CN110408657A - A kind of construction method of AFFT1 cell - Google Patents

A kind of construction method of AFFT1 cell Download PDF

Info

Publication number
CN110408657A
CN110408657A CN201910438763.7A CN201910438763A CN110408657A CN 110408657 A CN110408657 A CN 110408657A CN 201910438763 A CN201910438763 A CN 201910438763A CN 110408657 A CN110408657 A CN 110408657A
Authority
CN
China
Prior art keywords
cell
tcr
polypeptide
aff
afft1
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
Application number
CN201910438763.7A
Other languages
Chinese (zh)
Other versions
CN110408657B (en
Inventor
焦顺昌
张嵘
周子珊
解佳森
陈小彬
陈红利
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Original Assignee
Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Beijing Dingcheng Taiyuan Biotechnology Co Ltd filed Critical Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Publication of CN110408657A publication Critical patent/CN110408657A/en
Application granted granted Critical
Publication of CN110408657B publication Critical patent/CN110408657B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • 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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

This application involves a kind of preparation methods of AFFT1 cell; RFF cell is transformed with TCR-T technical principle; improved T cell compiles the knockout that technology of seizing carries out immunosupress target spot to it with gene again; it accurately protects specific killing T cell from inhibiting in vivo, improves T cell to the lethality of tumour cell.

