CN107164412A - A kind of safety-type anti-CEA Chimeric antigen receptors modify the preparation method and applications of T cell - Google Patents

A kind of safety-type anti-CEA Chimeric antigen receptors modify the preparation method and applications of T cell Download PDF

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CN107164412A
CN107164412A CN201710523596.7A CN201710523596A CN107164412A CN 107164412 A CN107164412 A CN 107164412A CN 201710523596 A CN201710523596 A CN 201710523596A CN 107164412 A CN107164412 A CN 107164412A
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cell
cea
safety
hsv
chimeric antigen
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CN107164412B (en
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刘明录
强邦明
张传鹏
姜夕锋
王立新
金海锋
韩国英
卢永灿
韩庆梅
万磊
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Jinan Xingyi Medical Technology Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention discloses the preparation method that a kind of safety-type anti-CEA Chimeric antigen receptors modify T cell, Leader scFv (CEA) CD8 CD137 CD3 ζ T2A HSV TK of synthesis genetic fragment is inserted into pLent C GFP carriers by the plasmid transfection 293T cells, it is packaged into the slow virus for carrying Leader scFv (CEA) CD8 CD137 CD3 ζ T2A HSV TK encoding genes, by the heterogeneous T lymphocytes of the slow-virus infection monocyte induction for carrying Leader scFv (CEA) CD8 CD137 CD3 ζ T2A HSV TK encoding genes, obtain the T cell of safety-type anti-CEA Chimeric antigen receptors modification, normal cell is exempted in the invention, " effect of missing the target " is reduced while curative effect is ensured, improve safety.

Description

A kind of preparation method of safety-type anti-CEA Chimeric antigen receptors modification T cell and its Using
Technical field
The present invention relates to and it is biological with new medical technology field, be a kind of safety-type anti-CEA chimeric antigens in particular Acceptor modifies the preparation method and applications of T cell.
Background technology
Carcinomebryonic antigen (carcinoembryonic antigen, CEA) is a kind of acidity of high expression Several Kinds of Malignancy The colorectal cancer of glycoprotein, such as expression more than 95%, 90% or so stomach cancer, cancer of pancreas, more than 80% non-cellule type Lung cancer, 50% or so breast cancer.Therefore CEA be acknowledged as one of tumor markers, make neoplasm targeted therapy and One of optimal target position of diagnosis.
Traditional treatment CEA oncology tools include operation, radiotherapy, chemotherapy etc. far from meeting wanting for numerous cancer patients Ask, the number dead because of CEA tumours is more than the 60% of whole tumor mortality numbers every year, is the serious prestige of human health The side of body.And T cell (CAR-T) mistake is modified with carcinomebryonic antigen Chimeric antigen receptor (chimeric antigen receptor CAR) The immunization therapy therapy rich in desired by as CEA oncotherapies is targetted after property.Current CAR-T technologies are in acute white blood There is significant treatment in the treatment of the Several Kinds of Malignancy such as disease, NHL, cancer of pancreas, breast cancer, kidney, colon cancer Effect, but also result in the adverse reactions such as " effect of missing the target " and " cytokine storm ".Therefore, CAR-T cell therapies effect is being ensured On the premise of fruit, the security for how improving CAR-T cells is the matter of utmost importance that current clinical practice faces.
The content of the invention
It is not enough in order to make up the above, provide a kind of safety-type anti-CEA Chimeric antigen receptors the invention provides the present invention and repair The T cell of decorations.
The solution of the present invention is：
A kind of safety-type anti-CEA Chimeric antigen receptors modify the preparation method of T cell, by Leader-scFv (CEA)- CD8-CD137-CD3 ζ-T2A-HSV-TK nucleotides synthesize, by Leader-scFv (the CEA)-CD8-CD137-CD3 ζ of synthesis- T2A-HSV-TK genetic fragment is inserted into pLent-C-GFP carriers (Invitrogen) NotI-AsiSI sites, through sequencing just After really, plasmid is built into, by the plasmid transfection 293T cells, is packaged into and carries Leader-scFv (CEA)-CD8- The slow virus of CD137-CD3 ζ-T2A-HSV-TK encoding genes, Leader-scFv (CEA)-CD8-CD137- is carried by described The heterogeneous T lymphocytes of the slow-virus infection monocyte induction of CD3 ζ-T2A-HSV-TK encoding genes, obtain safety-type anti- The T cell of CEA Chimeric antigen receptors modification.
