CN109294997A - A kind of LRFFT1 cell - Google Patents
A kind of LRFFT1 cell Download PDFInfo
- Publication number
- CN109294997A CN109294997A CN201811153215.1A CN201811153215A CN109294997A CN 109294997 A CN109294997 A CN 109294997A CN 201811153215 A CN201811153215 A CN 201811153215A CN 109294997 A CN109294997 A CN 109294997A
- Authority
- CN
- China
- Prior art keywords
- cell
- lrfft1
- polypeptide
- tcr
- epitope
- 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
-
- 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
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- 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
- C12N15/63—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
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Abstract
The present invention carries out ctDNA sequencing using source of people peripheral blood or tumor tissues carry out full exon sequencing, filters out mutational site and carries out Epitope prediction, connects and synthesize mutant polypeptide expressing gene sequence;Slow virus carrier is constructed simultaneously, pack slow virus, transfect APC cell, complete the transformation of specificity LV cell, the PBMC separated in vitro with from peripheral blood is co-cultured, effective polypeptide is filtered out, by the second Secondary Shocks of accurate effectively polypeptide stimulation, general T cell is transformed into the RFF cell for more precisely killing ability;TCR-T technical principle is recycled to be transformed;Improved T cell compiles the knockout that technology of seizing carries out immunosupress target spot to it with gene again, accurately protects specific killing T cell from inhibiting in vivo, improves T cell to the lethality of tumour cell, the LRFFT1 cell more being had conditions in both attack and defence.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of LRFFT1 cell and preparation method thereof.
Background technique
Currently, existing LAK, DC, CIK, DC-CIK cell and method are basic in terms of the specific active immunotherapy of tumour
It is proved to be invalid, and the cell technologies such as NK, CAR-NK, TIL need maturation, CAR-T cell is in safety and solid tumor
It is also defective in treatment.The prior art generally passes through transformation DC cell, generates specific killing by DC submission T cell.Some experiments
It is attempting to carry out transfection submission T cell, the specific killing of inducing T cell as the method for carrier with virus in room.We were also once
PBMC, inducing T cell are directly stimulated with mutation mixed polypeptide.There are also laboratories to utilize TCR-T technology, targets submission MAGE A3
Antigen.
The above treatment method is simultaneously immature, especially external evoked DC cell and DC cell loading tumour antigen technical know-how
Upper research is more, but there are many more problems in the specific implementation process, lack specific, tumour cell occurrence and development key letter
Number conduction path relevant molecule because tumour antigen is unknown and the immunosuppressive obstacle of tumor microenvironment, makes as inducing antigen
Realize that specific cell targeting immunization therapy is difficult to smoothly implement.Although not having in addition, what is had has carried out antigen in vitro impact
Carry out it is external cultivate altogether and amplification in vitro, allow more thin specific cell directly facing complicated tumour immunity microenvironment,
Therefore, it is difficult to play expected effect.Although also have can also external submission and total cultivation, target spot is single (MAGE-3),
Only work to individual cancer kinds such as non-small cell lung cancer.Transfection submission is carried out although also having and attempting the method that slow virus is carrier,
Safety, convenience are not so good as polypeptide mode.And the direct stimulation of polypeptide is simply mixed, although simple and convenient, efficiency is lower.It is special
The secondary stimulus of anisotropic precisely polypeptide is more direct not as good as the tumour specific antigen of T cell receptor transduction.Existing TCR-T is being controlled
In the solution for treating neoplastic hematologic disorder and entity tumor, lack the accurately TCR of covering more polyoma kind.
Above scheme does not account for the self-protection technology of T cell, so that the direct face of specific T-cells that quantity is few
To powerful tumour immunity microenvironment.It is accurately and effectively analyzed general lack of also lacking to patient's antigen.
Summary of the invention
The present invention carries out ctDNA sequencing using peripheral blood in patients or tumor tissues carry out full exon sequencing, filters out prominent
Displacement point carries out Epitope prediction, connects and synthesizes mutant polypeptide expressing gene sequence;Slow virus carrier is constructed simultaneously, is packed
Slow virus transfects APC cell, completes the transformation of specificity LV cell, and the PBMC separated in vitro with from peripheral blood is co-cultured, sieve
Effective polypeptide is selected, by the second Secondary Shocks of accurate effectively polypeptide stimulation, general T cell is transformed into have and is more precisely killed
Hurt the RFF cell of ability;TCR-T technical principle is recycled to be transformed;Improved T cell seizes technology with gene volume again
The knockout of immunosupress target spot is carried out to it, accurately protects specific killing T cell from inhibiting in vivo, it is thin to improve T
Born of the same parents are to the lethality of tumour cell, the LRFFT1 cell that is more had conditions in both attack and defence.
LRFFT1 cell provided by the invention can be widely applied to individuation and precisely treat entity tumor.
