CN109294982A - A kind of RFF2 cell - Google Patents

A kind of RFF2 cell Download PDF

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Publication number
CN109294982A
CN109294982A CN201811153229.3A CN201811153229A CN109294982A CN 109294982 A CN109294982 A CN 109294982A CN 201811153229 A CN201811153229 A CN 201811153229A CN 109294982 A CN109294982 A CN 109294982A
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cell
polypeptide
rff2
impact
pbmc
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CN109294982B (en
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焦顺昌
张嵘
周子珊
解佳森
王海燕
李营营
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Abstract

The present disclosure provides a kind of RFF2 cells, belong to field of biotechnology.The present invention, which discloses, provides a kind of RFF2 cell for cellular immunotherapy, be a kind of attacking and defending take into account, the specific T-cells with specific killing action, accuracy is good, killing rate is high.The cell is prepared by following methods: PBMC cell loading causes the polypeptide of Tumor mutations, then carries out polypeptide impact to the PBMC cell after load polypeptide；Expand culture after impact, to obtain FF cell；So that the polypeptide of Tumor mutations directly stimulates the FF cell to screen accurate polypeptide as antigen；PBMC cell is loaded with accurate polypeptide, then cultivates, and during the cultivation process, carries out repeat impact with accurate polypeptide；Continue to cultivate after impact, obtains RFF cell；It carries out inhibitive ability of immunity signaling molecule to gained RFF cell with antibody such as PD1 to close in vitro, to obtain RFF2 cell.Gained RFF2 cell can be used for cellular immunotherapy technology.

Description

A kind of RFF2 cell
Technical field
The present invention relates to field of biotechnology more particularly to a kind of for the RFF2 cell of cellular immunotherapy and its preparation Method.
Background technique
Tumour cell immunization therapy is a kind of emerging tumor treatment model, it acquires immunocyte from the patient, so In vitro culture and amplification are carried out afterwards, then is fed back in patient body, to excite and enhance the autoimmune function of body to treat Tumour.Tumour cell immunization therapy is the 4th kind of tumor therapeuticing method after operation, radiation and chemotherapy.
The human body cell transplanting or input patient's body, the cell newly inputted that normal or bioengineering was transformed can substitute Damaged cell or has the function of stronger immunologic cytotoxicity, to achieve the purpose that treat disease.
The cell specific process that bioengineering was transformed is transformed in incubation in vitro, its killing can be improved Property, it effectively kills except patient's body tumour cell.Such as, Chinese patent application CN201210194280.5 provide a kind of people's cell because The killing cell of son induction.It is thin that Chinese patent application CN201510034781.0 provides a kind of polyclonal T of tumor cell specific Born of the same parents.Chinese patent application CN201510013987.5 provides a kind of antitumor T cell and preparation method thereof.Chinese patent application CN201711060030.1 provides a kind of CAR-T cell and its preparation method and application treated AIDS and merge lymthoma.In State patent application CN201610824893.0 provides double T cells with antigenic specificity of a kind of antibody regulation and preparation method thereof and answers With.
Summary of the invention
The present invention is intended to provide a kind of new specific T-cells (being named as RFF2 cell) for cellular immunotherapy, The impact of joint polypeptide and cell expansion ex vivo, external closing, it is thin to provide a kind of T with specific killing action, attack-defense integrated Born of the same parents solve the problems, such as that immunocyte attacking and defending in cellular immunotherapy cannot take into account, and accuracy is good, killing rate is high.
The present invention provides a kind of RFF2 cell for cellular immunotherapy, wherein the preparation method packet of the RFF2 cell Include following steps:
S1) PBMC cell loading causes the polypeptide of Tumor mutations, then carries out polypeptide punching to the PBMC cell after load polypeptide It hits；
S2 expand culture after) impacting, to obtain FF cell；
S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；
S4 PBMC cell) is loaded with the accurate polypeptide of step S3 screening, is then cultivated, and during the cultivation process, with accurate Polypeptide carries out repeat impact；
S5 continue to cultivate after) impacting, obtain RFF cell；
S6 inhibitive ability of immunity signaling molecule closing) is carried out to RFF cell obtained by step S5 with monoclonal antibody medicine, it is thin to obtain RFF2 Born of the same parents, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD244)。
Further, in step s 2, expand culture after the impact, obtaining FF cell includes: after impacting polypeptide PBMC cell is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25；
After cultivating a period of time, it is transferred to and continues to train in the cell culture apparatus containing culture solution OKM-100+12%FBS It supports；
After cultivating a period of time, transfers in the cell culture apparatus containing culture solution OKM-200+5%FBS and continue to train It supports.