Description

A kind of construction method of AFFT1 cell
Technical field
The invention belongs to field of biotechnology, specifically, being related to a kind of AFFT1 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 Be proved to be invalid, and NK, CAR-NK, TIL, etc. cell technologies need maturation, CAR-T cell is in safety and entity It is also defective in tumor treatment.
The prior art generally passes through transformation DC cell, generates specific killing by DC submission T cell.It is attempting in some laboratories Transfection submission T cell, the specific killing of inducing T cell are carried out as the method for carrier with virus.We are also once mixed with mutation Closing polypeptide directly stimulates PBMC, inducing T cell.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 External cultivation and amplification in vitro altogether are carried out, allows more thin specific cell directly facing complicated tumor 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 to non- Individual cancer kinds such as Small Cell Lung Cancer work.Transfection submission, safety, side are carried out although also having and attempting the method that slow poison is carrier Just property is not so good as polypeptide mode.And the direct stimulation of polypeptide is simply mixed, although simple and convenient, efficiency is lower.Object is anisotropic precisely The secondary stimulus of polypeptide is more direct not as good as the tumour specific antigen of T cell receptor transduction.Existing TCR-T is swollen in treatment blood In tumor and the solution of 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 immune microenvironment.
Summary of the invention
The present invention is transformed by RFF cell with TCR-T technical principle, and improved T cell seizes skill with gene volume again Art carries out the knockout of immunosupress target spot to it, accurately protects specific killing T cell from inhibiting in vivo, improves T The lethality of cells against tumor cells.
Explanation for specific term:
A: DC technology is immortalized
FF: mixed polypeptide technology
R: accurate polypeptide secondary pulse technology
T:TCR-T technology
1: target spot knocks out guard technology
AFFT1 cell modification scheme
1. full exon sequencing
1) using source of people peripheral blood carry out ctDNA detection or commercially available engineering cell system (such as H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323B16F1, CRL-2539 4T1, U14 are small Mouse cervical cancer cell, the small glioma cell of BV-2 mouse, G422 mouse glioma cell etc.) carry out full exon survey Sequence；
2) sequencing information is analyzed using software: on the one hand obtains MHC type information；It on the other hand, will be entirely outer aobvious Sub- sequencing result filters out mutational site compared with the genome of normal cell；
2. Epitope prediction
1) centered on the amino acid sites of mutation, extend 10 amino acid to two sides, by the more of 21 amino acid of this section Peptide is used as " potentially antigenic epitope "；
2) using forecasting software analyze potentially antigenic epitope IC50 (recommend software: NetMHCpan 3.0, PickPocket, artificial neural networks (ANN), think this potentially antigenic epitope if IC50 < 1000nM For " epitope "；
3. synthesizing mutant polypeptide
Polypeptide is synthesized by technical service company；
1)
4. immortalizing DC
1) peripheral blood 100ml is extracted
2) Ficol density gradient centrifugation separates PBMC
3) U.S. day Ni Dendritic Cells separating kit isolating dendritic cells, are resuspended in culture medium.
4) the separated Dendritic Cells of TAX-GFP slow-virus infection, 37 DEG C of incubator static gas wave refrigerators, observation
5) after Cell-cloned growth, selected clone is cultivated respectively in 96 orifice plates
6) Phenotype is analyzed
7) it chooses ideal clone and is used as immortality DC；
5. immortality DC load sudden change polypeptide
1) prepare polypeptide solution: using step 3 synthesize mutant polypeptide prepared, every polypeptide final concentration of 10~ 100 μ g/mL, preferably 50 μ g/mL, it is spare；
2) the immortal DC of acquisition is collected by centrifugation, is resuspended with the polypeptide solution of preparation, it is more to be placed in progress in tissue culture plate Peptide load；
3) 37 DEG C of 5%CO2, 1~4h, preferably 4h are impacted, it is spare；
6. the DC and PBMC of load sudden change polypeptide are incubated for altogether
1) the pre- bed board of stimulating factor OKM-25,40 μ L OKM-25+4mL PBS, 2mL/ ware (4.5cm2) room temperature 4h, 4 DEG C are standby With；
2) it by the DC of load sudden change polypeptide and the PBMC extracted before, is mixed with the ratio of 1:1~1:50, preferably 1: 10, and be transferred in the tissue culture plate or culture bottle for overlaying OMK25；
3) even, 37 DEG C of 5%CO are shaken2Culture, is denoted as the 0th day；
4) co-cultured cell, at the 5th day, is transferred to big culture bottle according to cell density by observation co-cultured cell situation In, fresh culture solution OKM-100+12%FBS is mended, 20mL is in 75cm2In culture bottle；
5) it co-cultures the 7th day, adds the fresh OKM-100+12%FBS of 20mL；
6) it co-cultures the 10th day, culture solution OKM-200+5%FBS, by co-cultured cell from 75cm2Bottle is transferred to 175cm2In big bottle；Specific transfer method: 25mL culture solution OKM-200+5%FBS piping and druming, then it is transferred to big bottle, it is repeated 2 times；With Culture solution OKM-200+5%FBS complements to 200mL.
7) when culture was to 14-21 days, it can be obtained AFF Protocols Cell.
7. polypeptide directly stimulates T cell to screen accurate polypeptide as antigen:
1) the AFF Protocols Cell of acquisition is collected by centrifugation, 1500rpm is centrifuged 5min and collects T-cells, and 10mL PBS weight is added Outstanding cell simultaneously counts, and 1500rpm is centrifuged 5min, collects T-cells with 1640+10%FBS+200U/mL IL2 resuspension, counts and adjust It is whole to 1 × 106cells/mL；