Explanation for specific term:
L: slow-virus transfection technology
R: accurate polypeptide secondary pulse technology
FF: mixed polypeptide technology
T:TCR-T technology
1: target spot knocks out guard technology
Such as: LRFFT1 cell, i.e., it is final via above-mentioned L, R, FF, T, 1 every technical solution or technological means transformation
The cell of acquisition.
LRFFT1 cell modification scheme is summarized as follows:
1, Epitope prediction
1) using source of people peripheral blood carry out ctDNA sequencing or commercially available engineering cell system (such as H1299, H226, H358,
H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323 B16F1, CRL-2539 4T1, U14
Mouse carcinoma of uterine cervix cell, the small glioma cell of BV-2 mouse, G422 mouse glioma cell etc.) carry out the inspection of MHC type
It surveys and full exon sequencing detection RNA is mutated;
2) MHC type and gene mutation information prediction epitope are utilized: centered on the amino acid sites of mutation, to two
Side extends 8 amino acid, using the polypeptide of 17 amino acid of this section as potentially antigenic epitope;
3) IC50 that potentially antigenic epitope is analyzed using forecasting software, thinks this potentially antigenic table if IC50 < 1000nM
Position is epitope;
2, polypeptide connects
1) IC50 that any epitope is connected two-by-two at rear joint is analyzed using aforementioned software, when IC50 >=1000nM recognizes
To be weak immunogene, can connect;It is considered strongly immunogenic when IC50 < 1000nM, cannot connects;
2) according to the above results, the epitope of weak immunogene is linked together, joint IC50 is higher than two sides
The IC50 of epitope (namely joint, which avoids generating as far as possible, combines by force antigen);
3, the gene order of composite coding polypeptide
1) polypeptide after connection is reduced to nucleic acid sequence, and carries out codon optimization;
2) gene order of solid-phase synthesis synthetic antigen epitope peptide is used;Or it is synthesized by technical service company;
4, slow virus is packed
After the slow virus expression plasmid for the gene order building expression epitope peptide that upper step is synthesized, slow virus packet is carried out
Dress;
5, it transfects antigen presenting cell (APC) and is co-cultured with PBMC
1) using the slow-virus transfection antigen presenting cell of expression epitope peptide, (including but not limited to: peripheral blood is single
Nucleus, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage);
2) APC that processing is completed is collected, is mixed and is co-cultured with the ratio of APC:PBMC=1:5-20, obtain effector cell;
6, effective accurate polypeptide is screened, and stimulates T cell again using accurate polypeptide
1) T cell of above scheme acquisition is collected by centrifugation, polypeptide directly stimulates T cell to screen accurate polypeptide as antigen:
2) positive control: T cell+100ng/mL OKT3 is set;Negative control: T cell+1640+10%FBS+200U/
mL IL2;
3) accurate polypeptide judgment criteria:
A. positive control and negative control are normal, then illustrate that this data is credible;
B. it is effective accurate polypeptide that experimental group, which is noticeably greater than negative control group,;
4) the accurate polypeptide secondary pulse T cell to filter out;
7, TCR-T cell is constructed
1) dyeing of CD8, CD137, IFN-γ are carried out to post-stimulatory T cell, and are sorted with flow cytometer;
2) specific cell that can identify accurate polypeptide is sub-elected, is sequenced and is determined high frequency TCR sequence and expand;
3) tcr gene expression vector, packaging virus are constructed;
4) it knocks out original tcr gene in the periphery blood T cell, is transferred to the tcr gene of step building, cultivates to obtain the final product
TCR-T cell;
8, cell surface inhibitive ability of immunity signaling molecule is knocked out to get LRFFT1 cell
Cell surface inhibition signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,
2B4(CD244);
9, building specific antigen expression target cell and tumor model survival assay.
Beneficial effects of the present invention:
1. tumour antigen is mutant antigen, different from other tissues, target spot specificity is strong, is not susceptible to undershooting-effect, pacifies
Quan Xinggao;
2. the specific cell ratio obtained is high, the specific cell of tumour antigen usually can be identified, in point of PBMC
Cloth be 0.5% hereinafter, by LRFFT1 retrofit scheme cell, identify that specific T-cells (TCR+) ratio of tumour antigen is
50% or more;
3.LRFFT1 cell due to being knocked out to the inhibitive abilities of immunity target spot such as PD1, CTLA4, TIM3, LAG3, it is right
The killing ability of tumour is unrestricted, and killing-efficiency is higher.
Detailed description of the invention
Fig. 1: slow-virus transfection APC Efficiency testing;Wherein, 1A: control group, 1B: transfection group.
The detection of Fig. 2: LFF cell typing.
Fig. 3: the screening of accurate polypeptide.
Fig. 4: flow cytometer detection specific T-cells ratio;Wherein, 4A: control group, 4B:LRFF scheme.