Further, it in step S4, repeats polypeptide and impacts 3~4 times.
Further, in polypeptide impact, polypeptide impact is carried out with the polypeptide solution that concentration is 10 μ of μ g/mL~100 g/mL.
Further, in step S4, the PBMC cell after accurate polypeptide will be loaded and overlaying cell stimulation factor OKM-25 Cell culture apparatus in cultivate, with the accurate polypeptide of gained carry out polypeptide impact, 1~4h of attack time after cultivating a period of time；
It is transferred in the cell culture apparatus containing culture solution OKM-100+12%FBS (culture plate or culture bottle) and continues to train It supports, carried out polypeptide impact, 1~4h of attack time with identified accurate polypeptide again every 3~4 days.
Further, the polypeptide for causing Tumor mutations is synthesized to obtain by following methods:
1) exon is sequenced
Full exon sequencing is carried out to tumour cell；
By full exon sequencing result compared with the genome of normal cell, the amino acid sites of mutation are filtered out；
2) Epitope prediction
Centered on the amino acid sites of mutation, extends 10 amino acid to two sides, obtain the more of one section of 21 amino acid Peptide, in this, as potentially antigenic epitope；
IC50 < 1000nM then thinks that this potentially antigenic epitope is epitope；
3) synthesis polypeptide
Using polypeptide solid-state reaction method synthetic antigen epitope peptide.
Further, the tumour cell derive from engineering cell system, including H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323B16F1, CRL-2539 4T1, U14 Mouse Uterus Neck cancer cell, the small glioma cell of BV-2 mouse, G422 mouse glioma cell.
Further, in step S6, RFF cell obtained by step S5 is resuspended with OKM-200+5%FBS, then plus The monoclonal antibody medicine for entering final concentration of 50~200 μ g/mL, closes the inhibitive ability of immunity signaling molecule of RFF cell.
Further, in step S6, RFF cell obtained by step S5 is first washed once with phosphate buffer, 1000rpm from Heart 5min, is then resuspended with OKM-200+5%FBS.
The invention has the following beneficial effects:
The present invention provides a kind of new specific T-cells for cellular immunotherapy, i.e. RFF2 cell, is a kind of attacking and defending The super T cell having both.The present invention is by filtering out effective precisely polypeptide and it being carried out secondary polypeptide punching to PBMC cell It hits, is more there is the T cell of specific cytotoxicity, attack-defense integrated.
In the prior art, it is in T cell generally by DC cell delivery, generates the T cell of specific killing；Or it is made of virus For carrier, pass through the specific killing of slow-virus transfection technological guide T cell.In this application, skill is impacted by secondary polypeptide The specific killing of art inducing T cell.First time polypeptide impacts first, directly stimulates PBMC cell with mixed polypeptide；Then sieve Select accurate polypeptide；PBMC is stimulated with accurate polypeptide again, and PBMC cell is repeatedly stimulated during co-cultivation.By more General T cell is transformed into the super T cell for more accurately killing ability by secondary stimulation.Compared to slow-virus transfection, simply It is convenient, highly-safe.In this application, the impact of joint polypeptide and amplification in vitro improve T cell to complicated tumor microenvironment Adaptive faculty.In the application, joint antibody drug is closed guard technology in vitro and is closed in vitro to inhibitive ability of immunity signaling molecule, Its own protective capacities is improved on the basis of not reducing T cell lethality.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art To obtain other drawings based on these drawings.
Fig. 1 shows the flow diagram of the preparation method of RFF2 cell described in the embodiment of the present invention；
Fig. 2 shows antigen load Efficiency testings；
Fig. 3 shows accurate polypeptide the selection result；
Fig. 4 shows flow cytometer detection specific T-cells ratio；
Fig. 5 shows inhibitive ability of immunity signaling molecule encapsulation situations；
Fig. 6 shows the killing ability of flow cytometer detection RFF2 cells against tumor；
Fig. 7 shows the release of RFF2 cell by cell factor IFN-γ；
Fig. 8 shows tumor-bearing mice survivorship curve.
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.