2) T-cells is divided into 96 hole flat undersides with the volley of rifle fire, 200 holes μ L/, cell number is 2 × 105cells；Distinguish again The mutant polypeptide that the step 3 of 10 μ L 1mg/mL synthesizes, final concentration of 50 μ g/mL is added, 3 multiple holes are arranged in every polypeptide；
3) positive control: T-cells+100ng/mL OKT3 is set；Negative control: 1640+10%FBS+200U/mL IL2；Two T-cells controls are as background release detection, respectively first time plus T-cells, and last time plus T- cells；Using the difference that two backgrounds discharge as systematic error；
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)；
8. accurate polypeptide judgment criteria:
1) positive control and negative control are normal, then illustrate that this data is credible；
2) polypeptide is as antigen using T-cells as baseline；
3) every group of experiment is two baselines, high baseline and low baseline, and the difference of two baselines is systematic error, data analysis When, to detected value > low baseline, > high baseline and > high baseline+systematic error, it is labeled respectively；Detected value > high baseline+ Systematic error is effective accurate polypeptide；
9. preparing AFF cell with the accurate polypeptide of screening
1) preparation of accurate polypeptide A FF cell is carried out in method 4,5,6；
10. the culture and separation of mutant antigen specific killing T cell:
1) with polypeptide directly as antigenic stimulus, the AFF cell obtained to step 9 is stimulated, after stimulating 12~72h, It is spare；
2) dyeing of CD8, CD137, IFN-γ are carried out to post-stimulatory cell, and is sorted with flow cytometer, are selected Select CD8+CD137+ or CD8+IFN- γ+cell；
The clone of 11.CD8+T cell TCR frequency detecting and high frequency TCR:
1) sorting obtains cell, carries out the extraction of genome immediately；
2) genome carries out TCR sequencing analysis and determines the TCR sequence of high frequency according to TCR distribution frequency；
3) mRNA of PBMC is extracted, reverse transcription is at from DNA, and according to the sequence of high frequency TCR, design primer, amplification obtains TCR Gene；
4) tcr gene expression vector, packaging virus are constructed；
12. constructing the CRISPR carrier that inhibitive ability of immunity signaling molecule knocks out
1) surface PBMC inhibitive ability of immunity 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；
13. the CRISPR carrier that building knocks out 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) step 3)~5 in repetition methods 12) complete TCR knockout carrier building and virus packaging；
14. the TCR-T that building knocks out inhibitive ability of immunity signaling molecule:
1) recovery PBMC, it is spare with magnetic bead sorting CD8+ cell；
2) to acquire virus in method 12 and 13, the CD8+T cell filtered out by step 10 is infected, while carrying out original The knockout of TCR and the knockout of inhibitive ability of immunity signaling molecule；
3) after infecting, 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；
4) 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；
5) cell situation and cell density are observed and co-cultured cell is transferred to 75cm at the 5th day2In bottle, mend fresh Culture solution OKM-100+12%FBS；
6) by cell from 75cm2175cm is transferred in bottle2Big bottle, culture solution OKM-200+5%FBS；
7) when culture was to 14-21 days, the TCR-T of knockout inhibitive ability of immunity signaling molecule, i.e. AFFT1 cell can be harvested.
15. constructing 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 AFFT1 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.
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 AFFT1 retrofit scheme cell, identify that specific T-cells (TCR+) ratio of tumour antigen is 70% or more；
3.AFFT1 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
The micro- sem observation DC form of Fig. 1
The efficiency of Fig. 2 DC load polypeptide
The detection of Fig. 3 AFF cell typing
The screening of the accurate polypeptide of Fig. 4
Fig. 5 flow cytometer detection specific T-cells ratio
Fig. 6 TCR distribution frequency
The knockout situation of Fig. 7 inhibition target spot
The knockout Efficiency testing of the original TCR of Fig. 8
The expression efficiency of Fig. 9 specificity TCR
Figure 10 flow cytometer detection killing-efficiency
The release of Figure 11 ELISA detection cell factor IFN-γ
Figure 12 animal lotus knurl model survivorship curve
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
1. full exon sequencing
1) peripheral blood from patients with lung cancer is taken, the sequencing and the detection of HLA parting of ctDNA are carried out；
2) sequencing information is analyzed using software: by ctDNA sequencing result compared with the genome of normal cell, sieve Select mutational site；
2. Epitope prediction
1) centered on the amino acid sites of mutation, extend 10 amino acid to two sides, by the more of 21 amino acid of this section Peptide is used as " potentially antigenic epitope "；
2) 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 "；
3. synthesis polypeptide
Epitope peptide symthesis method uses polypeptide solid-state reaction method (being synthesized by technical service company)
4. immortalizing DC
1) peripheral blood 100ml is extracted
2) Ficol density gradient centrifugation separates PBMC
3) U.S. day Ni Dendritic Cells separating kit isolating dendritic cells, are resuspended in culture medium.
4) the separated Dendritic Cells of TAX-GFP slow-virus infection, 37 DEG C of incubator static gas wave refrigerators, observation
5) after Cell-cloned growth, selected clone is cultivated respectively in 96 orifice plates
6) Phenotype is analyzed
7) it chooses ideal clone and is used as immortality DC；
5. immortality DC load sudden change polypeptide
1) prepare polypeptide solution: using step 3 synthesize mutant polypeptide prepared, every polypeptide final concentration of 10~ 100 μ g/mL, preferably 50 μ g/mL, it is spare；