Fig. 5: TCR distribution frequency.
Fig. 6: the knockout situation of inhibition target spot;Wherein, 6A: before knockout, 6B: after knockout.
Fig. 7: original TCR knockout Efficiency testing;Wherein, 7A: before knockout, 7B: after knockout.
Fig. 8: the expression efficiency of specificity TCR;Wherein, 8A: before transfection, 8B: after transfection 7 days.
Fig. 9: LDH release detection killing-efficiency.
The release of Figure 10: ELISA detection cell factor IFN-γ.
Figure 11: animal lotus knurl model survivorship curve.
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
Details are as follows for technical solution:
1, Epitope prediction
1) ctDNA sequencing is carried out using peripheral blood from patients with lung cancer and HLA parting detects;
2) sequencing information is analyzed using software: by ctDNA sequencing result compared with the genome of normal cell, sieve
Select mutational site;
3) centered on the amino acid sites of mutation, extend 8 amino acid to two sides, by the polypeptide of 17 amino acid of this section
As potentially antigenic epitope;
4) using forecasting software analyze potentially antigenic epitope IC50 (recommend software: NetMHCpan 3.0,
PickPocket, artificial neural networks (ANN)), this potentially antigenic table is thought if IC50 < 1000nM
Position is epitope.
2, polypeptide connects
1) IC50 that any epitope is connected two-by-two at rear joint is analyzed using aforementioned software, when IC50 >=1000nM recognizes
To be weak immunogene, can connect;It is considered strongly immunogenic when IC50 < 1000nM, cannot connects and (be considered as 3 herein
The IC50 calculated result of forecasting software can just be considered weak immunogene as IC50 >=1000nM that >=2 software calculates, when
It can just be considered strongly immunogenic when the IC50 < 1000nM that >=2 software calculates);
2) according to the above results, epitope is linked together, joint IC50 is higher than two sides epitope
IC50 (namely joint, which avoids generating as far as possible, combines by force antigen);Weak immunogene peptide will be exempted from by force as link peptide when necessary
Epidemic focus peptide interval;Or patient's self amino acid is added to joint, for reducing a possibility that generating strong antigen.
3, the gene order of composite coding polypeptide
1) polypeptide after connection is reduced to nucleic acid sequence, and carries out codon optimization;
If the nucleic acid sequence shorter (< 100bp) after the completion of connection can suitably repeat amino acid sequence, but
It is, it should be noted that inverted repeat in gene order, directly repetition and mirror image should be avoided to repeat sequence as far as possible when being reduced into gene order
The appearance of column
2) gene order (being synthesized by technical service company) of solid-phase synthesis synthetic antigen epitope peptide is used.
4, slow virus is packed
1) the epitope peptide gene sequence of synthesis is cloned into pCDH-MSCV-MCS-EF1-copGFP plasmid, is constructed
Express the slow virus expression plasmid of epitope peptide;
2) slow virus is packed:
A. recovery 293T cell is packed after passing for two generations for slow virus;
B. cell transfecting: (T175 culture bottle)
A 15ml centrifuge tube (labeled as A) is taken, 4mL is lightly added in 400 μ L Lipofectamine 2000
It in DMEM, lightly mixes, is stored at room temperature culture 5min;
Two 15mL centrifuge tubes (labeled as B and C, B is control group, and C is experimental group) is separately taken, following reagent and gently is added
Ground mixes, and is stored at room temperature culture 5min;
A pipe liquid is averagely transferred in B pipe and C pipe, is lightly mixed, culture 20min is stored at room temperature.
Culture medium old in T175 is poured out, cell is washed one time using PBS, changes new 25mL DMEM into (without antibiosis
Element and serum), A, B or A, C mixed liquor is lightly added, gently shakes up, is placed in 37 DEG C, is cultivated in 5%CO2 incubator;
After transfecting 6h, the culture medium containing transfection composite is sucked, is changed to the fresh culture of 37 DEG C of preheatings;Culture
48h, and collect;
3) slow virus concentration and titer determination:
After slow virus is packed successfully, slow virus supernatant is collected, 4 DEG C, 4000g is centrifuged 10min;It is filtered with 0.45 μm of filter
Supernatant removes cell fragment;According to vial supernatant: the mixing of concentrated reagent=5:1 ratio, 4 DEG C of placement 2h or overnight;
By the mixed liquor being incubated in 4 DEG C, 4000g is centrifuged 30min, i.e., visible tube bottom has rice white precipitating;Carefully supernatant is removed (to be sure not
Touch sediment);The DMEM or PBS of appropriate volume is added, gently sediment is resuspended in piping and druming;Packing virus on demand, -80
DEG C save (note: slow virus never multigelation, every freeze thawing is primary, and lentivirus titers will decline 10%-20%);
Measurement the previous day is diluted to 5 × 10 after counting the good 293H cell dissociation of growth conditions496 holes are added in/mL
Plate, 100 holes μ L/ prepare 8-10 hole for each virus.37 DEG C are put into, is cultivated in 5%CO2 incubator
It takes a certain amount of virus liquid infection cell: doing 10 times of gradient dilutions in EP pipe.Dilution process is as follows: every kind of virus
Prepare 10 1.5mL EP pipes, every pipe is added 90 μ L culture solutions, 10 μ L virus stock solution useds are added into first pipe, are denoted as 10 °;It is mixed
After even, draw 10 μ L and second pipe mixing is added, be denoted as 10-1;And so on (10 ° -10-8), 10 are added in corresponding cell hole
Virus liquid that μ L has diluted simultaneously marks, and observes result after cultivating 48-72h;
Titre calculates: fluorescent technique method can be used to measure titre the slow virus with fluorescent marker;In fluorescence microscopy
Under the microscope as a result, and count most latter two have fluorescence fluorecyte clone number, it is assumed that be X and Y, then titre (TU/mL)=
The content (μ L) of the virus liquid in the hole (X+Y × 10) × 1000/2/X.