As shown in Figure 1, the embodiment of the present invention provides a kind of specific T-cells for cellular immunotherapy, it is named as RFF2 cell.The preparation method of the RFF2 cell the following steps are included:
S1) PBMC cell (peripheral blood mononuclear cell, peripheral blood mononuclear cells) load causes The polypeptide of Tumor mutations then carries out a polypeptide impact to the PBMC cell after load polypeptide；
S2 expand culture after) impacting, obtain FF cell；
S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；
S4 PBMC cell) is loaded with the accurate polypeptide of step S3 screening, is then cultivated, and during the cultivation process, with accurate Polypeptide carries out multiple polypeptide impact；
S5 continue to cultivate after) impacting, obtain RFF cell；
S6 inhibitive ability of immunity signaling molecule closing) is carried out to RFF cell obtained by step S5 with monoclonal antibody medicine, it is thin to obtain RFF2 Born of the same parents, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD244)。
Herein, " RFF2 " cell is the cell finally to be obtained of the embodiment of the present invention, that is, passes through joint mixed polypeptide Technology, accurate polypeptide secondary pulse technology, antibody drug are closed guard technology (RFF2 scheme) in vitro and are transformed by general T cell To have conditions in both attack and defence, the specific T-cells with specific killing action." FF " cell is indicated by FF scheme (not by essence Quasi- polypeptide secondary stimulus) obtained T cell." RFF " cell is indicated by RFF scheme (joint mixed polypeptide technology and accurate polypeptide Secondary pulse technology) obtained T cell.Wherein, " R " represents polypeptide secondary pulse technology；" FF " represents mixed polypeptide technology, " 2 " indicate external closing guard technology.
The embodiment of the invention provides a kind of T cell for cellular immunotherapy, filter out really effective accurate Polypeptide, and secondary polypeptide impact is carried out to PBMC cell with it, it is had conditions in both attack and defence, with the special of specific killing action Property T cell.
Before step S1, the polypeptide for causing Tumor mutations can be synthesized to obtain by following methods: 1) exon is sequenced； 2) Epitope prediction；3) synthesis polypeptide.
1) exon is sequenced
Full exon sequencing is carried out using engineering cell system, then sequencing information is analyzed using software, on the one hand Obtain MHC type information；On the other hand, full exon sequencing result is filtered out into mutation compared with the genome of normal cell Amino acid sites.
Exon sequencing in, engineering cell system include 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-2 mouse Small glioma cell, G422 mouse glioma cell.Engineering cell system refers to using technique for gene engineering or cell fusion Technology carries out modification transformation or recombination to the inhereditary material of host cell, obtains the cell with the unique shape for stablizing heredity System.
2) Epitope prediction
In Epitope prediction, centered on the amino acid sites of mutation, extend 10 amino acid to two sides, by this section The polypeptide of 21 amino acid is used as " potentially antigenic epitope ".(recommended soft using the IC50 that forecasting software analyzes potentially antigenic epitope Part: NetMHCpan 3.0, PickPocket, artificial neural networks (ANN)).If IC50 < 1000nM This potentially antigenic epitope is regarded as into " epitope ".
3) synthesis polypeptide
Using polypeptide solid-state reaction method synthetic antigen epitope peptide.
In step sl, with causing the polypeptide of Tumor mutations to carry out polypeptide impact to the PBMC cell after load polypeptide, directly PBMC cell after stimulation load polypeptide, carries out first time stimulation.
Specifically, PBMC cell loading causes the polypeptide of Tumor mutations, then carries out to the PBMC cell after load polypeptide more Peptide impact is specifically as follows:
Mixed polypeptide is dissolved with RPMI 1640+10%FBS (fetal calf serum) or OKM100+12%FBS, the end of polypeptide is dense Degree is 10~100 μ g/mL, and preferably 50 μ g/mL carry out polypeptide impact with this.
Further, the time of polypeptide impact can be 1~4h, such as preferably can be able to be 4h with 1h, 3h or 4h.
In step s 2, expand culture after impact to obtain FF cell.PBMC cell and cell after polypeptide is impacted pierce Swash factor OKM-25 to train altogether, to obtain FF cell.
Step S2 is specifically as follows:
PBMC cell after polypeptide is impacted is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25；
After cultivating a period of time, it is transferred to and continues to train in the cell culture apparatus containing culture solution OKM-100+12%FBS It supports；
It is transferred in the cell culture apparatus containing culture solution OKM-200+5%FBS after culture a period of time and continues to train It supports.
Specifically, FF cell composition can be obtained after expanding culture.It can be from the FF cell composition by centrifugation Separation and Extraction T cell (i.e. FF cell).
