CN109337873A

CN109337873A - A kind of LRFF cell

Info

Publication number: CN109337873A
Application number: CN201811153193.9A
Authority: CN
Inventors: 焦顺昌; 张嵘; 张天赋; 周子珊; 解佳森; 王海燕; 吴子明
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2019-02-15

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

A kind of LRFF cell

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of LRFF cell and preparation method thereof.

Background technique

Currently, existing LAK, DC, CIK, DC-CIK cell and method are basic in terms of the specific active immunotherapy of tumour It is proved to be invalid, and the cell technologies such as NK, CAR-NK, TIL need maturation, CAR-T cell is in safety and solid tumor It is also defective in treatment.The prior art generally passes through transformation DC cell, generates specific killing by DC submission T cell.Some experiments It is attempting to carry out transfection submission T cell, the specific killing of inducing T cell as the method for carrier with virus in room.We were also once PBMC, inducing T cell are directly stimulated with mutation mixed polypeptide.

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

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

In, the slow virus expression plasmid of building expression 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 ELISA Ancillary Reagent Kit；

Accurate polypeptide judgment criteria:

1) positive control and negative control are normal, then illustrate that this data is credible；

2) when experimental group is noticeably greater than negative control, illustrate that polypeptide is effective accurate polypeptide.

7, the accurate polypeptide secondary pulse T cell to filter out

1) when PBMC is cultivated with step 5 to the 2nd~14 day, 2 × 10 are taken⁷Effector cell, final concentration of 10 μ g/ is added The accurate polypeptide of the μ of mL~100 g/mL impacts 1-4h；

2) it after impacting 4h, is transferred in the 6 orifice plates of the pre- wrapper sheet of OKM25 or T25cm²Culture bottle mends OKM100+12%FBS, 37 DEG C of 5%CO₂Culture, according to cell growth status, is transferred in T75 culture bottle, and holding cell density as far as possible is 1 × 10⁶ cells/mL；

3) when entering in T175 culture bottle, culture medium OKM200+5%FBS, culture can be obtained precisely more for 10~14 days The T cell that peptide secondary pulse obtains；

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

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.

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

1. a kind of LRFF cell, which is characterized in that the LRFF cell is prepared by the following steps: 1) being carried out using source of people peripheral blood CtDNA sequencing or tumor tissues carry out full exon sequencing, filter out mutational site；2) epitope is carried out according to mutational site Prediction, synthesizes the gene order of mutant polypeptide；3) slow virus carrier of building expression mutant polypeptide, packs slow virus；4) it transfects Antigen presenting cell is simultaneously co-cultured with PBMC, obtains LFF cell；4) mutant polypeptide is as LFF cell described in antigenic stimulus, Filter out effective accurate polypeptide；5) the LFF cell is stimulated again using the accurate polypeptide as antigen, filtering out can know The specific cell of the not described accurate polypeptide is to get LRFF cell.

2. LRFF cell as described in claim 1, which is characterized in that the source of people peripheral blood is also possible to commercially available engineering cell System.

3. LRFF cell as described in claim 1, which is characterized in that the Epitope prediction is the amino acid position with mutation Centered on point, respectively extend 8 amino acid to two sides, using the polypeptide of 17 amino acid of this section as potentially antigenic epitope；Using pre- The IC50 that software analyzes potentially antigenic epitope is surveyed, thinks that this potentially antigenic epitope is epitope if IC50 < 1000nM.

4. LRFF cell as described in claim 1, which is characterized in that the antigen presenting cell includes: the single core of peripheral blood Cell, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage.