2, NKG2D, EGFRvIII, CS1, BCMA, Mesothelin Chimeric antigen receptor.
Preferably, the φt cell receptor is targeting HBV, HPV E6, NY-ESO, mNY-ESO, WT1, MART-1, MAGE-
A3, MAGE-A4, P53, Thyroglobulin, Tyrosinase φt cell receptor.
The construction method of universal CART/TCRT cells with antibody drug resistance, using CRISPR technologies in allosome T
φt cell receptor α, β chain and CD52 molecules on intracellular specific knockdown T cell surface.
Preferably, use sgRNA and crRNA sequences used in CRISPR technologies for：
SgRNA-TCR α are 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 are 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 are 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 are 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)
Preferably, use sgRNA and crRNA sequences used in CRISPR technologies for：
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)。
Relative to prior art, advantages of the present invention is as follows,
The universal CART/TCRT cells with antibody drug resistance of the present invention realize the progress of TCR and CD52 molecules
Double knockouts, had both remained the targeting of tumour specific antigen, and TCR knockout eliminates GvHD；CD52 antibody drug simultaneously
Alemtuzumab use removes the T cell in host, and so as to prevent allogeneic T cells to be ostracised, and CD52 molecules strike
The allogeneic T cells removed are because can over a long time survive resistant to Alemtuzumab plays anti-tumor activity；Can conduct
A kind of universal simple, low cost, the use of high activity CART/TCRT cell preparations.
The present invention the universal CART/TCRT cells with antibody drug resistance construction method, using Cas9 with
TCR and CD52 genes in Cpf1 Technique on T cells carry out double knockouts, and caused double negative cells group respectively reaches 40.1% He
48.3%, this cell mass loses GvHD activity and weakens the susceptibility to antibody drug Alemtuzumab simultaneously；Pass through single
The negative sortings of TCR/CD3, TCR negative cells groups reach 99%.
The present invention edits the intracellular TCR and CD52 molecules of CART using CRISPR, can produce without GvHD, but simultaneously
With the CART cells to CD52 antibody drug Alemtuzumab resistances；When being treated to patient, use
The T cell that Alemtuzumab removes patient's body prevents rejection, and CD52 molecules knockout has Alemtuzumab resistances
Universal CART cells do not cause GvHD while tumour is treated, so as to greatly reduce into production and treatment cost.
Brief description of the drawings
The primary T cells surface TCR/CD3 compounds of Fig. 1 .Cas9 and Cpf1 mediation knock out；1a is situated between for CRISPR/Cas9
The TCR/CD3 compounds led knock out, and 1b is that the TCR/CD3 compounds of CRISPR/Cpf1 mediations knock out.
The primary T cells surface C D52 molecules of Fig. 2 .Cas9 and Cpf1 mediation knock out.
The primary T cells surface TCR/CD52 molecules of Fig. 3 .Cas9 and Cpf1 mediation are double to be knocked out；3a is situated between for CRISPR/Cas9
The double knockouts of TCR/CD52 molecules led, 3b, which is that the TCR/CD52 molecules of CRISPR/Cpf1 mediations are double, to be knocked out.
The CART cell surface TCR/CD52 molecules of Fig. 4 .Cas9 and Cpf1 mediation are double to be knocked out.
Fig. 5 .TCR/CD52 are double to knock out CART/TCRT gene expression detections；5aTCR/CD52 is double to knock out CART gene expressions,
5b, which is that TCR/CD52 is double, knocks out TCRT gene expressions.
Fig. 6 .TCR/CD52 are double to knock out CART/TCRT cell cytokine secretions；6a is that the double knockout CART of TCR/CD52 are thin
Born of the same parents' cytokine secretion, 6b, which is that TCR/CD52 is double, knocks out TCRT cell cytokine secretions.
Fig. 7 .TCR/CD52 are double to knock out the detection of CART/TCRT cell tumours killing activity；7a, which is that TCR/CD52 is double, to be knocked out
The anti-tumor activity detection of CART cells, 7b are the double anti-tumor activity detections for knocking out TCRT cells of TCR/CD52.
Fig. 8 are using different sgRNA and crRNA to T cell TCR α, TCR β and the editorial efficiency of CD52 molecules.
Embodiment
Embodiment 1：
Primary T cells expand.Primary human T-Cells are being contained into 10%FBS, 100U/ml penicillin, 100 μ g/ml sulfate chains
Mycin, cultivate in 10mM Hepes RPMI 1640, and using coupling CD3/CD28 antibody magnetic bead according to 1：3 ratio is entered
Line activating stimulates.Cell is counted, every 2 days addition culture mediums, T is collected on appropriate opportunity by cell size and growth parameter(s)
Cell, carry out functional examination or freezen protective.
CRISPR design and implementation
Sequence in sgRNA and crRNA targeting TCR α constant region exons 1, TCR β constant regions 1 and 2 exons 1s share
Sequence, CD52 exons 1 sequence, and be cloned into the carrier containing T7 promoters.These plasmids restriction enzyme line
Property.Transcribed using MEGAscript T7Transcription kits (Life Technologies, Carlsbad, CA)
SgRNA/crRNA, RNA is stored in -80 DEG C.
SgRNA the and crRNA sequences of Select to use of the present invention are as follows：
SgRNA-TCR α are 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 are 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 are 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 are 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, sgRNA the and crRNA sequences preferably used are 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：First, T cell was at the 0th day, by being coupled CD3/
The magnetic bead of CD28 antibody enters line activating, and in the transfection that the 2nd~5 day carries out CRISPR, gene editing was collected at the 9th to the 11st day
T cell；
For the gene editing of CART/TCRT cells, in addition to except the 1st day plus virus carried out CAR/TCR transfections to T cell, its
He is same as above step.
The operating procedure of electroporation method approximately as：
1. using Opti-MEM cleaning T cells 3 times, each 300g, centrifuge 10 minutes and remove supernatant；
2. T cell is resuspended in Opti-MEM, make its final concentration of 10^6~10^8 cells/mL；
3. 20~100ug Cas9/Cpf1 albumen and 20~100ug sgRNA/crRNA mixed at room temperature are incubated 10 minutes；
4. albumen is mixed with sgRNA/crRNA mixtures with 100ul cell suspensions
5. carry out electricity using electroporation to turn.BTX-ECM830 parameter is 200~500V, 100us~3ms.(electroporation bag
Include BTX, Biorad, Lonza etc.)
6. after electroporation, cell is immediately placed in 2mL pre-temperature culture mediums, 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
Because the TCR on allogeneic T cells can cause GvHD to react, while allogeneic T cells can be clear by host T cell identification
Remove, present invention employs TCR the and CD52 molecules for knocking out T cell surface simultaneously to prepare universal allogeneic T cells.TCR's strikes
Except eliminating GvHD；CD52 antibody drug Alemtuzumab use removes the T cell in host simultaneously, so as to anti-
Stop allogeneic T cells to be ostracised, and the allogeneic T cells that CD52 molecules knock out are because resistant to Alemtuzumab can grow
Period survival plays anti-tumor activity.
We carry out gene knockout using CRISPR/Cas9 to TCR α, the β chain on allogeneic T cells first, find TCR α, β
The knockout of chain destroys the formation of the TCR/CD3 complexs on T cell surface, and the editorial efficiency of TCR α, β chains has respectively reached 90%
With 85% (Fig. 1 a).CRISPR/Cpf1 is a kind of new gene editing system, and its identification module (PAM) is different from Cas9's
NGG, and it is TTTN, the analysis tool case of gene editing is expanded, possibility is provided for operation AT enriched DNA fragments.To realize
Gene editing in primary T cells, we are cut using Cpf1 to TCR α, β chains, and it knocks out efficiency and respectively reached
82% and 80% (Fig. 1 b).