5, slow-virus transfection antigen presenting cell (APC) and with PBMC co-culture
1) it is prepared using RPMI-1640 and contains 300U/mL rIL-2, the full cell culture medium of 10%FBS is denoted as RPMI-
10-IL-2;APC cell concentration is adjusted to 1 × 10 using RPMI-10-IL-26/mL;Use the slow disease of expression epitope peptide
With MOI=5-20 infection APC, (including but not limited to: peripheral blood mononuclear cells, Dendritic Cells, neutrophil leucocyte, B drench poison
Bar cell, macrophage);It is put into 37 DEG C of culture 72h;
4) APC that processing is completed is collected, is mixed with the ratio of APC:PBMC=1:5-20, PBMC is about 5 × 107, it is added
50mL OKM100 culture medium is into T75 culture bottle;It is put into 30-37 DEG C of cell incubator and cultivates 14 days, i.e. acquisition LFF scheme
Effector cell.
6, effective accurate polypeptide is screened
Polypeptide is as the direct accurate polypeptide of stimulating effect cell screening of antigen:
1) the above LFF Protocols Cell is collected by centrifugation, 1500rpm is centrifuged 5min and collects T cell, and 10mL PBS is added and is resuspended carefully
Born of the same parents simultaneously count, and 1500rpm is centrifuged 5min, collect T cell with 1640+10%FBS+200U/mL IL2 resuspension, counting is adjusted to 1
×106cells/mL;
2) T cell is divided into 96 hole flat undersides with the volley of rifle fire, 200 holes μ L/, cell number is 2 × 105cells;Add respectively again
Enter the mutant polypeptide of 10 μ L 1mg/mL, final concentration of 50 μ g/mL, 3 multiple holes are arranged in every polypeptide;
3) positive control: T cell+100ng/mL OKT3 is set;Negative control: T cell+1640+10%FBS+200U/
mL IL2;
4) 37 DEG C, 5%CO2After stimulation for 24 hours, 1500rpm is centrifuged 10min, shifts 140 μ L supernatants into 96 new orifice plates;
5) 96 orifice plates are centrifuged again, 1500rpm 10min, take sample carry out ELISA detection (or by sample as-
80 DEG C of preservations);
Detect the ELISA system of IFN-γ:
1) testing the ELISA kit that can be used for detecting IFN-γ has Biolegend:LEGEND MAX Human at present
IFN-γ ELISA Kit with Pre-coated Plates (article No.: 430107) He Dake are as follows: Human IFN-γ ELISA
Kit (article No.: DKW12-1000-096), is please operated in strict accordance with shop instruction;
2) the manual wrapper sheet system of ELISA (15 blocks of plates): 15 plate of Human IFN-gamma DuoSet (article No.:
DY285B) × 2 (article No.: DY008) × 31, DuoSet ELISA Ancillary Reagent Kit;
Accurate polypeptide judgment criteria:
1) positive control and negative control are normal, then illustrate that this data is credible;
2) when experimental group is noticeably greater than negative control, illustrate that polypeptide is effective accurate polypeptide.