It is used as preferred technical scheme, the base of Leader-scFv (the CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK Because fragment is the nucleotide sequence shown in sequence table SEQ .ID.NO.1.
Prepared present invention also offers a kind of heterogeneous T lymphocytes for inducing monocyte as follows：Take outside autologous patient All blood, separating peripheral blood mononuclear cells after the culture medium Fiber differentiation 24 hours of recombinant interferon alpha-2a, add restructuring white Cytokine 2, OKT-3 and 5% autologous patient blood plasma induction continue to cultivate 24 hours；Multiple proportions liquid feeding, is cultivated extremely every three days 14th day, the positive expression rate of CD3+, CD56+ in Flow cytometry T cell；CD3+ positive rates>80%, CD3+CD56 + bis- positive rates>20%, obtain the heterogeneous T lymphocytes of monocyte induction.
Present invention also offers one kind by the plasmid transfection 293T cells, it is packaged into and carries Leader-scFv (CEA) method of the slow virus of-CD8-CD137-CD3 ζ-T2A-HSV-TK encoding genes：By slow virus package cell line 293T It is inoculated in containing in DMEM+10%FBS 10cm culture dishes, 37 DEG C, 5% CO2Under the conditions of cultivate, adherent rate is 70%-80% Transfected afterwards, the plasmid transfects 293T cells using calcium phosphate transfection method, and after transfection after 24h, cell is significantly increased, in ball Shape, nucleus becomes big, is rounded, and adherent ability declines and easy to fall off；It was observed that intracellular have under inverted fluorescence microscope after 48h Egfp expression；After 72h, supernatant is collected, filtration sterilization obtains slow virus, preserves standby in -80 DEG C of low temperature refrigerators With.
The present invention also provides a kind of medicine for treating tumour, contains the safety-type anti-CEA chimeric antigens described in claim 1 The T cell of acceptor modification.
By adopting the above-described technical solution, a kind of safety-type anti-CEA Chimeric antigen receptors modify the preparation side of T cell Method, Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK nucleotides is synthesized, by the Leader-scFv of synthesis (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK genetic fragment is inserted into pLent-C-GFP carriers (Invitrogen) NotI-AsiSI sites, after sequencing correctly, are built into plasmid, by the plasmid transfection 293T cells, are packaged into and carry The slow virus of Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK encoding genes, carries described The slow-virus infection monocyte induction of Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK encoding genes Heterogeneous T lymphocytes, obtain the T cell of safety-type anti-CEA Chimeric antigen receptors modification.
The advantage of the invention：
The medium affinity restructuring Chimeric antigen receptor of active " switch ", including medium affinity carcinomebryonic antigen are combined Area, CD8 Hinge areas and transmembrane region, CD137 and CD3 ζ intracellular signal area, in the 3' ends insertion of restructuring Chimeric antigen receptor Active " switch " element.
Restructuring Chimeric antigen receptor of the present invention, active " switch " element is phonetic by suicide gene herpe simplex thymus gland Pyridine kinases is constituted.It is connected by autothermic cracking peptide T 2A with the restructuring other functional domains of Chimeric antigen receptor.Connected by T2A Gene normally can be translated and expressed after T cell is transferred to, and expression is suitable.It is chimeric containing restructuring after GCV induction The dead "Off" to realize GCV control of T cell cracking of antigen receptor is acted on, i.e., prevented by injecting GCV With treatment CAR-T adverse reaction.
Restructuring Chimeric antigen receptor of the present invention, it is single-stranded anti-that medium affinity antigen binding domain is selected from medium affinity Body.The killing tumor cell for enabling CAR-T cells to be selected according to the difference of antigen density, and normal cell is exempted, ensureing " effect of missing the target " is reduced while curative effect, safety is improved.