In order to produce the T cell resistant to antibody drug Alemtuzumab, the present invention enters to allogeneic T cells simultaneously
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 knockouts of Cas9 and Cpf1 mediations have respectively reached 83% and 73.5% (Fig. 2).
Universal allogeneic T cells are built by CRISPR gene knockouts
By the present invention in that operated with Cas9 and Cpf1 to TCR the and CD52 genes in T cell simultaneously, structure is realized
Build universal allogeneic T cells purpose.
Double knockouts, caused double negative cells group point are carried out to TCR the and CD52 genes in T cell using Cas9 and Cpf1
Do not reach 40.1% and 48.3% (Fig. 3), this cell mass loses GvHD activity and weakened to antibody drug Alemtuzumab simultaneously
Susceptibility.Sorted by the way that single TCR/CD3 is negative, TCR negative cells groups reach 99%.
Universal allosome CART/TCRT cells are built by CRISPR gene knockouts
The present invention is by combining virus transfection external source CAR/TCR genes and using Cas9/Cpf1 simultaneously in CART cells
TCR and CD52 genes operated, realize the universal allosome CART/TCRT cell purposes of structure.
CAR/TCR transfections are carried out to primary T cells by integrating virus, then using Cas9 and Cpf1 in T cell
TCR and CD52 genes carry out double knockouts, and caused double negative cells group respectively reaches 35.3% and 35.2% (Fig. 4), this cell
Group loses GvHD activity and weakens the susceptibility to antibody drug Alemtuzumab simultaneously.The T cell of caused gene editing
CAR/TCR expression rates are respectively 86.5% and 90.1% (Fig. 5), can be used as universal CART/TCRT cells.
Use flow cytomery T cell surface gene expression
The monoclonal antibody and reagent used.BD Biosciences (San Jose, CA)：APC-anti-CD3
(555335), FITC-anti-CD8 (555366), 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 versions 7.6.1 (Tree Star,
Inc.Ashland, OR) analysis.
The function assessment research of universal CAR/TCRT cells
It is of the invention respectively to its targets neoplastic cells toxicity in order to verify the function of universal CAR/TCRT cells, and
Tested in terms of cytokine secretion.
The double CAR/TCRT cells knocked out of TCR and CD52 have the anti-tumor activity equal with wild type CAR/TCRT；
And secrete the cell factor IL2 and IFNr (Fig. 6,7) of peer-level.
CD107a staining procedures approximately as：In the RPMI culture mediums of 96 orifice plates, with effector cell：T cell ratio is
1：1 (1 × 10^5 effector cell:1 × 10^5 target) plating cells.CD107 antibody is added, and plate is incubated at 37 DEG C
Educate 1 hour, then add Golgi stop, and plate is incubated in addition 2.5~3 hours.Then addition CD8 and CD3 antibody, and
37 DEG C incubate 30 minutes.After incubation terminates, cell is washed with FACS buffer solution, passes through flow cytometer showed.
Cell factor ELISA measure approximately as：In the complete medium of the RPMI 1640 containing 10% hyclone
Middle washing target tumour cell, and suspended with 1 × 10^6 cell/ml.The 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 cells combine with the target cell in designation hole.Plate is incubated 18 to 20 hours at 37 DEG C.After incubation terminates, collect
Supernatant simultaneously carries out ELISA measure.
Cytotoxic T cell killing activity detection based on luciferase.By the tumour cell of targeted expression luciferase
Wash in complete medium, and be resuspended with 1 × 10^5 cell/ml, then by 100ul tumour cells and the T of different proportion
Cell (such as 30:1,15:1 etc.) it is incubated, is cultivated 18 to 20 hours at 37 DEG C together.After incubation terminates, substrate is added to carefully
In born of the same parents and measure is luminous immediately, and result of calculation.
It should be noted that above-described embodiment is only presently preferred embodiments of the present invention, it is not used for limiting the present invention's
Protection domain, the equivalent substitution or replacement made on the basis of the above belong to protection scope of the present invention.