2) the immortal DC of acquisition is collected by centrifugation, is resuspended with the polypeptide solution of preparation, and is more as being carried out in tissue culture plate Peptide load；
3) 37 DEG C of 5%CO2, 1~4h, preferably 4h are impacted, it is spare；
6. the DC and PBMC of load sudden change polypeptide are incubated for altogether
1) the pre- bed board of stimulating factor OKM-25,40 μ L OKM-25+4mL PBS, 2mL/ ware (4.5cm2) room temperature 4h, 4 DEG C are standby With；
2) it by the DC and PBMC of load sudden change polypeptide, is mixed, preferably 1:100, and is turned with the ratio of 1:50~1:500 It moves to and overlays in the tissue culture plate or culture bottle of OMK25；
3) even, 37 DEG C of 5%CO are shaken2Culture, is denoted as the 0th day；
4) co-cultured cell, at the 5th day, is transferred to big culture bottle according to cell density by observation co-cultured cell situation In, fresh culture solution OKM-100+12%FBS is mended, 20mL is in 75cm2In culture bottle；
5) it co-cultures the 7th day, adds the fresh OKM-100+12%FBS of 20mL；
6) it co-cultures the 10th day, culture solution OKM-200+5%FBS, by co-cultured cell from 75cm2That transfer in bottle To 175cm2In big bottle；Transfer method: 25mL culture solution OKM-200+5%FBS piping and druming, then it is transferred to big bottle, it is repeated 2 times；With training Nutrient solution OKM-200+5%FBS complements to 200mL.
8) when culture was to 14-21 days, it can be obtained AFF Protocols Cell.
7. polypeptide directly stimulates T cell to screen accurate polypeptide as antigen:
1) the AFF Protocols Cell of acquisition is collected by centrifugation, 1500rpm is centrifuged 5min and collects T-cells, and 10mL PBS weight is added Outstanding cell simultaneously counts, and 1500rpm is centrifuged 5min, collects T-cells with 1640+10%FBS+200U/mL IL2 resuspension, counts and adjust It is whole to 1 × 106cells/mL；
2) T-cells is divided into 96 hole flat undersides with the volley of rifle fire, 200 holes μ L/, cell number is 2 × 105cells；Distinguish again The mutant polypeptide synthesized in the step 3 of 10 μ L 1mg/mL, final concentration of 50 μ g/mL is added, 3 multiple holes are arranged in every polypeptide；
3) positive control: T-cells+100ng/mL OKT3 is set；Negative control: 1640+10%FBS+200U/mL IL2；Two T-cells controls are as background release detection, respectively first time plus T-cells, and last time plus T- cells；Using the difference that two backgrounds discharge as systematic error；
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)；
8. accurate polypeptide judgment criteria:
1) positive control and negative control are normal, then illustrate that this data is credible；
2) polypeptide is as antigen using T-cells as baseline；
3) every group of experiment is two baselines, high baseline and low baseline, and the difference of two baselines is systematic error, data analysis When, to detected value > low baseline, > high baseline and > high baseline+systematic error, it is labeled respectively；Detected value > high baseline+ Systematic error is effective accurate polypeptide；
9. preparing AFF cell with the accurate polypeptide of screening
1) preparation of accurate polypeptide A FF cell is carried out in method 4,5,6；
10. the culture and separation of mutant antigen specific killing T cell:
1) with the accurate polypeptide that filters out directly as antigenic stimulus, the AFF cell obtained to step 9 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；
The clone of 11.CD8+T cell TCR frequency detecting and high frequency TCR:
1) sorting obtains cell, carries out the extraction of genome immediately；
2) genome carries out TCR sequencing analysis and determines the TCR sequence of high frequency according to TCR distribution frequency；
3) mRNA of PBMC is extracted, reverse transcription is at from DNA, and according to the sequence of high frequency TCR, design primer, amplification obtains TCR Gene；
4) tcr gene expression vector, packaging virus are constructed；
12. constructing the CRISPR carrier that inhibitive ability of immunity signaling molecule knocks out
1) surface PBMC inhibitive ability of immunity 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；
13. the CRISPR carrier that building knocks out 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) step 3)~5 in repetition methods 12) complete TCR knockout carrier building and virus packaging；
14. the TCR-T that building knocks out inhibitive ability of immunity signaling molecule:
1) recovery PBMC, it is spare with magnetic bead sorting CD8+ cell；
2) to acquire virus, the CD8+T cell that infection step 10 obtains in method 12 and 13, while original TCR is carried out Knockout and inhibitive ability of immunity signaling molecule knockout；
3) after infecting, 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；
4) 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；
5) 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；
6) by cell from 75cm2175cm is transferred in bottle2Culture solution is OKM-200+5%FBS after big bottle；
7) when culture was to 14-21 days, the TCR-T of knockout inhibitive ability of immunity signaling molecule, i.e. AFFT1 cell can be harvested.
15. constructing 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 100-120mm3 or so, grouping feeds back cell, 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 AFFT1 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.
Experimental result
1. mutational site and Epitope prediction
Table 1 be the mutational site that detects of sequencing and Epitope prediction as a result, underscore mark be mutation amino Acid；
1 Epitope prediction of table
2. immortality DC morphologic observation
After inducing DC mature, microscope first observes form, sees it can be observed that apparent Dendritic Cells (Fig. 1)；
3.DC antigen load Efficiency testing
According to the mutant antigen of the synthesis prediction of table 1, and the label of biotin is carried out, after antigen load DC, with PE label Affine streptomysin detects biotin in the distribution situation of cell surface, to detect the efficiency of DC deduction polypeptide antigen；As a result such as Fig. 2 Shown: dark color is, without the testing result for loading labeling polypeptide, light color is the testing result of load biotinyl polypeptide, as a result table Bright: the load efficiency of DC is 99.4%；
