CN110157745A - A kind of construction method of HAFFT1 cell - Google Patents

A kind of construction method of HAFFT1 cell Download PDF

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CN110157745A
CN110157745A CN201910438772.6A CN201910438772A CN110157745A CN 110157745 A CN110157745 A CN 110157745A CN 201910438772 A CN201910438772 A CN 201910438772A CN 110157745 A CN110157745 A CN 110157745A
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cell
tcr
polypeptide
hafft1
construction method
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CN110157745B (en
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焦顺昌
张嵘
张天赋
周子珊
陈小彬
李营营
彭刚
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Abstract

The invention belongs to field of biotechnology, and in particular to a kind of construction method of HAFFT1 cell.This method will be by being transformed by the small numerator modified post-stimulatory cell of polypeptide with TCR-T technology; improved T cell carries out the knockout of immunosupress target spot again; it accurately protects specific killing T cell from inhibiting in vivo, improves T cell to the lethality of tumour cell.By the cell of HAFFT1 retrofit scheme, identify that specific T-cells (TCR+) ratio of tumour antigen is 80% or more.

Description

A kind of construction method of HAFFT1 cell
Technical field:
The invention belongs to field of biotechnology, specifically, being related to a kind of construction method of HAFFT1 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.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 is carried out although also having and attempting the method that slow virus is carrier, safety, Convenience is not so good as polypeptide mode.And the direct stimulation of polypeptide is simply mixed, although simple and convenient, efficiency is lower.Specific essence The secondary stimulus of quasi- polypeptide is more direct not as good as the tumour specific antigen of T cell receptor transduction.Existing TCR-T is in treatment blood In tumour 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 tumor microenvironment.
Summary of the invention:
In order to solve the above-mentioned technical problem, the present invention will provide a kind of construction method of HAFFT1 cell, and this method will be through It crosses and is transformed by the small numerator modified post-stimulatory cell of polypeptide with TCR-T technology, improved T cell is exempted from again Epidemic disease inhibits the knockout of target spot, accurately protects specific killing T cell from inhibiting in vivo, it is thin to tumour to improve T cell The lethality of born of the same parents.
The construction method of the HAFFT1 cell, including following key step: 1) extracting peripheral blood in patients, carries out ctDNA Exon sequencing, or full exon sequencing is carried out with tumor tissues, mutational site is filtered out, Epitope prediction is carried out, is closed At mutant polypeptide and carry out small numerator modified；2) DC is immortalized using peripheral blood preparation, and loaded described through small numerator modified mistake Mutant polypeptide, be incubated for altogether with PBMC, obtain HAFF cell；3) use the mutant polypeptide through small numerator modified mistake as antigen The HAFF cell is stimulated, screening obtains accurate polypeptide；4) DC cell is immortalized with the accurate polypeptide load to incubate altogether with PBMC It educates, prepares HAFF ' cell；5) using the accurate polypeptide as antigenic stimulus HAFF ' cell, screening, which obtains, can identify the essence The specific T-cells of quasi- polypeptide obtain the high frequency TCR of specific cell by sequencing, knock out original TCR and carry out high frequency TCR Expression, construct TCR-T cell；6) knockout that above-mentioned TCR-T cell is carried out to inhibition signaling molecule, is prepared HAFFT1 Cell.