Brief description of the drawings
Fig. 1 is Chimeric antigen receptor Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV- of the present invention The design drawing of TK fusion fragment；
Fig. 2 expresses for slow virus Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK of the present invention The schematic diagram of plasmid；
The efficiency that Fig. 3 is the expression CAR of CAR-T cells of the present invention is 18 ± 1.56；
Fig. 4 is the growth of suppression CEA positive colorectal cancers cell line SW480 cells in CAR-T bodies of the present invention, and CAR-T Activity is controlled by suicide gene system；
Fig. 5 is the result figure of the different tumour cell of 4 kinds of CEA expression quantity of CAR-T cell killings of different affinity；
Embodiment
In order to make up, the above is not enough, the invention provides a kind of T cell of safety-type anti-CEA Chimeric antigen receptors modification, The problem of to solve in above-mentioned background technology.
Embodiment 1 expresses the structure and virus bag of the slow virus plasmid of the Chimeric antigen receptor albumen of nucleic acid coding of the present invention Dress
1. fusion fragment Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK are inserted into slow virus Expression vector pLent-C-GFP.
Fig. 1 (Complete Nucleotides are shown in Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK CAR modules signal Sequence is shown in annex SEQ ID NO.1).
Anti-CEA each sequence of modules of CAR
(1) sub- Leader nucleic acid artificial sequence (SEQ ID NO.2) is guided
(2)Anti-carcinoembryonic antigen antibody single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO.3)
(3) CD8Hinge areas nucleic acid artificial sequence (SEQ ID NO.4)
(4) CD8 transmembrane regions nucleic acid artificial sequence (SEQ ID NO.5)
(5) CD137 intracellular regions nucleic acid artificial sequence (SEQ ID NO.6)
(6) CD3 ζ intracellular regions nucleic acid artificial sequence (SEQ ID NO.7)
(7) autothermic cracking peptide T 2A nucleic acid artificial sequence (SEQ ID NO.8)
(8) HSV-TK nucleic acid artificial sequence (SEQ ID NO.9)
Respectively by nucleic acid artificial sequence, Anti-CEA nucleic acid artificial sequence, the CD8Hinge areas core for guiding sub- Leader Sour artificial sequence, the nucleic acid artificial sequence of CD8 transmembrane regions, CD137 nucleic acid artificial sequence, CD3 ζ nucleic acid artificial sequence, from Cracking peptide T 2A nucleic acid artificial sequences, HSV-TK nucleic acid artificial sequence (SEQ ID NO.10) student on commission's work bioengineering (on Sea) Co., Ltd synthesizes its whole expression cassette, insertion pLent-C-GFP carrier (Invitrogen) NotI-AsiSI sites (see Fig. 2), E.coli (DH5 α) is transformed into, after sequencing correctly, extracts and purifies using the plasmid purification kit of Qiagen companies Plasmid, obtains the high-quality plasmid of recombinant expression carrier.
2. slow virus is packed, titre detection
Slow virus package cell line 293T is inoculated in containing in DMEM+10%FBS 10cm culture dishes, 37 DEG C, 5% CO2Under the conditions of cultivate, adherent rate be 70%-80% after transfected.Recombinant plasmid and empty plasmid are packed with slow virus respectively Plasmid uses calcium phosphate transfection method cotransfection 293T cells, specific method reference molecule clone.After transfection after 24h, cell is obvious Increase, spherical in shape, nucleus becomes big, is rounded, and adherent ability declines and easy to fall off.Observed after 48h under inverted fluorescence microscope There is egfp expression to intracellular.After 72h, supernatant is collected, filtration sterilization is saved backup in -80 DEG C of low temperature refrigerators. According to Lenti-XTMGo StixTMKit (ocean Science and Technology Ltd. of Beijing China product) determines virus titer, as a result table It is bright, the titre 2.66 × 10 of recombinant slow virus6Pfu/mL, the titre 2.78 × 10 of unloaded slow virus6pfu/ml。
The slow-virus infection T cell of embodiment 2
1. the preparation of heterogeneous T cell