7, the accurate polypeptide secondary pulse T cell to filter out
1) when PBMC is cultivated with step 5 to the 2nd~14 day, 2 × 10 are taken7Effector cell, final concentration of 10 μ g/ is added
The accurate polypeptide of the μ of mL~100 g/mL impacts 1-4h;
2) it after impacting 4h, is transferred in the 6 orifice plates of the pre- wrapper sheet of OKM25 or T25cm2Culture bottle mends OKM100+12%FBS,
37 DEG C of 5%CO2Culture, according to cell growth status, is transferred in T75 culture bottle, and holding cell density as far as possible is 1 × 106
cells/mL;
3) when entering in T175 culture bottle, culture medium OKM200+5%FBS, culture can be obtained precisely more for 10~14 days
The T cell that peptide secondary pulse obtains, i.e. LRFF cell;
8, the culture and separation of mutant antigen specific killing T cell
1) with the accurate polypeptide that filters out directly as antigenic stimulus, the LRFF cell obtained to step 7 is stimulated, and is pierced
It is spare after swashing 12~72h;
2) dyeing of CD8, CD137, IFN-γ are carried out to post-stimulatory T cell, and is sorted with flow cytometer, are selected
Select CD8+CD137+ or CD8+IFN- γ+cell;
9, the clone of CD8+T cell TCR frequency detecting and high frequency TCR
1) CD8+CD137+ the or CD8+IFN- γ+cell genome for extracting sorting, carries out the detection of TCR frequency,
Determine the TCR sequence of high frequency;
2) mRNA of sorting cell is extracted, reverse transcription is at from DNA, and according to the sequence of high frequency TCR, design primer is expanded
To tcr gene;
3) tcr gene expression vector, packaging virus are constructed;
10, the CRISPR carrier that building inhibitive ability of immunity signaling molecule knocks out
1) cell surface inhibition signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA,
CD160,2B4(CD244);
2) exon for analyzing inhibition signaling molecule, finds the area CDS of the mRNA of gene on pubmed, respectively will be every
A exon knock out the prediction of target spot;
3) forward primer and reverse primer needed for design synthesis sgRNA, forward primer and reverse primer 1:1 are mixed
Afterwards, 95 DEG C of 5~60min of processing, then slow cooling is carried out, form the DNA sequence dna of sgRNA;
4) CRISPR Lentiviral is subjected to double digestion, and double-stranded DNA corresponding with sgRNA is attached, turned
Enter and clone competent cell, after 12h, picking monoclonal is sequenced, and retains sequencing correctly clone;
5) the CRISPR slow virus carrier plasmid for carrying sgRNA corresponding DNA sequence is extracted, viral packaging is carried out;
11, building knocks out the CRISPR carrier of TCR
1) area CDS of the mRNA of tcr gene is found on pubmed, and analyzes the conserved region of TCR, and conserved region is struck
Except the prediction of target spot;
2) according to the step 3) in step 10~5) complete TCR knockout carrier building and virus packaging;
12, building knocks out the TCR-T of inhibitive ability of immunity signaling molecule:
1) virus to be obtained in step 10 and 11, the CD8+T cell that infection step 8 obtains, while carrying out original TCR
Knockout and inhibitive ability of immunity signaling molecule knockout;
2) after knocking out, after CD8+T cell cultivates 0-5 days in the medium, preferably 3 days, then it is transferred to the TCR expression load of building
Body;
3) metainfective CD8+T cell is resuspended with OKM100+12%FBS, is placed in overlaying for stimulating factor OKM-25
On plate, it is denoted as the 0th day;
4) it observes cell situation and co-cultured cell was transferred in big culture bottle by cell density at the 5th day, mend fresh
Culture solution OKM-100+12%FBS;
5) by cell from 75cm2175cm is transferred in bottle2Culture solution is OKM-200+5%FBS after big bottle;
6) when culture was to 14-21 days, the TCR-T of knockout inhibitive ability of immunity signaling molecule, i.e. LRFFT1 cell can be harvested.
13, building specific antigen expression target cell and tumor model survival assay
1) building can be with the slow virus carrier of the accurate polypeptide (specific antigen) of expression screening.
2) specific antigen expression slow virus carrier is packaged into lentiviral particle, the infection suitable tumour of HLA distribution type is thin
Born of the same parents stablize and are overexpressed specific antigen, flow cytometer detection expression and expression intensity.
3) stablize the tumor cell line inoculation NGS mouse for being overexpressed specific antigen peptide, do dystopy tumor-bearing model.It will
5×105The tumour cell of expression specificity antigen is suspended from 100 μ l physiological saline, is subcutaneously injected respectively to 30 NSG mouse
Right side side of body rib portion is subcutaneous, while mouse being numbered.
4) in tumour growth to 100-120mm3Grouping feeds back cell when left and right, according to gross tumor volume size, by animal mould
Type is randomly divided into three groups, and every group of 5-6 mouse, one group is given placebo physiological saline, gives for one group and does not carry out any heredity
The T cell (control group) 1 × 10 of operation7, one group is given LRFFT1 cell 1 × 107, carried out after injecting cell 7 days for the first time second
Injection, third time injection cell, is observed continuously 60 days after 7 days, counts survival data, draws survivorship curve.
Test result:
1, mutational site and Epitope prediction
Table 1 is the mutational site and Epitope prediction result that sequencing detects.