In step S3, directly stimulate T cell to screen accurate polypeptide using polypeptide as antigen.
The screening criteria of accurate polypeptide are as follows:
Using FF cell as baseline；Two independent to repeat, and detected value is high for high baseline, low for low baseline；
The difference of two baselines is systematic error, when data are analyzed, to detected value > low baseline, > high baseline and > Gao Ji Line+systematic error, is labeled respectively；Detected value > high baseline+systematic error is effective accurate polypeptide.
Step S3 can specifically include:
1) it is centrifuged FF cell composition obtained, 1500rpm is centrifuged 5min and collects T cell, and 10mL PBS is added and is resuspended Cell simultaneously counts, 1500rpm be centrifuged 5min, collect T cell with 1640+10%FBS+200U/mL IL2 resuspension, counting adjust to 1×106A/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 × 105It is a；It is separately added into again 3 multiple holes are arranged in mutant polypeptide 10 μ L, the final concentration of 50 μ g/mL of 1mg/mL, every polypeptide；
3) positive control: T cell+100ng/mL OKT3 (CD3 monoclonal antibody) is set；Negative control: RPMI 1640+ 10%FBS+200U/mL IL2 (interleukin 2)；Two T cells are compareed as background release detection, are respectively added for the first time T cell, and last time plus T cell；Using the difference that two backgrounds discharge as systematic error；
4) 37 DEG C, 5%CO2After stimulation for 24 hours, 1500rpm is centrifuged 10min, shifts 140 μ L supernatants into 96 new orifice plates；
5) 96 orifice plates are centrifuged again, 1500rpm 10min, sample is taken to carry out ELISA (enzyme linked immunosorbent assay (ELISA)) Detection (or saving sample as -80 DEG C)；
6) accurate polypeptide is screened:
Polypeptide is as antigen using FF cell as baseline；
Every group of experiment is two baselines, high baseline and low baseline, and the difference of two baselines is systematic error, data analysis When, to detected value > low baseline, > high baseline and > high baseline+systematic error, it is labeled respectively；Detected value > high baseline+ Systematic error is effective accurate polypeptide.
In step S4, PBMC cell is loaded with the accurate polypeptide of step S3 screening, is then cultivated, and during the cultivation process, Multiple polypeptide impact is carried out with accurate polypeptide, carrying out second stimulates.Have by stimulating for general T cell to be transformed into twice More accurately kill the super T cell of ability.
Compared to step S1, impacted in step s 4 using the accurate polypeptide of dosage.That is, in this step to PBMC Cell carries out multiple polypeptide impact stimulation.Polypeptide can be specifically repeated to impact 3~4 times.
In incubation, culture scheme is similar to load the expansion culture of the PBMC cell after polypeptide in step S2.First exist It overlays and cultivates a period of time in the device of cell stimulation factor OKM-25, be then successively transferred to OKM-100+12%FBS culture Continue to cultivate in base, OKM-200+5%FBS culture medium.By the impact of joint polypeptide and amplification in vitro, T cell is improved to complexity Tumor microenvironment adaptive faculty.
It can specifically include following steps:
The PBMC cell for loading accurate polypeptide is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25, Polypeptide impact is carried out with identified accurate polypeptide after culture a period of time, the attack time can be 1~4h；
It is transferred in the cell culture apparatus containing culture solution OKM-100+12%FBS (culture plate or culture bottle) and continues to train It supports, carried out polypeptide impact with identified accurate polypeptide again every 3~4 days, the attack time can be 1~4h.
In step S5, continue to cultivate after impact, obtains RFF cell.Applicable culture medium can be OKM200+5%FBS. After step S4, it is transferred to after polypeptide impact and continues to cultivate in the device containing culture solution OKM200+5%FBS, 37 DEG C, 5% CO2Lower culture.
In step S6, inhibitive ability of immunity signaling molecule closing is carried out to RFF cell obtained by step S5 with monoclonal antibody medicine, to obtain RFF2 cell, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT、2B4(CD244)。
In one embodiment, RFF cell obtained by step S5 is resuspended with OKM-200+5%FBS, is then added final concentration of 50 The monoclonal antibody medicine such as PD-1 antibody etc. of~200 μ g/mL, preferably 50~150 μ g/mL, to the inhibitive ability of immunity signaling molecule of RFF cell It is closed.
In another embodiment, RFF cell obtained by step S5 is first washed once with PBS (phosphate buffer), 1000rpm from Heart 5min, is then resuspended with OKM-200+5%FBS, can be resuspended to 1 × 107A/mL.Then it is added final concentration of 50 The monoclonal antibody medicine of~200 μ g/mL, preferably 150 μ g/mL, 0~37 DEG C of closing 1~4h, preferably 37 DEG C closing 1h.
During the present invention discloses, on the basis of RFF cell, external self-isolation operation is carried out using a small amount of monoclonal antibody medicine, Its own protective capacities is improved on the basis of not reducing specific T cell responses power.
It is thin that the present invention by a kind of cells in vitro preparation method provides a kind of new RFF2 for cellular immunotherapy Born of the same parents' (super T cell having conditions in both attack and defence obtained after being transformed by ordinary cells).During the preparation process, really effective essence is filtered out Quasi- polypeptide simultaneously carries out secondary pulse to PBMC cell with it, is more had the RFF2 cell of specific cytotoxicity, killing-efficiency is more It is high.During the preparation process, protection and amplification in vitro are closed in vitro by antibody drug, improves gained RFF2 cell to patient The adaptability of the tumor microenvironment of intracorporal complexity.
Hereafter the RFF2 cell further described in the embodiment of the present invention for cellular immunotherapy is described in detail.
(1) full exon sequencing
1) full exon sequencing is carried out using LLC Lewis lung cancer cells；
2) sequencing information is analyzed using software: on the one hand obtains MHC type information；It on the other hand, will be entirely outer aobvious Sub- sequencing result filters out mutational site compared with the genome of normal cell.
(2) Epitope prediction
1) centered on the amino acid sites of mutation, extend 10 amino acid to two sides, by the more of 21 amino acid of this section Peptide is used as " potentially antigenic epitope "；
2) using forecasting software analyze potentially antigenic epitope IC50 (recommend software: NetMHCpan3.0, PickPocket, artificial neural networks (ANN)), this potentially antigenic table is thought if IC50 < 1000nM Position is " epitope ".
(3) synthesis polypeptide
Epitope peptide symthesis method uses polypeptide solid-state reaction method
1) it is anchored: first amino acid is anchored on solid-phase resin；
2) deprotect: the amino acid of protection removes the blocking group of amino using basic solvent；
3) it activates: activating amino acid carboxyl to be connected using activator；
4) bonded: the carboxyl of the activation amino exposed with previous amino acid reacts, and forms peptide
5) step 2-4 is repeated, whole epitope peptide chain complete synthesis is made.
(4) PBMC load sudden change polypeptide
1) it prepares polypeptide solution: polypeptide being dissolved with 1640+10%FBS or OKM100+12%FBS, the end of every polypeptide is dense Degree is 50 μ g/mL, spare；
2) PBMC shifts to an earlier date 1 day and recovers, and blows and beats cell, draws 15mL, and count, and is centrifuged；
3) PBMC is resuspended with the polypeptide solution of preparation, and as being impacted in tissue culture plate；
4) 37 DEG C of 5%CO2, 4h is impacted, it is spare.
(5) expansion culture of the PBMC after polypeptide impacts
1) the pre- bed board of stimulating factor OKM-25,40 μ L OKM-25+4mL PBS, 2mL/ ware (4.5cm2) room temperature 4h, 4 DEG C are standby With；
2) PBMC after impact is transferred in the culture bottle for overlaying OMK25；
3) even, 37 DEG C of 5%CO are shaken2Culture, is denoted as the 0th day；
4) co-cultured cell situation is observed, cell is transferred in big culture bottle at the 5th day according to cell density, is mended new Fresh culture solution OKM-100+12%FBS, 20mL is in 75cm2In culture bottle；
5) it co-cultures the 7th day, adds the fresh OKM-100+12%FBS of 20mL；
6) it co-cultures the 10th day, culture solution OKM-200+5%FBS, by co-cultured cell from 75cm2That transfer in bottle To 175cm2In big bottle；
7) 25mL culture solution OKM-200+5%FBS is blown and beaten, then is transferred to big bottle, is repeated 2 times；With culture solution OKM-200+5% FBS complements to 200mL；
8) when culture was to 14-21 days, it can be obtained FF cell composition.