Specific preparation process is as follows for HAFFT1 cell:
1, full exon sequencing
It carries out ctDNA sequencing using source of people peripheral blood or carries out full exon sequencing with tumor tissues to survey, sequencing is tied Fruit filters out mutational site compared with the genome of normal cell；
The peripheral blood is also possible to commercially available engineering cell system, as H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323B16F1, CRL-2539 4T1, U14 mouse carcinoma of uterine cervix cell, BV- The small glioma cell of 2 mouse, G422 mouse glioma cell etc., carry out full exon sequencing；
2, Epitope prediction
(1) centered on the amino acid sites of mutation, extend 8 amino acid to two sides, by the more of 17 amino acid of this section Peptide is used as " potentially antigenic epitope "；
(2) IC50 of potentially antigenic epitope is analyzed using forecasting software, the potentially antigenic epitope of IC50 < 1000nM is true It is set to " epitope "；
3, it synthesis polypeptide and modifies
(1) " epitope " is synthesized into mutant polypeptide；
(2) modified small molecule (H4) and polypeptide antigen (P) using following on-link mode (OLM) (aminoterminal to c-terminus: N-C): H-P, P-H, H-P-H；
4, DC load sudden change polypeptide is immortalized
(1) it by the Dendritic Cells in peripheral blood, is infected using TAX-GFP slow virus, and chooses ideal clone As immortal DC；
(2) above-mentioned immortality DC will be loaded by small numerator modified antigen polypeptide；
5, the DC and PBMC of load sudden change polypeptide are incubated for altogether
The DC of load sudden change polypeptide and PBMC is incubated for altogether, can be obtained HAFF cell；
6, accurate polypeptide is screened
The T cell in HAFF cell is collected, T cell is stimulated using the small numerator modified mutant polypeptide that step 3 obtains, passes through Check that accurate polypeptide is screened in the secretion of IFN-γ；
7, HAFF ' cell is prepared with the accurate polypeptide of screening
Accurate polypeptide HAFF ' cell is prepared with accurate polypeptide substitution mutant polypeptide repetition step 3,4 and 5；
8, the determination of specific cell high frequency TCR and expression vector establishment
(1) to stimulate by small numerator modified accurate polypeptide HAFF ' cell, post-stimulatory cell is carried out The dyeing of CD8, CD137, IFN-γ select CD8+CD137+ or CD8+IFN- γ+T cell；Extract genome and to TCR It carries out sequencing analysis and the TCR sequence of high frequency is determined according to TCR distribution frequency；
(2) according to the sequence of high frequency TCR, design primer expands and obtains tcr gene；Tcr gene expression vector is constructed, and Packaging virus；
9, the CRISPR carrier that building inhibitive ability of immunity signaling molecule knocks out, and carry out viral packaging；
10, building knocks out the CRISPR carrier of TCR, and carries out viral packaging；
11, TCR is transformed
To acquire virus in step 9 and 10, the CD8+T cell filtered out by step 8 is infected, striking for original TCR is carried out It removes and the knockout of inhibitive ability of immunity signaling molecule；It is transferred to the TCR expression vector of step 8 building again；It is prepared HAFFT1 cell；
The inhibition signaling molecule can be PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160 or 2B4 (CD244)、TIGIT。
The utility model has the advantages that
1. HAFFT1 cell provided by the invention, using tumour antigen as mutant antigen, different from other tissues, target spot is single-minded Property is strong, is not susceptible to undershooting-effect, highly-safe；
2. the specific cell ratio that the present invention obtains is high, the specific cell of tumour antigen usually can be identified, PBMC be distributed as 0.5% hereinafter, by HAFFT1 retrofit scheme cell, identify the specific T-cells (TCR of tumour antigen +) ratio be 80% or more；
3. the HAFFT1 cell that the present invention obtains is due to carrying out the inhibitive abilities of immunity target spot such as PD1, CTLA4, TIM3, LAG3 It knocks out, therefore, unrestricted to the killing ability of tumour, 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 LDH release detection killing-efficiency；
The release of Figure 11 ELISA detection cell factor IFN-γ；
The existence of Figure 12 HAFFT1 cells against tumor tumor-bearing mice improves situation.
Specific embodiment:
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Base Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts it is all its His embodiment, shall fall within the protection scope of the present invention.
Explanation for specific term HAFFT1, wherein H: small numerator modified polypeptide antigen technology；A: DC skill is immortalized Art；FF: mixed polypeptide stimulating technology；T:TCR-T technology；1: target spot knocks out guard technology.HAFFT1 cell is indicated using above-mentioned The cell that technical tie-up prepares.
