CN109136268A - A kind of construction method of LRFF cell - Google Patents
A kind of construction method of LRFF cell Download PDFInfo
- Publication number
- CN109136268A CN109136268A CN201811153227.4A CN201811153227A CN109136268A CN 109136268 A CN109136268 A CN 109136268A CN 201811153227 A CN201811153227 A CN 201811153227A CN 109136268 A CN109136268 A CN 109136268A
- Authority
- CN
- China
- Prior art keywords
- cell
- lrff
- polypeptide
- epitope
- construction method
- 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.)
- Withdrawn
Links
- 238000010276 construction Methods 0.000 title claims description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 103
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 69
- 229920001184 polypeptide Polymers 0.000 claims abstract description 57
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 55
- 241000700605 Viruses Species 0.000 claims abstract description 28
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 25
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- 238000012163 sequencing technique Methods 0.000 claims abstract description 11
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 8
- 239000011886 peripheral blood Substances 0.000 claims abstract description 8
- 230000000869 mutational effect Effects 0.000 claims abstract description 6
- 239000000427 antigen Substances 0.000 claims description 27
- 108091007433 antigens Proteins 0.000 claims description 27
- 102000036639 antigens Human genes 0.000 claims description 27
- 210000000612 antigen-presenting cell Anatomy 0.000 claims description 18
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 230000000890 antigenic effect Effects 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 210000004443 dendritic cell Anatomy 0.000 claims description 4
- 210000002540 macrophage Anatomy 0.000 claims description 4
- 210000004493 neutrocyte Anatomy 0.000 claims description 4
- 239000002574 poison Substances 0.000 claims description 3
- 231100000614 poison Toxicity 0.000 claims description 3
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 210000004698 lymphocyte Anatomy 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims 2
- 239000008280 blood Substances 0.000 claims 2
- 210000005087 mononuclear cell Anatomy 0.000 claims 1
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 32
- 210000004881 tumor cell Anatomy 0.000 abstract description 10
- 238000000338 in vitro Methods 0.000 abstract description 4
- 230000009466 transformation Effects 0.000 abstract description 4
- 231100000225 lethality Toxicity 0.000 abstract description 2
- 230000035939 shock Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 13
- 102100037850 Interferon gamma Human genes 0.000 description 11
- 108010074328 Interferon-gamma Proteins 0.000 description 11
- 238000000034 method Methods 0.000 description 9
- 238000001890 transfection Methods 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- 239000012636 effector Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000009650 gentamicin protection assay Methods 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 108010008038 Synthetic Vaccines Proteins 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 238000013528 artificial neural network Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 206010027457 Metastases to liver Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 230000009948 RNA mutation Effects 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000003116 impacting effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- 230000008466 key signal transduction pathway Effects 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
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
- 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
- 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
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention carries out ctDNA sequencing using peripheral blood in patients 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, and filters out effective accurate polypeptide, the second Secondary Shocks stimulated by accurate polypeptide, general T cell is transformed into the LRFF cell for more precisely killing ability, improves T cell to the lethality of tumour cell.The LRFF cell that invention provides can be widely applied to individuation and precisely treat entity tumor.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of construction method of LRFF cell.
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.
The above treatment method has limitation, especially external evoked DC cell and DC cell loading tumour antigen technology reason
It is more by upper research, but there are many more problems in the specific implementation process, lack specific, tumour cell occurrence and development key
Signal transduction pathway relevant molecule is as inducing antigen, because tumour antigen is unknown and the immunosuppressive obstacle of tumor microenvironment,
Make to 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
There are progress external cultivation and amplification in vitro altogether, allows more thin specific cell directly facing complicated tumour immunity micro-loop
Border, therefore, it is difficult to play expected effect.Although also have can also external submission and total cultivation, the single (MAGE- of target spot
3) it, only works 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,
But 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.
Above scheme is accurately and effectively analyzed general lack of to patient's antigen, and more efficient way should should consider to pacify
Full property problem, takes into account antigen submission efficiency again, can be only achieved the purpose precisely treated in this way.
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 accurate polypeptide is selected, the second Secondary Shocks stimulated by accurate polypeptide, general T cell, which is transformed into, to be had more precisely
The LRFF cell of killing ability improves T cell to the lethality of tumour cell.
LRFF 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
Such as: LRFF cell is finally obtained via above-mentioned L, R, FF items technical solution or technological means transformation
Cell.