1 Epitope prediction of table
2, slow-virus transfection APC Efficiency testing
1) it is prepared using RPMI-1640 and contains 300U/mL rIL-2, the full cell culture medium of 10%FBS is denoted as RPMI-
10-IL-2;APC cell concentration is adjusted to 1 × 10 using RPMI-10-IL-26/mL;Use the slow disease of expression epitope peptide
With MOI=5-20 infection APC, (including but not limited to: peripheral blood mononuclear cells, Dendritic Cells, neutrophil leucocyte, B drench poison
Bar cell, macrophage);It is put into 37 DEG C of culture 72h;
2) using GFP positive ratio (as shown in Figure 1) in flow cytomery APC.
3, LFF cell typing detects
After LFF Protocols Cell culture, the parting detection of CD4+ and CD8+ cell is carried out, as a result as shown in Figure 2: CD8+
T cell is that 89.1%, CD4+T cell is 8.11%.
4, with the accurate polypeptide of LFF cell screening
The T cell for being stimulated culture respectively with 12 polypeptides is detected effective polypeptide, tied by detecting the secretion of IFN-γ
Fruit is as shown in Figure 3: burst size > negative control burst size of IFN-γ caused by No. 3 and No. 7 polypeptides, belongs to effectively precisely more
Peptide.
5, to the identification and sorting of the T cell of accurate polypeptid specificity
With No. 3 of screening and No. 7 polypeptides, LFF Protocols Cell is stimulated, it is thin with T of the flow cytometer detection to accurate polypeptid specificity
Born of the same parents' ratio, as a result as shown in figure 4, FL1+ is specific T-cells: LRFF Protocols Cell, No. 3 and the caused release of No. 7 polypeptides
The cell proportion of IFN-γ, hence it is evident that higher than not having irritant cell (control), illustrate, LRFF scheme, can obtain to precisely more
The specific T-cells of peptide;The sorting of CD8+IFN- γ+cell (FL1+) is carried out with flow cytometer simultaneously.
6, the identification of high frequency TCR and clone
Sorting is obtained into cell and carries out the extraction of genome and the sequencing of TCR, the distribution situation of TCR is (high as shown in Figure 5
20) TCR6 distribution frequency is higher for frequency division cloth preceding, illustrates that this TCR and mutant antigen are closely related, according to TCR6 sequence, to TCR
It is expanded, constructs Lentiviral.
The sequence situation of 2 TCR β chain CDR3 of table
Known TCR- α:
Amino acid sequence:
MMKSLRVLLV ILWLQLSWVW SQQKEVEQNS GPLSVPEGAI ASLNCTYSDR
GSQSFFWYRQ YSGKSPELIM FIYSNGDKED GRFTAQLNKA SQYVSLLIRD SQPSDSATYL
CAVNFGGGKL IFGQGTELSV KPN
Base sequence:
Known TCR- β:
Amino acid:
MRIRLLCCVA FSLLWAGPVI AGITQAPTSQ ILAAGRRMTL RCTQDMRHNA
MYWYRQDLGL GLRLIHYSNT AGTTGKGEVP DGYSVSRANT DDFPLTLASA
VPSQTSVYFC ASSLSFGTEA FFGQGTRLTV V
Horizontal line is CDR3 sequence, the sequence for needing to be replaced
Replaced TCR- β:
Horizontal line is the CDR3 sequence of replacement
7, inhibition target spot knocks out the detection of efficiency
Using CRISPR technology, the inhibition target spot PD-1 on PBMC is knocked out, sgRNA sequence is shown in Table 3, inhibition
The knockout efficiencies of target spot are as shown in Figure 6: the knockout efficiency highest of sgRNA1 can be effectively blocked inhibition signaling molecule
The expression of PD-1;SgRNA preferred sgRNA1, sgRNA2;The method can also be used, to Tim-3, LAG3, CTLA-4, BTLA,
The inhibitions signaling molecules such as VISTA, CD160,2B4 (CD244) are knocked out;The table of inhibition signaling molecule can be effectively blocked
It reaches.
3 inhibition target spot sgRNA sequence of table
8, original TCR knocks out the detection of efficiency
Using CRISPR technology, original TCR on PBMC is knocked out, as a result as shown in Figure 7: can effectively reduce
The expression of original TCR, at this point, the transfection of expression specificity TCR slow virus can be carried out.
9, the detection of specificity TCR expression
To pack the slow-virus transfection PBMC of specificity TCR, at the 7th day, with the expression efficiency of flow cytometer detection TCR,
As a result as shown in Figure 8: the TCR of building can be 67.4% with normal expression, the cell proportion of TCR+.
10, lethal effect of the LRFFT1 cell to target cell
Carry out the inspection of killing-efficiency to the target cell in mutant antigen epitope source with control cell and LRFFT1 cell respectively
It surveys, using non-treated cell as control (Mock), imitates target ratio and be set as 40:1, as a result as shown in figure 9, compared with the control group,
LRFFT1 cell has stronger fragmentation effect to target cell.