75ml autologous patient peripheral bloods are taken, with TBD sample rates separating liquid (biological purchased from the foreign China Tech of Tianjin Hao), separation is outer All blood mononuclear cells.(it is purchased from the culture medium of the recombinant interferon alpha-2a (being purchased from the pharmacy of the Shenyang three lives) containing 1000IU/ml CORNING companies, 88-551-CM) Fiber differentiation is after 24 hours, and the recombinant interleukin 2 for adding 1000IU/ml (is purchased from Shen Positive three lives pharmacy), the induction of 50ng/ml OKT-3 and 5% autologous patient blood plasma continues to cultivate 24 hours.Multiple proportions every three days Liquid feeding, was cultivated to the 14th day, the positive expression rate of CD3+, CD56+ in Flow cytometry T cell (CD3-FITC, CD16/CD56-PE antibody is purchased from BECKMAN companies, A07735).CD3+ positive rates>The double positive rates of 80%, CD3+CD56+> 20%, it is considered as induced t cell success, and leaves and takes the T cell and treats viral infection
2. the amplification cultivation of T cell after slow-virus infection T cell and infection
T cell is infected with MOI=5 respectively with above-mentioned restructuring and unloaded slow virus.Metainfective 37 DEG C of cell, 5%CO2Training After supporting in case culture 8 hours, cell is collected, virus liquid is rejoined, 1000g, 32 DEG C, after centrifuging 90 minutes again, 37 DEG C, 5%CO2Continue to cultivate in incubator, multiple infection is so repeated, the efficiency of infection of T cell is improved.Suction is abandoned in 2ml cultures Clearly, 2ml fresh CORNING culture mediums are added, continue to expand culture, culture is expanded to enough consumptions to cell in 17 days.It is logical Fluorescence microscope detection Chimeric antigen receptor expression is crossed, because GFP and CAR is co-expressed, detection GFP positive cell is expression The positive cell (Fig. 3) of Chimeric antigen receptor.Using the T lymphocytes that are uninfected by as negative control, recombinant slow virus infection T is thin Its positive rate 18 ± 1.56% of born of the same parents, its positive rate 19.33 ± 1.38% of unloaded slow-virus infection T cell.
Embodiment 3：CAR-T cell killing activities research with suicide gene system
The colorectal cancer cell system SW480 conducts for taking the CEA of stable expressing luciferase (Fire-Luciferase) positive Target cell, effector cell is CAR-T cells and unloaded slow-virus infection T.
1. control of the suicide gene system to CAR-T cytoactives
By CAR-T cells according to density 1 × 105Individual/ml is inoculated with 96 orifice plates, per hole 100ul, is placed in 5%CO2, 37 DEG C of trainings Support case culture 24h；Add 10ng GCV (derivant of suicide gene system, the limited public affairs of Wuhan Hai Te bio-pharmaceuticals shares Department), every 12 hours are once, totally 3 times.Not add the CAR-T cells of GCV to be used as negative control.Detected with luciferase and be Luciferase contents in remaining cell in system kit E4550 (Nanjing Sheng Xing Bioisystech Co., Ltd) detection orifice plates, from Genic system is killed to the active control rate of T cell after modification=(1- adds GCV culture hole fluorescence intensity/do not add GCV Culture hole fluorescence intensity) × 100%.Suicide gene system is 98.34% ± 2.03% to the control rate of CAR-T cytoactives； As a result illustrate that the CAR-T cytoactives that the present invention is designed are controlled by suicide gene system.
2.CAR-T cells are analyzed CEA positive tumor cells system killing activity
It is inoculated with 100 μ l 1 × 104The target cell SW480 in/hole is into 96 porocyte culture plates, according to 1:1 effect target is than adding Effector cell is CAR-T cells, is placed in 5%CO2, 37 DEG C of incubator culture 24h.To add zero load slow-virus infection T as right According to group.Utilize residue in Luciferase Assay System kit E4550 (Nanjing Sheng Xing Bioisystech Co., Ltd) detection orifice plates Intracellular Luciferase contents, CAR-T cell lines are to the Cytotoxicity in vitro efficiency of target cell=(1-CAR-T cells target cell is total to Culture hole fluorescence intensity/zero load slow-virus infection T cell target cell co-cultures hole fluorescence intensity) × 100%.Effector T cell pair The killing-efficiency of CEA positive tumor cells system SW480 cells is 82.34% ± 1.97%；As a result illustrate what the present invention was designed CAR-T cells are acted on very High Fragmentation CEA positive tumor cells system.