LRFF 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-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 MHC type detection
Detection RNA mutation is sequenced with full exon；
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, filters out the specificity that can identify the accurate polypeptide
Cell is to get LRFF cell；
7, building specific antigen expression target cell and tumor model survival assay.
Beneficial effects of the present invention:
1. novel specific LRFF cell of the present invention finds out accurate polypeptide and carries out secondary stimulus on the basis of LV cell,
Having better solved patient's immunocyte cannot accurately identify and the problem of killing tumor cell, at the same compensate for LV cell and
The deficiency of RFF cell, the killing-efficiency of RFF are not so good as LRFF cell, and the specificity of LV cell is not so good as LRFF cell.
2. tumour antigen is mutant antigen, different from other tissues, target spot specificity is strong, is not susceptible to undershooting-effect, pacifies
Quan Xinggao；
3. 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 LRFF retrofit scheme cell, identify that specific T-cells (TCR+) ratio of tumour antigen is
10% or more.
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: LRFF 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: LDH release detection cell killing efficiency.
The release of Fig. 6: ELISA detection cell factor IFN-γ.
Fig. 7: 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 100；It is mixed
After even, draw 10 μ L and second pipe mixing is added, be denoted as 10-1；And so on (100-10-8), 10 μ are added in corresponding cell hole
Virus liquid that 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): Human IFN-gamma DuoSet 15plate (article No.:
DY285B) × 2 (article No.: DY008) × 31, DuoSet ELISAAncillary 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；
8, 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 LRFF cell 1 × 107, second, which is carried out, after injecting cell 7 days for the first time infuses
It penetrates, 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+, 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 group), illustrate, LRFF scheme can be obtained to accurate
The specific T-cells of polypeptide；
6, lethal effect of the LRFF cell to target cell
Carry out the detection of killing-efficiency to the target cell in mutant antigen epitope source with control cell and LRFF cell respectively,
Using non-treated cell as control (Mock), effect target ratio is set as 40:1, as a result as shown in figure 5, compared with the control group, LRFF
Cell has stronger fragmentation effect to target cell.
7, the detection of LRFF 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, Fig. 6 LRFF
When cell and tumour cell 1:1 are co-cultured, the detection of the IFN-γ of release, the results showed that generated with effector cell itself
IFN-γ compares (T cells only), and after co-culturing with tumour cell, LRFF cell can produce this result of a large amount of IFN-γ
Consistent with killing experiments result to illustrate: the specific T-cells that accurate polypeptide stimulates again can more effectively improve antitumor energy
Power.
8, 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 (Fig. 7),
The existence of LRFF cells against tumor tumor-bearing mice, which improves to have, significantly affects effect.
9, clinical case:
Certain male: 57 years old
Medical diagnosis on disease: left kidney neoplasms；
First course for the treatment of: monthly LRFF cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year LRFF cell, quantity 1 × 109A cell, totally 2 times；
After administration, 22 months Progression free survivals；
Other cases:
| Patient code | Medical diagnosis on disease | The Progression free survival time |
| 1 | Sdenocarcinoma of stomach hepatic metastases | 2015.4- so far |
| 2 | Gastric cancer | 2015.4- so far |
| 3 | Lung cancer | 2015.5- so far |
| 4 | Adenocarcinoma of lung | 2015.5- so far |
| 5 | Adenocarcinoma of lung | 2015.5- so far |
| 6 | The cancer of the esophagus | 2015.6- so far |
Note: containing for " so far " is meant " on the day before the applying date ".
Claims (4)
1. a kind of construction method of LRFF cell, which is characterized in that 1) construction method includes the following steps: using outside source of people
All blood carries out ctDNA sequencing or tumor tissues carry out full exon sequencing, filters out mutational site；2) it is carried out according to mutational site
Epitope prediction synthesizes the gene order of mutant polypeptide；3) slow virus carrier of building expression mutant polypeptide packs slow disease
Poison；4) it transfects antigen presenting cell and is co-cultured with PBMC, obtain LFF cell；4) mutant polypeptide is as antigenic stimulus institute
LFF cell is stated, effective accurate polypeptide is filtered out；5) it stimulates the LFF cell again using the accurate polypeptide as antigen, sieves
The specific cell that can identify the accurate polypeptide is selected to get LRFF cell.
2. the construction method of LRFF cell as described in claim 1, which is characterized in that the source of people peripheral blood is also possible to city
Sell engineering cell system.