Sequence table
<110>Nanjing Bei Heng bio tech ltd
<120>Universal CART/TCRT cells and its construction method with antibody drug resistance
<160> 43
<170> SIPOSequenceListing 1.0
<210> 1
<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
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
cttcaagagc aacagtgctg 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
tggaataatg ctgttgttga 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
taggcagaca gacttgtcac 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
tggatttaga gtctctcagc 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
tctctcagct ggtacacggc 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
agagtctctc agctggtaca 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gagcagccgc ctgagggtct 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
gagctggtgg gtgaatggga 20
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
gctgtcaagt ccagttctac 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
ggctctcgga gaatgacgag 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
ggagaatgac gagtggaccc 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ggcgctgacg atctgggtga 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
gttgcggggg ttctgccaga 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
gcagtatctg gagtcattga 20
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
ggcacaccag tgtggccttt 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
ggctcaaaca cagcgacctc 20
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
cctactcacc atcagcctcc 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
gcatccagca acataagcgg 20
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
gttatggtac agatacaaac 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
caccatcagc ctcctggtta 20
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
gttatggtac agatacaaac 20
<210> 26
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
gagtctctca gctggtacac ggc 23
<210> 27
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
catgtgcaaa cgccttcaac aac 23
<210> 28
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
cacatgcaaa gtcagatttg ttg 23
<210> 29
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
ttgctccagg ccacagcact gtt 23
<210> 30
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
tctgtgatat acacatcaga atc 23
<210> 31
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
tgacacattt gtttgagaat caa 23
<210> 32
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
tttgagaatc aaaatcggtg aat 23
<210> 33
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
agaatcaaaa tcggtgaata ggc 23
<210> 34
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
attctcaaac aaatgtgtca caa 23
<210> 35
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
ggtgtgggag atctctgctt ctg 23
<210> 36
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
cctcggtgtc ctaccagcaa ggg 23
<210> 37
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
gccctatcct gggtccactc gtc 23
<210> 38
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
agccatcaga agcagagatc tcc 23
<210> 39
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 39
gctggtgtcg ttttgtcctg aga 23
<210> 40
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 40
tcctgagagt ccagtttgta tct 23
<210> 41
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 41
tatctgtacc ataaccagga ggc 23
<210> 42
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 42
cttttcttcg tggccaatgc cat 23
<210> 43
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 43
ttcgtggcca atgccataat cca 23
Claims (6)
1. the universal CART/TCRT cells with antibody drug resistance, it is characterised in that the universal CART/TCRT is thin
Born of the same parents are the allogeneic T cells for having Chimeric antigen receptor and φt cell receptor, and φt cell receptor α, β chain and CD52 molecules are struck
Remove.
2. universal CART/TCRT cells as claimed in claim 1, it is characterised in that 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, GD2, NKG2D, EGFRvIII, CS1, BCMA,
Mesothelin Chimeric antigen receptor.
3. universal CART/TCRT cells as claimed in claim 1, it is characterised in that the φt cell receptor is targeting HBV,
HPV E6, NY-ESO, mNY-ESO, WT1, MART-1, MAGE-A3, MAGE-A4, P53, Thyroglobulin, Tyrosinase
φt cell receptor.
4. the construction method of the universal CART/TCRT cells with antibody drug resistance, its feature as described in claim 1-3
It is, the knockout technology of φt cell receptor α, β chain and CD52 molecules is CRISPR technologies.