The detection of 4.AFF Protocols Cell parting
After AFF Protocols Cell culture, the parting detection of CD4+, CD8+, NK and NKT cell is carried out, as a result such as Fig. 3 institute Show: it be 19.16%, NKT cell be 14.2%, NKT cell is 2.46% that CD8+T cell, which is 83.2%, CD4+T cell,；
5. with the accurate polypeptide of AFF cell screening
The T cell for being stimulated culture respectively with 10 polypeptides is detected effective polypeptide, tied by detecting the secretion of IFN-γ Fruit is as shown in Figure 4: the burst size of IFN-γ caused by No. 6 polypeptides > high baseline+systematic error, belongs to effective accurate polypeptide；
6. the identification and sorting of the T cell of pair accurate polypeptid specificity
With No. 6 polypeptides of screening, AFF Protocols Cell is stimulated, with flow cytometer detection to the T cell ratio of accurate polypeptid specificity Example discharges IFN- caused by No. 6 polypeptides as a result as shown in figure 5, (P5) is specific T-cells: AFF Protocols Cell in black box The cell proportion of γ, hence it is evident that higher than not having irritant cell (control), illustrate, AFF scheme can obtain the spy to accurate polypeptide Specific T cell；The sorting of CD8+IFN- γ+cell (in black box) is carried out with flow cytometer simultaneously；
7. identification and the clone of high frequency TCR
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 6 20) TCR3 distribution frequency is higher for frequency division cloth preceding, illustrates, this TCR and mutant antigen are closely related, according to TCR 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:
Base sequence:
Known TCR- β:
Amino acid:
Horizontal line is CDR3 sequence, the sequence for needing to be replaced
Replaced TCR- β:
Horizontal line is the CDR3 sequence of replacement
8. the detection of inhibition target spot knockout 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 7: the knockout efficiency highest of sgRNA1 can be effectively blocked inhibition signaling molecule The expression of PD-1；SgRNA preferred sgRNA1, sgRNA2, sgRNA3 and sgRNA4；The method can also be used, to Tim-3, LAG3, The inhibitions signaling molecules such as CTLA-4, BTLA, VISTA, CD160,2B4 (CD244) are knocked out；
3 inhibition target spot sgRNA sequence of table
9. the detection of inhibition target spot knockout efficiency
Using CRISPR technology, the inhibition target spot on PBMC is knocked out, as a result as shown in Figure 7: can effectively hinder The expression of disconnected inhibition signaling molecule；
10. the detection that original TCR knocks out efficiency
Using CRISPR technology, original TCR on PBMC is knocked out, as a result as shown in Figure 8: can effectively reduce The expression of original TCR, at this point, the transfection of expression specificity TCR slow virus can be carried out；
11. 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 9: the TCR of building can be 76.5% with normal expression, the cell proportion of TCR+
Lethal effect of the 12.AFFT1 cell to target cell
Killing effect is carried out with the target cell of AFF cell, AFFT cell and AFFT1 cell to mutant antigen epitope source respectively The detection of rate, using non-treated cell as control (Mock), the results are shown in Figure 10, compared with the control group, AFF cell, AFFT cell and AFFT1 cell have certain fragmentation effect to target cell, and to the killing-efficiency of tumour (in red block) AFFT1 > AFFT cell > AFF cell；Illustrate the T cell of expression specificity TCR, in addition the closing of inhibition target spot can be improved effectively To the killing-efficiency of tumour cell；
The detection of 13.AFFT1 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 in antitumor action, and Figure 11 is difference Training method cell, when being co-cultured with tumour cell, the detection of the IFN-γ of release, the results showed that produced with effector cell itself Raw IFN-γ compare (T cells only), with tumour cell co-culture after, AFF Protocols Cell, AFFT Protocols Cell and AFFT1 cell can produce a large amount of IFN-γ, especially AFFT and AFFT1 cell, due to expressing specificity TCR (AFFT), while inhibition signal has been knocked (AFFT1), the releasable more IFN-γ of effector cell, this result and killing Experimental result unanimously illustrates: the T cell of expression specificity TCR, in conjunction with inhibition target spot knockout can more effectively improve it is anti- Tumour ability；
14. constructing 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 12), The existence of AFFT1 cells against tumor tumor-bearing mice, which improves to have, significantly affects effect.
15. clinical case:
Certain male: 61 years old
Medical diagnosis on disease: double lung poorly differentiated adenocarcinomas；Left pulmonary tuberculosis
First course for the treatment of: monthly AFFT1 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year AFFT1 cell, quantity 1 × 109A cell, totally 2 times；
After administration, 20 months Progression free survivals；
Other cases:
Patient code Medical diagnosis on disease The CDR3 region sequence of high frequency TCR The Progression free survival time
1 Intrahepatic cholangiocarcinoma CATSRGTVSYEQYF 2016.4- so far
2 Oophoroma CASSQEGAFYGYTF 2016.5- so far
3 Oophoroma CASSIDHVSSSYNSPLHF 2017.4- so far
4 Sdenocarcinoma of stomach hepatic metastases CASSEGTESSYEQYF 2017.5- so far
5 Gastric cancer CASSIDGTATYEQYF 2017.11- so far
6 Lung cancer CASSYLSETYEQYF 2017.8- so far
7 The cancer of the esophagus CASSSRLAGGTDTQYF 2018.1- so far
8 Adenocarcinoma of lung CATSRDWLSNGNTEAFF 2018.3- so far
9 Adenocarcinoma of lung CATSIYSGETQYF 2018.3- so far
10 Gastric cancer CASSITEGSPLHF 2018.4- so far
Note: containing for " so far " is meant " on the day before the applying date "