3. the construction method of LRFF cell as described in claim 1, which is characterized in that the Epitope prediction is to be mutated
Amino acid sites centered on, respectively extend 8 amino acid to two sides, using the polypeptide of 17 amino acid of this section as potentially antigenic table
Position；The IC50 that potentially antigenic epitope is analyzed using forecasting software thinks that this potentially antigenic epitope is anti-if IC50 < 1000nM
Former epitope.
4. the construction method of LRFF cell as described in claim 1, which is characterized in that the antigen presenting cell includes: outer
All blood mononuclear cells, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811153227.4A CN109136268A (en) | 2018-09-30 | 2018-09-30 | A kind of construction method of LRFF cell |
| CN201910443232.7A CN110093371B (en) | 2018-09-30 | 2019-05-24 | Construction method of LRFF cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811153227.4A CN109136268A (en) | 2018-09-30 | 2018-09-30 | A kind of construction method of LRFF cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109136268A true CN109136268A (en) | 2019-01-04 |
Family
ID=64813682
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811153227.4A Withdrawn CN109136268A (en) | 2018-09-30 | 2018-09-30 | A kind of construction method of LRFF cell |
| CN201910443232.7A Active CN110093371B (en) | 2018-09-30 | 2019-05-24 | Construction method of LRFF cell |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910443232.7A Active CN110093371B (en) | 2018-09-30 | 2019-05-24 | Construction method of LRFF cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN109136268A (en) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015533473A (en) * | 2012-07-12 | 2015-11-26 | ペルシミューン，インコーポレイテッド | Individual cancer vaccine and adaptive immune cell therapy |
| CN103242448B (en) * | 2013-05-27 | 2015-01-14 | 郑州大学 | Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof |
| US20160320408A1 (en) * | 2013-12-19 | 2016-11-03 | Opexa Therapeutics, Inc. | Methods of t cell epitope profiling, making t cell compositions, and treating diseases |
| CN104099297A (en) * | 2014-07-24 | 2014-10-15 | 南通大学附属医院 | Polygene transfection tumor cell strain and fusion vaccine thereof, as well as preparation methods |
| CA2963362A1 (en) * | 2014-10-02 | 2016-04-07 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of isolating t cell receptors having antigenic specificity for a cancer-specific mutation |
-
2018
- 2018-09-30 CN CN201811153227.4A patent/CN109136268A/en not_active Withdrawn
-
2019
- 2019-05-24 CN CN201910443232.7A patent/CN110093371B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110093371B (en) | 2021-06-18 |
| CN110093371A (en) | 2019-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110499297A (en) | A kind of novel oncolytic virus and its preparation method and application | |
| CN109553686A (en) | The novel regulatable double Chimeric antigen receptor T cells of one kind and its construction method and application | |
| CN110205341A (en) | A kind of construction method of LRFFT2 cell | |
| CN110093376A (en) | A kind of construction method of LRFFT1 cell | |
| CN105367661B (en) | Chimeric antigen receptor and its gene and recombinant expression carrier, the NKT cell of engineering HER1 targeting and its application | |
| 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 | |
| CN119752807A (en) | A monoclonal cell expressing HPV18 E6 protein and its application in constructing HPV tumor animal model | |
| CN109136268A (en) | A kind of construction method of LRFF cell | |
| CN109337873A (en) | A kind of LRFF cell | |
| CN109679917A (en) | A kind of LRFFT2 cell | |
| CN108823247A (en) | A kind of humanization CD-19 Chimeric antigen receptor T lymphocyte carrier and its application | |
| CN109136278A (en) | A kind of MRFFT1 cell | |
| CN110157745A (en) | A kind of construction method of HAFFT1 cell | |
| CN109294982A (en) | A kind of RFF2 cell | |
| CN109295097A (en) | A kind of MRFFT2 cell | |
| CN109136279A (en) | A kind of construction method of MRFFT2 cell | |
| CN109136281A (en) | A kind of construction method of MRFFT1 cell | |
| CN110129371A (en) | A kind of construction method of RFFT2 cell | |
| CN109294983A (en) | A kind of LFF2 cell | |
| CN110129276A (en) | A kind of construction method of LFF1 cell | |
| CN114569709B (en) | Preparation method and application of melanoma autologous tumor vaccine with high ADAM-28 expression | |
| CN109679916A (en) | A kind of LFF1 cell | |
| CN110093317A (en) | A kind of construction method of LFF2 cell | |
| CN110129373A (en) | A kind of construction method of RFF1 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 | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190104 |