11, the detection of LRFFT1 cell cytokine release
When tumour cell and effector cell co-culture, due to effector cell, can with mutant antigen on tumor cell, because
This, can generate a series of cell factor, and IFN-γ is one of most important cell factor, Tu10Wei in antitumor action
When LRFFT1 cell and tumour cell 1:1 are co-cultured, the detection of the IFN-γ of release, the results showed that produced with effector cell itself
Raw IFN-γ compares (T cells only), and after co-culturing with tumour cell, LRFFT1 cell can produce a large amount of IFN-γ
This result is consistent with killing experiments result to be illustrated: the T cell of expression specificity TCR, can be more in conjunction with the knockout of inhibition target spot
It is effective to improve anti-tumor capacity.
12, building specific antigen expression target cell and tumor model survival assay
Specific antigen expression tumour target cell system is successfully constructed, establishes tumor-bearing model, as the result is shown (Figure 11),
The existence of LRFFT1 cells against tumor tumor-bearing mice, which improves to have, significantly affects effect.
13, clinical case:
Certain female: 58 years old
Medical diagnosis on disease: oophoroma and breast cancer;
First course for the treatment of: monthly LRFFT1 cell, quantity 1 × 109A cell, totally 2 times;
Second course for the treatment of: every half a year LRFFT1 cell, quantity 1 × 109A cell, totally 2 times;
After administration, 22 months Progression free survivals;
Other cases:
Note: containing for " so far " is meant " on the day before the applying date ".
Claims (6)
1. a kind of LRFFT1 cell, which is characterized in that the LRFFT1 cell is prepared by the following steps: 1) using source of people peripheral blood
It carries out ctDNA sequencing or tumor tissues carries out full exon sequencing, filter out mutational site;2) antigen is carried out according to mutational site
Antigen Epitope Prediction synthesizes the gene order of mutant polypeptide;3) slow virus carrier of building expression mutant polypeptide, packs slow virus;4)
It transfects antigen presenting cell and is co-cultured with PBMC, obtain LFF cell;4) mutant polypeptide is as LFF described in antigenic stimulus
Cell filters out effective accurate polypeptide;5) using the accurate polypeptide as LFF cell described in antigenic stimulus, filtering out can
The specific cell for identifying the accurate polypeptide is sequenced and obtains the high frequency tcr gene of specific cell;6) peripheral blood T is knocked out
Original tcr gene in cell, be transferred to step acquisition can obtain TCR-T with the tcr gene in conjunction with accurate polypeptid specificity
Cell;7) cell surface inhibitive ability of immunity signaling molecule is knocked out to get LRFFT1 cell.
2. LRFFT1 cell as described in claim 1, which is characterized in that it is thin that the source of people peripheral blood is also possible to commercially available engineering
Born of the same parents system.
3. LRFFT1 cell as described in claim 1, which is characterized in that the Epitope prediction is the amino acid with mutation
Centered on site, respectively extend 8 amino acid to two sides, using the polypeptide of 17 amino acid of this section as potentially antigenic epitope;It uses
Forecasting software analyzes the IC50 of potentially antigenic epitope, thinks that this potentially antigenic epitope is epitope if IC50 < 1000nM.
4. LRFFT1 cell as described in claim 1, which is characterized in that original TCR base in the knockout periphery blood T cell
Cause and/or the method for cell surface inhibitive ability of immunity signaling molecule are CRISPR technology.