Embodiment 4：CAR-T cells are acted on colorectal cancer BALB/C mice Tumor growth inhibition
18-22g female KM mices (being purchased from Traditional Chinese Medicine University Of Guangzhou) raise (23 ± 2 DEG C of room temperature, humidity 50% in Animal House ± 10%), the colorectal cancer cell system SW480 cells of logarithmic phase are collected, phosphate buffer (PBS) is diluted to 2 × 105Individual/ mL.Under aseptic condition, the left oxter inoculation 0.2mL colorectal cancer SW480 cell suspending liquids of mouse observe 3-5d, treat that oxter occurs The harder tubercle of grain of rice size is used as the successful standard of modeling.
(size that slide measure measures hypodermic tumour tissue block is 90- to C57BL/6 colorectal cancer xenografts model mice 100mm3) 4 groups are randomly divided into, every group 20, start to inject Experiment on therapy.Experimental group is respectively：
A. control group, the physiological saline of tail vein injections equal volume；
B. one group, tail vein injections 2 × 10 are treated6An individual cell/CAR-T cell, carries out for 7 days again after injecting first Secondary same dosage injection；
C. two groups, tail vein injections 2 × 10 are treated6An individual cell/CAR-T cell, carries out for 7 days again after injecting first Secondary same dosage injection, and after the 12h for the treatment of injection first, daily tail vein injections GCV (8mg/kg) is purged CAR-T cells, altogether lasting 14d.
D. three groups, tail vein injections 2 × 10 are treated6An individual cell/CAR-T cell, carries out for 7 days again after injecting first Secondary same dosage injection, and after Retreatment injection 12h, daily tail vein injections GCV (8mg/kg) is purged CAR-T cells, altogether lasting 7d.
Each experimental mice hypodermic tumour tissue block is measured greatly by slide measure daily, and recorded, lump average is used Tumor growth curve figure is drawn, as a result as shown in Figure 4.Inject after CAR-T cells the 3rd day, the tumour of 40% mouse starts to diminish, At 14 days, 65% mouse was almost touched less than tumour.Works of the CAR-T to tumour is almost completely inhibit after injection GCV With illustrating that GCV can eliminate CAR-T cells, prevent the collateral line damage to normal tissue and organ.As a result this is shown The CAR-T cells of invention design have obvious inhibitory action to BALB/C mice tumour growth, and activity is by suicide gene system Control.
Embodiment 5：CAR-T cell clinical application methods：
For the treatment of CEA tumour patients, treatment method is local tumor injection and venous re-transfusion 2 × 106Individual CAR-T is thin Born of the same parents, once in a week, continuous immunity two weeks.If there is the generation of adverse reaction, local tumor injection and venous re-transfusion GCV (5-10mg/kg), continuous 7-14d.
Embodiment 6：With the CAR-T cells of the different affinity of two kinds of Flow cytometry it is different to CEA expression quantity 4 The killing activity research of group tumour cell
1. the analysis of different type tumor cell surface CEA expression quantity
It is 1 × 10 to harvest first and adjust cell concentration6/ ml is in Eppendorf pipes, plus people 2mg/L and anti-CEA people mouse Chimeric antibodies T84.66 (IgG1) (moral Thailand biological product), is mixed in 1h on ice, after PBS is washed, on ice lh fluorescent labeled antibodies, After PBS is washed again, it is suspended in 1.0ml FACSFlow solution, uses flow cytometer FACSCalibur (for U.S. company BD Product) detection and analysis tumor cell surface CEA expression quantity, using human IgG as control antibodies.With recombinant antibodies T84.66 (IgG1) Obvious cell line is specifically bound for colorectal cancer cell system SW480；With reference to slightly weak for lung cancer cell line KNS-62；It is few Amount combine for Colon cancer cell line HT-29：；The cell line not combined with CEA specific antibodies is gastric carcinoma cell lines MKN-74.