5. construction method as claimed in claim 4, it is characterised in that using sgRNA and crRNA used in CRISPR technologies
Sequence is：
SgRNA-TCR α are 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 are 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 are 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 are 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)。
6. construction method as claimed in claim 4, it is characterised in that using sgRNA and crRNA used in CRISPR technologies
Sequence is：
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)。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711082916.6A CN107828730B (en) | 2017-11-07 | 2017-11-07 | Universal CART/TCRT cell and its construction method with antibody drug resistance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711082916.6A CN107828730B (en) | 2017-11-07 | 2017-11-07 | Universal CART/TCRT cell and its construction method with antibody drug resistance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107828730A true CN107828730A (en) | 2018-03-23 |
| CN107828730B CN107828730B (en) | 2019-04-30 |
Family
ID=61653837
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711082916.6A Active CN107828730B (en) | 2017-11-07 | 2017-11-07 | Universal CART/TCRT cell and its construction method with antibody drug resistance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107828730B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109722437A (en) * | 2018-12-29 | 2019-05-07 | 广州百暨基因科技有限公司 | A kind of universal CAR-T cell and its preparation method and application |
| WO2019210280A1 (en) * | 2018-04-27 | 2019-10-31 | Casebia Therapeutics Limited Liability Partnership | Anti-bcma car-t-cells for plasma cell depletion |
| CN111218448A (en) * | 2020-04-23 | 2020-06-02 | 南京北恒生物科技有限公司 | Preparation and use of engineered immune cells |
| WO2020259707A1 (en) * | 2019-06-28 | 2020-12-30 | 科济生物医药（上海）有限公司 | Cell for resisting transplant reaction and method |
| CN113490502A (en) * | 2019-03-21 | 2021-10-08 | 艾洛基治疗公司 | Methods for increasing the efficiency of TCR α β + cell depletion |
| WO2022012591A1 (en) * | 2020-07-15 | 2022-01-20 | 南京北恒生物科技有限公司 | Engineered immune cell for allotransplantation |
| CN114729028A (en) * | 2019-08-29 | 2022-07-08 | 克莱格医学有限公司 | Cells and methods for resisting transplant response |
| WO2023025207A1 (en) * | 2021-08-24 | 2023-03-02 | 赛斯尔擎生物技术（上海）有限公司 | T cell product and use thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022215982A1 (en) * | 2021-04-05 | 2022-10-13 | 주식회사 셀렌진 | Guide rna complementary to trac gene and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647871A (en) * | 2016-01-27 | 2016-06-08 | 苏州佰通生物科技有限公司 | Chimeric antigen receptor T cell capable of conducting allograft and preparation method |
| CN106191062A (en) * | 2016-07-18 | 2016-12-07 | 广东华南联合疫苗开发院有限公司 | A kind of TCR/PD 1 double negative t cells and construction method thereof |
-
2017
- 2017-11-07 CN CN201711082916.6A patent/CN107828730B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647871A (en) * | 2016-01-27 | 2016-06-08 | 苏州佰通生物科技有限公司 | Chimeric antigen receptor T cell capable of conducting allograft and preparation method |
| CN106191062A (en) * | 2016-07-18 | 2016-12-07 | 广东华南联合疫苗开发院有限公司 | A kind of TCR/PD 1 double negative t cells and construction method thereof |
Non-Patent Citations (2)
| Title |
|---|
| LAURENT POIROT,ET AL.: "Multiplex genome edited T-cell manufacturing platform for"off-the-shelf", 《AMERICAN ASSOCIATION FOR CANCER RESEARCH》 * |
| MARK J OSBORN1,ET AL.: "Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases", 《THE AMERICAN SOCIETY OF GENE & CELL THERAPY》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019210280A1 (en) * | 2018-04-27 | 2019-10-31 | Casebia Therapeutics Limited Liability Partnership | Anti-bcma car-t-cells for plasma cell depletion |
| CN112512557A (en) * | 2018-04-27 | 2021-03-16 | 克里斯珀医疗股份公司 | anti-BCMA CAR-T-cells for plasma cell depletion |