Embodiment 1
The present embodiment will be by taking patients with lung cancer as an example, and providing has targetedly HAFFT1 cell and preparation method thereof:
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 8 amino acid to two sides, by the polypeptide of 17 amino acid of this section 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
Entrust technical service company that " epitope " is synthesized mutant polypeptide；
4. small numerator modified polypeptide
1) small molecule (H4) and the on-link mode (OLM) of polypeptide antigen (P) are following (aminoterminal is to c-terminus: N-C): H-P, P-H, H-P-H；
2) by small numerator modified polypeptide H-P or P-H or H-P-H, the culture T cell in a manner of mixed polypeptide stimulation is obtained In the T cell obtained, polypeptide antigen (P) individually impact culture is higher than to the ratio of the T cell of antigen polypeptid specificity；
5. immortalizing DC
1) peripheral blood in patients 100ml is extracted；
2) Ficol density gradient centrifugation separates PBMC；
3) U.S. day Ni Dendritic Cells separating kit isolating dendritic cells are used, 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；
6. immortality DC load it is small numerator modified after mutant polypeptide；
1) it prepares polypeptide solution: being prepared using the mutant polypeptide after step 4 modification, final concentration of the 10 of every polypeptide ~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；
7. 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 It is spare；
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 75cm2It is transferred in bottle 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 culture Liquid OKM-200+5%FBS complements to 200mL.
8) when culture was to 14-21 days, it can be obtained HAFF Protocols Cell.
8. polypeptide directly stimulates T cell to screen accurate polypeptide as antigen:
1) the HAFF Protocols Cell of acquisition is collected by centrifugation, 1500rpm is centrifuged 5min and collects T cell, and 10mL PBS weight is added Outstanding cell simultaneously counts, and 1500rpm is centrifuged 5min, collects T cell, with 1640+10%FBS+200U/mL IL2 resuspension, counts and adjusts It is whole 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 small numerator modified mutant polypeptide in the step 4 of 10 μ L 1mg/mL, final concentration of 50 μ g/mL, every polypeptide setting 3 is answered Hole；
3) positive control: T cell+100ng/mL OKT3 is set；Negative control: 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 sample is placed in- 80 DEG C of preservations)；
9. accurate polypeptide judgment criteria:
1) positive control and negative control are normal, then illustrate that this data is credible, on the contrary then need to re-start test；
2) it is effective accurate polypeptide that experimental group, which is significantly higher than negative control group,；
10. preparing HAFF ' cell with the accurate polypeptide of screening
1) preparation of accurate polypeptide HAFF ' cell is carried out with step 4,5,6,7；
11. the culture and separation of mutant antigen specific killing T cell:
1) with the accurate polypeptide that filters out directly as antigenic stimulus, the HAFF ' cell obtained to step 10 is stimulated, It is spare after stimulating 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+T cell；
The clone of 12.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 cDNA, and according to the sequence of high frequency TCR, design primer, amplification obtains TCR Gene；
4) tcr gene expression vector, packaging virus are constructed；
13. 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；
14. 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 13) complete TCR knockout carrier building and virus packaging；
15. 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 11 obtains in method 13 and 14, 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 of step 12 building Expression vector；
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 for knocking out inhibitive ability of immunity signaling molecule can be harvested, acquisition HAFFT1 is thin Born of the same parents；
16. 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 HAFFT1 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 is the mutational site and Epitope prediction result that sequencing detects；
1 Epitope prediction of table
2. immortality DC morphologic observation
After inducing DC mature, microscope first observes form, it can be observed that apparent Dendritic Cells (Fig. 1)；
3. flow cytometer detection DC antigen load efficiency
According to the mutant antigen of the synthesis prediction of table 1, and FITC label is carried out, after antigen load DC, uses flow cytometer The efficiencies that detection DC offers polypeptide antigen show: the load efficiency of DC is 99.6% (such as Fig. 2)；
The detection of 4.HAFF Protocols Cell parting
After HAFF Protocols Cell culture, the parting detection of CD4+ and CD8+ cell is carried out, as a result as shown in Figure 3: CD8 + T cell is that 68%, CD4+T cell is 9.45%；
5. with the accurate polypeptide of HAFF 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 No. 5 polypeptides stimulation T cell secretion of gamma-IFN as shown in Figure 4 is significantly higher than the IFN-γ of control group secretion release, is determined as having Imitate polypeptide.