Claims (5)

1. a kind of preparation method of AFFT1 cell, which is characterized in that the preparation of the AFFT cell the following steps are included:
1) mutational site is screened: by carrying out outside complete to peripheral blood in patients progress ctDNA exon sequencing, or with tumor tissues Aobvious son sequencing, filters out mutational site；
2) it synthesizes mutant polypeptide: Epitope prediction being carried out according to mutational site, synthesizes mutant polypeptide；
The Epitope prediction is to extend 10 amino acid to two sides, as potential centered on the amino acid sites of mutation Epitope；The potentially antigenic epitope of IC50 < 1000nM is determined as epitope by the IC50 for analyzing potentially antigenic epitope；
3) it obtains AFF cell: the Dendritic Cells in peripheral blood being infected using TAX-GFP slow virus, and chooses ideal Clone be used as immortality DC, and load the mutant polypeptide, be incubated for altogether with PBMC, acquisition AFF cell；
4) it obtains accurate polypeptide: using the mutant polypeptide as AFF cell described in antigenic stimulus, screening obtains accurate polypeptide；
5) it obtains AFF ' cell: AFF ' cell is prepared with the accurate polypeptide load DC cell；
6) using the accurate polypeptide as AFF ' cell described in antigenic stimulus, screening obtains the spy that can identify the accurate polypeptide Specific cell obtains the TCR β chain CDR3 region sequence of specific cell by sequencing, and by the known area a pair of TCR β chain CDR3 It is substituted for the CDR3 sequence；
7) it obtains AFFT1 cell: knocking out original tcr gene and surface inhibitive ability of immunity letter in the peripheral blood in patients T cell Number molecule, and be transferred to obtained in step 6) it is thin that AFFT1 can be prepared with the tcr gene in conjunction with accurate polypeptid specificity Born of the same parents.
2. preparation method described in claim 1, which is characterized in that the peripheral blood in patients is also possible to commercially available engineering cell system.
3. preparation method described in claim 2, which is characterized in that the engineering cell system be H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323 B16F1, CRL-2539 4T1, U14 Mouse Uterus Neck cancer cell, the small glioma cell of BV-2 mouse or G422 mouse glioma cell.
4. preparation method described in claim 1, which is characterized in that the tcr gene for knocking out patient peripheral's haemocyte and immune The method of inhibition signaling molecule is preferably CRISPR technology.
5. preparation method described in claim 1, which is characterized in that the surface inhibitive ability of immunity signaling molecule include: PD-1, Tim-3、LAG3、CTLA-4、BTLA、VISTA、CD160、2B4(CD244)、TIGIT。
CN201910438763.7A 2018-09-30 2019-05-24 Construction method of AFFT1 cells Active CN110408657B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811153159 2018-09-30
CN2018111531591 2018-09-30