2. point of the killing ability of the CAR-T cells of the two kinds of different affinity 4 group tumour cell different to CEA expression quantity Analysis
By different target cell SW480, KNS-62, HT-29, MKN-74 with 100 μ l 1 × 104/ hole is respectively inoculated into In 96 porocyte culture plates, according to 1:1 effect target is than medium affinity CAR-T cells and high-affinity CAR-T cell (antibody respectively Sequence is shown in SEQ ID NO.10, and building process is ibid), it is placed in 5%CO2,37 DEG C of incubator culture 24h.To automate XCELLigence PTCA DP instruments (Roche Products) monitor CAR-T killing ability in real time, as a result as shown in Figure 5.Medium parent With difference of the power CAR-T cells according to antigen, specific killing CEA height expression colorectal cancer cell system SW480 and lung cancer cell line KNS-62, and killing ability is obviously reduced with the reduction of antigen presentation amount, to the colon carcinoma cell line of a small amount of expression of CEA HT-29 and the gastric carcinoma cell lines MKN-74 not expressed do not have lethality.High-affinity CAR-T cells, except the stomach do not expressed CEA Cancerous cell line MKN-74 does not have outside lethality, to remaining each group effective killing ability.As a result medium affinity is shown The killing tumor cell that CAR-T cells can be selected according to the difference of antigen density, and normal cell is exempted, ensureing curative effect Reduction " effect of missing the target " simultaneously, improves safety.
Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, various modifications and replacement can be carried out to those details, these change the guarantor in the present invention Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.
Sequence table
<110>Shandong Xing Rui bio tech ltd
<120>A kind of safety-type anti-CEA Chimeric antigen receptors modify the preparation method and applications of T cell
<130> 2017
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 2678
<212> DNA
<213>Anti-CEA CAR artificial sequences
<400> 1
cgcatggccc tgcctgtgac agccctgctg ctgcctctgg ctctgctgct gcatgccgct 60
agacccgaca ttgtgctgac ccaatctcca gcttctttgg ctgtgtctct tgggcagagg 120
gccaccatgt cctgcagagc cggtgaaagt gttgatattt ttggcgttgg gtttttgcac 180
tggtaccagc agaaaccagg acagccaccc aaactcctca tctatcgtgc atccaaccta 240
gaatctggga tccctgtcag gttcagtggc actgggtcta ggacagactt caccctcatc 300
attgatcctg tggaggctga tgatgttgcc acctattact gtcagcaaac taatgaggat 360
ccgtacacgt tcggaggggg gaccaagctg gaaataaaag gaggaggagg aagcggagga 420
ggaggaagcg gaggaggagg aagcgaggtt cagctgcagc agtctggggc agagcttgtg 480
gagccagggg cctcagtcaa gttgtcctgc acagcttctg gcttcaacat taaagacacc 540
tatatgcact gggtgaagca gaggcctgaa cagggcctgg aatggattgg aaggattgat 600
cctgcgaatg gtaatagtaa atatgtcccg aagttccagg gcaaggccac tataacagca 660
gacacatcct ccaacacagc ctacctgcag ctcaccagcc tgacatctga ggacactgcc 720
gtctattatt gtgctccgtt tggttactac gtgtctgact atgctatggc ctactggggt 780
caaggaacct cagtcaccgt ctcctccgct agctagcacc acgacgccag cgccgcgacc 840
accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg 900
gccagcggcg gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat 960
ctgggcgccc ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta 1020
ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt 1080
acaaactact caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg 1140
atgtgaactg agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca 1200
gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa 1260
gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg 1320
cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa 1380
aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac 1440
caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgcgaag gccgagggag 1500
cctgctgaca tgtggcgatg tggaggaaaa cccaggacca atggcttcgt acccctgcca 1560
tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc ggccataaca accgacgtac 1620
ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc cgcctggagc agaaaatgcc 1680
cacgctactg cgggtttata tagacggtcc tcacgggatg gggaaaacca ccaccacgca 1740
actgctggtg gccctgggtt cgcgcgacga tatcgtctac gtacccgagc cgatgactta 1800
ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc tacaccacac aacaccgcct 1860
cgaccagggt gagatatcgg ccggggacgc ggcggtggta atgacaagcg cccagataac 1920