6. the identification and sorting of the T cell of pair accurate polypeptid specificity
With No. 5 polypeptides of screening, HAFF ' Protocols Cell is stimulated, with flow cytometer detection to the T cell ratio of accurate polypeptid specificity Example, as a result as shown in figure 5, being specific T-cells: HAFF ' Protocols Cell in box, release IFN-γ caused by No. 5 polypeptides Cell proportion, hence it is evident that higher than not having irritant cell (control), illustrate, HAFF ' scheme can be obtained to the special of effective polypeptide Property T cell；The sorting of CD8+IFN- γ+T cell (in 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 10) TCR2 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:
MMKSLRVLLV ILWLQLSWVW SQQKEVEQNS GPLSVPEGAI ASLNCTYSDR GSQSFFWYRQ
YSGKSPELIM FIYSNGDKED GRFTAQLNKA SQYVSLLIRD SQPSDSATYL CAVNFGGGKL
IFGQGTELSV KPN
Base sequence:
Known TCR- β:
Amino acid:
MRIRLLCCVA FSLLWAGPVI AGITQAPTSQ ILAAGRRMTL RCTQDMRHNA MYWYRQDLGL
GLRLIHYSNT AGTTGKGEVP DGYSVSRANT DDFPLTLASA VPSQTSVYFC ASSLSFGTEA
FFGQGTRLTV V (horizontal line is CDR3 sequence, the sequence for needing to be replaced)
Replaced TCR- β:
MRIRLLCCVA FSLLWAGPVI AGITQAPTSQ ILAAGRRMTL RCTQDMRHNA MYWYRQDLGL
GLRLIHYSNT AGTTGKGEVP DGYSVSRANT DDFPLTLASA VPSQTSVYFCASSQGSSGRAKNIQYF
GQGTRLTVV (the CDR3 sequence that horizontal line is replacement)
9. the detection of inhibition target spot knockout efficiency
Using CRISPR technology, the inhibition target spot PD-1 on CD8+T cell is knocked out, sgRNA sequence is shown in Table 3, The knockout efficiencies of inhibition target spot are as shown in Figure 7: the knockout efficiency highest of sgRNA1 can be effectively blocked inhibition signal The expression of molecule PD-1；The method can also be used, to Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,2B4 (CD244) etc. inhibitions signaling molecule is knocked out；
3 inhibition target spot sgRNA sequence of table
10. the detection that original TCR knocks out efficiency
Using CRISPR technology, original TCR is knocked out, as a result as shown in Figure 8: can effectively reduce original TCR Expression, 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 CD8+T cell of specificity TCR, at the 7th day, with the expression of flow cytometer detection TCR Efficiency, as a result as shown in Figure 9: the TCR of building can be 88.4% with normal expression, the cell proportion of TCR+.
Lethal effect of the 12.HAFFT cell to target cell
It is killed respectively with the target cell of HAFF ' cell, HAFFT cell and HAFFT1 cell to mutant antigen epitope source Hurt the detection of efficiency, effect target ratio is set as 40:1, and using non-treated cell as control (Mock), the results are shown in Figure 10, with Control group is compared, and HAFF ' cell, HAFFT cell and HAFFT1 cell have certain fragmentation effect to target cell, and to tumour Killing-efficiency HAFFT1 > HAFFT cell > HAFF ' cell；Illustrate the T cell of expression specificity TCR, in addition inhibition target spot Knock out the killing-efficiency that can effectively improve to tumour cell；
The detection of 13.HAFFT1 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 with effector cell itself (T Cell) it is compared with the IFN-γ for effector cell (MOCK) generation for not doing any transformation, after being co-cultured with tumour cell, HAFF ' Protocols Cell, HAFFT Protocols Cell and HAFFT1 cell can produce a large amount of IFN-γ, especially HAFFT and HAFFT1 is thin Born of the same parents, due to expressing specificity TCR (HAFFT), while inhibition signal has been knocked (HAFFT1), and effector cell is releasably more More IFN-γ, this result is consistent with killing experiments result to be illustrated: the T cell of expression specificity TCR, in conjunction with inhibition target spot Closing can more effectively improve anti-tumor capacity；
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 HAFFT1 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 HAFFT1 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year HAFFT1 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 CASSSNDNIPYNEQFF 2016.8- so far
2 Oophoroma CASSTERPEQYF 2016.7- so far
3 Oophoroma CSAPSGLAGALFYEQYF 2016.9- so far
4 Sdenocarcinoma of stomach hepatic metastases CASTSLDYEQYF 2017.1- so far
5 Gastric cancer CASSFGRERGFYNEQFF 2017.5- so far
6 Lung cancer CASSPRDRGLTNYGYTF 2017.5- so far
7 The cancer of the esophagus CASSQEILGGGTDTQYF 2017.9- so far
8 Adenocarcinoma of lung CASSSGKWGTEAFF 2018.1- so far
9 Adenocarcinoma of lung CASSDWPLNEQFF 2018.3- so far
10 Gastric cancer CASSPGTSRDNEQFF 2018.4- so far
Note: containing for " so far " is meant " on the day before the applying date ".