Publications (2)

Publication Number Publication Date
CN110408657A true CN110408657A (en) 2019-11-05
CN110408657B CN110408657B (en) 2021-03-16

Family

ID=68357795

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910438763.7A Active CN110408657B (en) 2018-09-30 2019-05-24 Construction method of AFFT1 cells

Country Status (1)

Country Link
CN (1) CN110408657B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026328A1 (en) * 1996-01-17 1997-07-24 Imperial College Innovations Limited Immunotherapy using cytotoxic t lymphocytes (ctl)
CN106645677A (en) * 2016-11-15 2017-05-10 恒瑞源正（深圳）生物科技有限公司 Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026328A1 (en) * 1996-01-17 1997-07-24 Imperial College Innovations Limited Immunotherapy using cytotoxic t lymphocytes (ctl)
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
CN106645677A (en) * 2016-11-15 2017-05-10 恒瑞源正（深圳）生物科技有限公司 Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
姚堃等: "EBV-LMP2A 重组腺病毒体外转染树突状细胞激发特异性CTL 的研究", 《中国免疫学杂志》 *
朱登彦等: "负载k-ras抗原的树突状细胞诱导细胞毒性T淋巴细胞对肺癌细胞的体内杀伤作用", 《中华实验外科杂志》 *

Also Published As

Publication number Publication date
CN110408657B (en) 2021-03-16

Similar Documents

Publication Publication Date Title
CN110093376A (en) A kind of construction method of LRFFT1 cell
CN109136284A (en) A kind of AFFT2 cell
CN110157745A (en) A kind of construction method of HAFFT1 cell
CN110408657A (en) A kind of construction method of AFFT1 cell
CN109337930A (en) A kind of AFFT1 cell
CN109136278A (en) A kind of MRFFT1 cell
CN110205341A (en) A kind of construction method of LRFFT2 cell
CN110129373A (en) A kind of construction method of RFF1 cell
CN109679917A (en) A kind of LRFFT2 cell
CN109294997A (en) A kind of LRFFT1 cell
CN109294982A (en) A kind of RFF2 cell
CN110093373A (en) A kind of construction method of AFFT2 cell
CN109295106A (en) A kind of HAFFT1 cell
CN109294998A (en) A kind of RFF1 cell
CN110129371A (en) A kind of construction method of RFFT2 cell
CN110129372A (en) A kind of construction method of RFFT1 cell
CN110093374A (en) A kind of construction method of MRFFT1 cell
CN109294996A (en) A kind of RFFT2 cell
CN109136279A (en) A kind of construction method of MRFFT2 cell
CN110129374A (en) A kind of construction method of RFFT cell
CN109295097A (en) A kind of MRFFT2 cell
CN109294995A (en) A kind of RFFT1 cell
CN110129269A (en) A kind of construction method of RFF2 cell
CN109280643A (en) A kind of RFFT cell
CN110093316A (en) A kind of construction method of AFF cell

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