aatgggcatg ccttatgccg tgaccgacgc cgttctggct cctcatatcg ggggggaggc 1980
tgggagctca catgccccgc ccccggccct caccctcatc ttcgaccgcc atcccatcgc 2040
cgccctcctg tgctacccgg ccgcgcgata ccttatgggc agcatgaccc cccaggccgt 2100
gctggcgttc gtggccctca tcccgccgac cttgcccggc acaaacatcg tgttgggggc 2160
ccttccggag gacagacaca tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct 2220
tgacctggct atgctggccg cgattcgccg cgtttacggg ctgcttgcca atacggtgcg 2280
gtatctgcag ggcggcgggt cgtggcggga ggattgggga cagctttcgg ggacggccgt 2340
gccgccccag ggtgccgagc cccagagcaa cgcgggccca cgaccccata tcggggacac 2400
gttatttacc ctgtttcggg cccccgagtt gttggccccc aacggcgacc tgtataacgt 2460
gtttgcctgg gccttggacg tcttggccaa acgcctccgt cccatgcacg tctttatcct 2520
ggattacgac caatcgcccg ccggctgccg ggacgccctg ctgcaactta cctccgggat 2580
ggtccagacc cacgttacca ccccaggctc cataccgacg atctgcgacc tggcgcgcac 2640
gtttgcccgg gagatggggg aggctaacgc gcggccgc 2678
<210> 2
<211> 63
<212> DNA
<213>Guide sub- Leader artificial sequences
<400> 2
atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60
ccc 63
<210> 3
<211> 751
<212> DNA
<213>Anti-carcinoembryonic antigen antibody single chain Fv antibody nucleic acid Artificial sequence
<400> 3
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctcttgggca gagggccacc 60
atgtcctgca gagccggtga aagtgttgat atttttggcg ttgggttttt gcactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180
gggatccctg tcaggttcag tggcactggg tctaggacag acttcaccct catcattgat 240
cctgtggagg ctgatgatgt tgccacctat tactgtcagc aaactaatga ggatccgtac 300
acgttcggag gggggaccaa gctggaaata aaaggaggag gaggaagcgg aggaggagga 360
agcggaggag gaggaagcga ggttcagctg cagcagtctg gggcagagct tgtggagcca 420
ggggcctcag tcaagttgtc ctgcacagct tctggcttca acattaaaga cacctatatg 480
cactgggtga agcagaggcc tgaacagggc ctggaatgga ttggaaggat tgatcctgcg 540
aatggtaata gtaaatatgt cccgaagttc cagggcaagg ccactataac agcagacaca 600
tcctccaaca cagcctacct gcagctcacc agcctgacat ctgaggacac tgccgtctat 660
tattgtgctc cgtttggtta ctacgtgtct gactatgcta tggcctactg gggtcaagga 720
acctcagtca ccgtctcctc cgctagctag c 751
<210> 4
<211>135
<212> DNA
<213>CD8 Hinge areas nucleic acid artificial sequence
<400> 4
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 5
<211> 72
<212> DNA
<213>CD8 transmembrane region nucleic acid artificial sequences
<400> 5
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 6
<211> 126
<212> DNA
<213>CD137 intracellular region nucleic acid artificial sequences
<400> 6
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 7
<211> 336
<212> DNA
<213>CD3 ζ intracellular region nucleic acid artificial sequences
<400> 7
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 8
<211> 54
<212> DNA
<213>Autothermic cracking peptide T 2A nucleic acid artificial sequences
<400> 8
gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg acca 54
<210> 9
<211> 1128
<212> DNA
<213>HSV-TK nucleic acid artificial sequences
<400>9
atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60
ggccataaca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120
cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 180
gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240
gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 300
tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360
atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420
cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480
ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 540
agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600
acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660
cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720
ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga ggattgggga 780
cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840
cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gttggccccc 900
aacggcgacc tgtataacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960
cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020
ctgcaactta cctccgggat ggtccagacc cacgttacca ccccaggctc cataccgacg 1080
atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaac 1128
<210> 10
<211> 860
<212> DNA
<213>High-affinity Anti-carcinoembryonic antigen antibody single chain Fv Antibody nucleic acid artificial sequences
<400> 10
atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctcttgggca gagggccacc 120
atgtcctgca gagccggtga aagtgttgat atttttggcg ttgggttttt gcactggtac 180
cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 240
gggatccctg tcaggttcag tggcactggg tctaggacag acttcaccct catcattgat 300
cctgtggagg ctgatgatgt tgccacctat tactgtcagc aaactaatga ggatccgtac 360
acgttcggag gggggaccaa gctggaaata aaacggagga ggaggaagcg gaggaggagg 420
aagcggagga ggaggaagca tgaaatgcag ctgggttatc ttcttcctga tggcagtggt 480
tacaggggtc aattcagagg ttcagctgca gcagtctggg gcagagcttg tggagccagg 540
ggcctcagtc aagttgtcct gcacagcttc tggcttcaac attaaagaca cctatatgca 600
ctgggtgaag cagaggcctg aacagggcct ggaatggatt ggaaggattg atcctgcgaa 660
tggtaatagt aaatatgtcc cgaagttcca gggcaaggcc actataacag cagacacatc 720
ctccaacaca gcctacctgc agctcaccag cctgacatct gaggacactg ccgtctatta 780
ttgtgctccg tttggttact acgtgtctga ctatgctatg gcctactggg gtcaaggaac 840
ctcagtcacc gtctcctcag 860

Claims (6)

1. a kind of safety-type anti-CEA Chimeric antigen receptors modify the preparation method of T cell, it is characterised in that：By Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK nucleotides is synthesized, by Leader-scFv (CEA)-CD8-CD137- of synthesis CD3 ζ-T2A-HSV-TK genetic fragment is inserted into pLent-C-GFP carriers (Invitrogen) NotI-AsiSI sites, through surveying After sequence is correct, plasmid is built into, by the plasmid transfection 293T cells, is packaged into and carries Leader-scFv (CEA)-CD8- The slow virus of CD137-CD3 ζ-T2A-HSV-TK encoding genes, Leader-scFv (CEA)-CD8-CD137- is carried by described The heterogeneous T lymphocytes of the slow-virus infection monocyte induction of CD3 ζ-T2A-HSV-TK encoding genes, obtain safety-type anti- The T cell of CEA Chimeric antigen receptors modification.
2. a kind of safety-type anti-CEA Chimeric antigen receptors as claimed in claim 1 modify the preparation method of T cell, its feature It is：The genetic fragment of Leader-scFv (the CEA)-CD8-CD137-CD3 ζ-T2A-HSV-TK is sequence table Nucleotide sequence shown in SEQ.ID.NO.1.
3. a kind of safety-type anti-CEA Chimeric antigen receptors as claimed in claim 1 modify the preparation method of T cell, its feature It is, the heterogeneous T lymphocytes preparation of the monocyte induction is as follows：Autologous patient peripheral blood is taken, separation peripheral blood is single Nucleus, after the culture medium Fiber differentiation 24 hours of recombinant interferon alpha-2a, adds recombinant interleukin 2, OKT-3 and 5% Autologous patient blood plasma induction continue cultivate 24 hours；Multiple proportions liquid feeding, is cultivated to the 14th day, Flow cytometry every three days The positive expression rate of CD3+, CD56+ in T cell；CD3+ positive rates>The double positive rates of 80%, CD3+CD56+>20%, obtain single The heterogeneous T lymphocytes of nucleus induction.
4. a kind of safety-type anti-CEA Chimeric antigen receptors as claimed in claim 1 modify the preparation method of T cell, its feature It is, by the plasmid transfection 293T cells, is packaged into and carries Leader-scFv (CEA)-CD8-CD137-CD3 ζ-T2A- The method of the slow virus of HSV-TK encoding genes：Slow virus package cell line 293T is inoculated in containing DMEM+10%FBS In 10cm culture dishes, 37 DEG C, 5% CO2Under the conditions of cultivate, adherent rate is to be transfected after 70%-80%, plasmid use Calcium phosphate transfection method transfects 293T cells, after transfection after 24h, and cell significantly increases, spherical in shape, and nucleus becomes big, is rounded, adherent Ability decline and it is easy to fall off；It was observed that intracellular have egfp expression under inverted fluorescence microscope after 48h；After 72h, Supernatant is collected, filtration sterilization obtains slow virus, saved backup in -80 DEG C of low temperature refrigerators.
5. a kind of medicine for treating tumour, it is characterised in that：Containing the safety-type anti-CEA chimeric antigens described in claim 1 by The T cell of body modification.
6. the T cell of the safety-type anti-CEA Chimeric antigen receptors modification described in claim 1 is used to prepare treatment malignant tumour The purposes of medicine.
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WO2023000685A1 (en) * 2021-07-20 2023-01-26 卡瑞济(北京)生命科技有限公司 Armored car-t cell that increases survivin expression and anti-tumor use thereof

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