Claims (7)

1. a kind of construction method of HAFFT1 cell, which comprises the following steps: loaded with accurate polypeptide and immortalize DC Cell, and be incubated for altogether with PBMC, prepare HAFF ' cell；Using accurate polypeptide as antigenic stimulus HAFF ' cell, screening obtains energy The specific T-cells for enough identifying the accurate polypeptide obtain the high frequency TCR of specific cell by sequencing, knock out original TCR simultaneously The expression of high frequency TCR is carried out, TCR-T cell is constructed；Above-mentioned TCR-T cell is carried out to the knockout of inhibition signaling molecule, preparation Obtain HAFFT1 cell；
The accurate polypeptide the preparation method is as follows:
(1) sequencing of ctDNA exon is carried out by human peripheral blood, or carries out full exon sequencing with tumor tissues, filtered out Mutational site carries out Epitope prediction, synthesizes mutant polypeptide and carries out small numerator modified；
Small molecule (H) and polypeptide antigen (P) is modified (aminoterminal is to c-terminus: N-C) using following on-link mode (OLM): H-P, P-H,H-P-H；
(2) it by the Dendritic Cells in peripheral blood, is infected using TAX-GFP slow virus, and chooses ideal clone's conduct Immortal DC, and the mutant polypeptide through small numerator modified mistake is loaded, it is incubated for altogether with PBMC, obtains HAFF cell；
(3) use the mutant polypeptide through small numerator modified mistake as HAFF cell described in antigenic stimulus, by checking IFN-γ Secretion screening obtain accurate polypeptide.
2. the construction method of HAFFT1 cell as described in claim 1, which is characterized in that the peripheral blood in patients is also possible to city Sell engineering cell system.
3. the construction method of HAFFT1 cell as claimed 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 carcinoma of uterine cervix cell, the small glioma cell of BV-2 mouse or G422 mouse glioma cell.
4. the construction method of HAFFT1 cell as described in claim 1, which is characterized in that the prediction of the epitope is with prominent Centered on the amino acid sites of change, extend 8 amino acid to two sides, as potentially antigenic epitope；Analyze potentially antigenic epitope The potentially antigenic epitope of IC50 < 1000nM is determined as epitope by IC50.
5. the construction method of HAFFT1 cell as described in claim 1, which is characterized in that the determination method of high frequency TCR is as follows: with HAFF ' cell is stimulated by small numerator modified accurate polypeptide, CD8, CD137, IFN- are carried out to post-stimulatory cell The dyeing of γ selects CD8+CD137+ or CD8+IFN- γ+T cell；It extracts genome and sequencing analysis, root is carried out to TCR According to TCR distribution frequency, the TCR sequence of high frequency is determined.
6. the construction method of HAFFT1 cell as described in claim 1, which is characterized in that knock out the TCR of patient peripheral's haemocyte The method of gene is preferably CRISPR technology.
7. the construction method of HAFFT1 cell as described in claim 1, which is characterized in that the inhibitive ability of immunity signaling molecule packet It includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,2B4 (CD244), TIGIT.
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