| CN109722437A (en) * | 2018-12-29 | 2019-05-07 | 广州百暨基因科技有限公司 | A kind of universal CAR-T cell and its preparation method and application |
| CN109722437B (en) * | 2018-12-29 | 2020-01-07 | 广州百暨基因科技有限公司 | Universal CAR-T cell and preparation method and application thereof |
| CN113490502A (en) * | 2019-03-21 | 2021-10-08 | 艾洛基治疗公司 | Methods for increasing the efficiency of TCR α β + cell depletion |
| WO2020259707A1 (en) * | 2019-06-28 | 2020-12-30 | 科济生物医药（上海）有限公司 | Cell for resisting transplant reaction and method |
| EP3992204A4 (en) * | 2019-06-28 | 2023-09-27 | CRAGE medical Co., Limited | Cell for resisting transplant reaction and method |
| CN114729028A (en) * | 2019-08-29 | 2022-07-08 | 克莱格医学有限公司 | Cells and methods for resisting transplant response |
| CN111218448A (en) * | 2020-04-23 | 2020-06-02 | 南京北恒生物科技有限公司 | Preparation and use of engineered immune cells |
| WO2022012591A1 (en) * | 2020-07-15 | 2022-01-20 | 南京北恒生物科技有限公司 | Engineered immune cell for allotransplantation |
| WO2023025207A1 (en) * | 2021-08-24 | 2023-03-02 | 赛斯尔擎生物技术（上海）有限公司 | T cell product and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107828730B (en) | 2019-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107746831B (en) | Universal CART/TCRT cell and its construction method with chemotherapeutic drug resistance | |
| CN107828730B (en) | Universal CART/TCRT cell and its construction method with antibody drug resistance | |
| AU2015249655B2 (en) | Chimeric antigen receptors (CAR) for use in therapy and methods for making the same | |
| JP7190096B2 (en) | Gene-edited T cells and uses thereof | |
| JP7142571B2 (en) | Methods for high-level and stable gene transfer in lymphocytes | |
| Stroun et al. | Circulating nucleic acids in higher organisms | |
| BR112021003670A2 (en) | genetically modified hematopoietic stem cells and their uses | |
| CN107630006A (en) | It is a kind of to prepare TCR and the method for the T cell of the dual-gene knockouts of HLA | |
| CN107949641A (en) | CRISPR/CAS9 compounds for genome editor | |
| CN114729367A (en) | Compositions and methods for CLL1 modification | |
| CN114787352A (en) | Compositions and methods for CD123 modification | |
| CN109055380A (en) | A kind of preparation method of universal CAR-T cell | |
| CN113122503B (en) | Universal CAR-T for targeting T cell lymphoma cells as well as preparation method and application of universal CAR-T | |
| CN114423865A (en) | Compositions and methods for CD33 modification | |
| Blaeschke et al. | Modular pooled discovery of synthetic knockin sequences to program durable cell therapies | |
| JP2023540277A (en) | Compositions and methods for CD123 modification | |
| CN112105720A (en) | Genetically engineered immune lymphocyte and preparation method thereof | |
| EP4086341A1 (en) | Method for purifying ucart cell and use thereof | |
| US20240110189A1 (en) | Compositions and methods for cll1 modification | |
| CN113122504A (en) | Method for purifying UCART cells and application | |
| CN117165529A (en) | Genetically modified T cell and preparation method and application thereof | |
| CN114901813A (en) | Universal CAR-T of targeted T cell lymphoma cell and preparation method and application thereof | |
| CN116850210A (en) | Compositions and methods for treating acute myeloid leukemia | |
| 刘晓娟 et al. | CRISPR-Cas9-Mediated Multiplex Gene Editing in CAR-T Cells | |
| Solloa | Treating Diffuse Large B Cell Lymphoma Using HLA Class I Molecule Deficient Anti CD19 CAR-NK Cells Suraj Das Gene Editing and CRISPR Technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: B1-2, building 16, Shuwu, No. 73, tanmi Road, Jiangbei new district, Nanjing, 211500, Jiangsu Province Patentee after: NANJING BIOHENG BIOTECHNOLOGY Co.,Ltd. Address before: No. 821, room F7, No. 9, 9 weatia Road, Qixia District, Qixia, Jiangsu Patentee before: NANJING BIOHENG BIOTECHNOLOGY Co.,Ltd. |