5. LRFFT1 cell as described in claim 1, which is characterized in that the cell surface inhibition signaling molecule includes:
PD-1、Tim-3、LAG3、CTLA-4、BTLA、VISTA、CD160、2B4(CD244)。
6. LRFFT1 cell as described in claim 1, which is characterized in that the antigen presenting cell includes: that peripheral blood is single
Nucleus, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811153215.1A CN109294997B (en) | 2018-09-30 | 2018-09-30 | LRFFT1 cell |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811153215.1A CN109294997B (en) | 2018-09-30 | 2018-09-30 | LRFFT1 cell |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109294997A true CN109294997A (en) | 2019-02-01 |
CN109294997B CN109294997B (en) | 2020-09-25 |
Family
ID=65161386
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811153215.1A Active CN109294997B (en) | 2018-09-30 | 2018-09-30 | LRFFT1 cell |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109294997B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113403275A (en) * | 2021-07-01 | 2021-09-17 | 北京百益宁医学科技有限责任公司 | Preparation method of new antigen of specific target cancer species |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104926944A (en) * | 2015-05-22 | 2015-09-23 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | Preparation method and application of multiple target complex antigen-loaded CD8<+> cytotoxic T lymphocyte |
CN105451759A (en) * | 2013-05-10 | 2016-03-30 | 拜恩科技股份公司 | Predicting immunogenicity of t cell epitopes |
WO2016174085A1 (en) * | 2015-04-27 | 2016-11-03 | Cancer Research Technology Limited | Method for treating cancer |
CN107074932A (en) * | 2014-10-02 | 2017-08-18 | 美国卫生和人力服务部 | Separate the method that the φt cell receptor with antigentic specificity is mutated to cancer specific |
CN107541498A (en) * | 2016-06-27 | 2018-01-05 | 复旦大学附属肿瘤医院 | A kind of preparation method and its usage of the CD8+T Memorability stem cells of tcr gene modification |
CN107557333A (en) * | 2017-10-16 | 2018-01-09 | 北京鼎成肽源生物技术有限公司 | A kind of neoantigen for cell therapy is in the preparation method of delivery cell |
CN107735492A (en) * | 2015-03-13 | 2018-02-23 | 深圳源正细胞医疗技术有限公司 | Use the method for activating T cell treatment cancer |
-
2018
- 2018-09-30 CN CN201811153215.1A patent/CN109294997B/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105451759A (en) * | 2013-05-10 | 2016-03-30 | 拜恩科技股份公司 | Predicting immunogenicity of t cell epitopes |
CN107074932A (en) * | 2014-10-02 | 2017-08-18 | 美国卫生和人力服务部 | Separate the method that the φt cell receptor with antigentic specificity is mutated to cancer specific |
CN107735492A (en) * | 2015-03-13 | 2018-02-23 | 深圳源正细胞医疗技术有限公司 | Use the method for activating T cell treatment cancer |
WO2016174085A1 (en) * | 2015-04-27 | 2016-11-03 | Cancer Research Technology Limited | Method for treating cancer |
CN104926944A (en) * | 2015-05-22 | 2015-09-23 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | Preparation method and application of multiple target complex antigen-loaded CD8<+> cytotoxic T lymphocyte |
CN107541498A (en) * | 2016-06-27 | 2018-01-05 | 复旦大学附属肿瘤医院 | A kind of preparation method and its usage of the CD8+T Memorability stem cells of tcr gene modification |
CN107557333A (en) * | 2017-10-16 | 2018-01-09 | 北京鼎成肽源生物技术有限公司 | A kind of neoantigen for cell therapy is in the preparation method of delivery cell |
Non-Patent Citations (3)
Title |
---|
LONG SHI等: "CRISPR knock out CTLA-4 enhances the anti-tumor activity of cytotoxic T lymphocytes", 《GENE》 * |
宋琪: "突变新抗原特异性T细胞治疗方法的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
魏智民等: "免疫检查点抑制剂在肺癌治疗中的应用研究进展", 《中华老年多器官疾病杂志》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113403275A (en) * | 2021-07-01 | 2021-09-17 | 北京百益宁医学科技有限责任公司 | Preparation method of new antigen of specific target cancer species |
Also Published As
Publication number | Publication date |
---|---|
CN109294997B (en) | 2020-09-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110093376A (en) | A kind of construction method of LRFFT1 cell | |
CN105384826A (en) | Cord blood nucleated cell for expressing chimeric antigen receptor and application of cord blood nucleated cell | |
CN109294997A (en) | A kind of LRFFT1 cell | |
CN109136284A (en) | A kind of AFFT2 cell | |
CN109679917A (en) | A kind of LRFFT2 cell | |
CN109136278A (en) | A kind of MRFFT1 cell | |
CN110157745A (en) | A kind of construction method of HAFFT1 cell | |
CN110205341A (en) | A kind of construction method of LRFFT2 cell | |
CN110093374A (en) | A kind of construction method of MRFFT1 cell | |
CN110129373A (en) | A kind of construction method of RFF1 cell | |
CN110093371A (en) | A kind of construction method of LRFF cell | |
CN109294982A (en) | A kind of RFF2 cell | |
CN110093375A (en) | A kind of construction method of MRFFT2 cell | |
CN109294998A (en) | A kind of RFF1 cell | |
CN109295097A (en) | A kind of MRFFT2 cell | |
CN109337873A (en) | A kind of LRFF cell | |
CN109337930A (en) | A kind of AFFT1 cell | |
CN110408657A (en) | A kind of construction method of AFFT1 cell | |
CN109295106A (en) | A kind of HAFFT1 cell | |
CN110129371A (en) | A kind of construction method of RFFT2 cell | |
CN110129374A (en) | A kind of construction method of RFFT cell | |
CN110129372A (en) | A kind of construction method of RFFT1 cell | |
CN109055317A (en) | A kind of AFP combines CIK immunocyte and its preparation method and application with the DC of the dual anti-protogene modification of HBsAg | |
CN110093373A (en) | A kind of construction method of AFFT2 cell | |
CN117024605B (en) | Chimeric antigen receptor, microglia expressing chimeric antigen receptor and application thereof |
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 |