(6) polypeptide directly stimulates T cell to screen accurate polypeptide as antigen:
1) T cell in FF cell composition is the T cell (FF cell) that FF scheme obtains.The FF of acquisition is collected by centrifugation Cell composition, 1500rpm are centrifuged 5min and collect T cell, 10mL PBS are added, cell is resuspended and counts, 1500rpm centrifugation 5min collects T-cells with 1640+10%FBS+200U/mL IL2 resuspension, and counting is adjusted to 1 × 106cells/mL；
2) FF cell is divided into 96 hole flat undersides with the volley of rifle fire, 200 holes μ L/, cell number is 2 × 105cells；Distinguish again The mutant polypeptide of 10 μ L 1mg/mL, final concentration of 50 μ g/mL is added, 3 multiple holes are arranged in every polypeptide；
3) positive control: T cell+100ng/mL OKT3 is set；Negative control: 1640+10%FBS+200U/mL IL2； Two T cell controls are as background release detection, respectively first time plus T cell, and last time plus T cell；With two sheets The difference of bottom release is as systematic error；
4) 37 DEG C, 5%CO2After stimulation for 24 hours, 1500rpm is centrifuged 10min, shifts 140 μ L supernatants into 96 new orifice plates；
5) 96 orifice plates are centrifuged again, 1500rpm 10min, take sample carry out ELISA detection (or by sample as- 80 DEG C of preservations).
(7) accurate polypeptide judgment criteria:
1) positive control and negative control are normal, then illustrate that this data is credible；
2) polypeptide is as antigen using T cell as baseline；
3) every group of experiment is two baselines, high baseline and low baseline, and the difference of two baselines is systematic error, data analysis When, to detected value > low baseline, > high baseline and > high baseline+systematic error, it is labeled respectively；Detected value > high baseline+ Systematic error is effective accurate polypeptide.
(8) RFF cell is prepared with the accurate polypeptide of screening
1) load the PBMC of accurate polypeptide with the culture of FF cell composition culture scheme (the pre- bed board of stimulating factor OKM-25, 40 μ L OKM-25+4mL PBS, 2mL/ ware (4.5cm2) room temperature 4h, 4 DEG C are spare；37 DEG C of condition of culture, 5%CO2) to the 2nd day extremely At the 14th day, preferably the 3rd day, impacting again for accurate polypeptide is carried out；
2) 2 × 10 are taken7T cell, the polypeptide of final concentration of 50 μ g/mL is added, impacts 4h；
3) after impacting 4h, it is transferred to 25cm2Culture bottle mends OKM100+12%FBS, 37 DEG C of 5%CO2Culture, it is raw according to cell Long situation, is transferred to 75cm2In culture bottle, holding cell density as far as possible is 1 × 106A/mL；
4) it when cultivating the 7th day, the 10th day and the 14th day, repeats the impact of accurate polypeptide and repeats step 2) and 3)；
5) cell enters 175cm2When in culture bottle, culture medium OKM200+5%FBS, culture can be obtained for 10~21 days The T cell that accurate polypeptide secondary pulse obtains, i.e. RFF cell.
(9) closing of inhibitive ability of immunity signaling molecule
1) cultured RFF cell, 1000rpm are centrifuged 5min；
2) it being washed once with PBS, 1000rpm is centrifuged 5min, TCR-T is resuspended with OKM-200+5%FBS, and adjust to 1 × 107/mL；
3) the monoclonal antibody medicine PD1 antibody of inhibition signaling molecule is added, final concentration of 150 μ g/mL, 37 DEG C of closing 1h are obtained RFF2 cell.
(10) tumor-bearing mice survival assay
1) it takes tumor cell line to be inoculated with NSG mouse, does dystopy tumor-bearing model.By 5 × 105Expression specificity antigen Tumour cell is suspended from 100 μ L physiological saline, is subcutaneously injected respectively subcutaneous while right to the right side side of body rib portion of 30 NSG mouse Mouse is numbered.
2) 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 mouse, one group is given placebo physiological saline, gives for one group and does not carry out any heredity behaviour The T cell (control group) 1 × 10 of work7, one group is given RFF2 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 50 days after 7 days, counts survival data, draws survivorship curve.
Experimental result
1. mutational site and Epitope prediction
Table 1 be the mutational site that detects of sequencing and Epitope prediction as a result, underscore mark be mutation amino Acid.
1 Epitope prediction of table
2. antigen load Efficiency testing
According to the mutant antigen of the synthesis prediction of table 1, and the label of biotin is carried out, after antigen load PBMC, is marked with PE Affine streptomysin detection biotin cell surface distribution situation, with detect PBMC deduction polypeptide antigen efficiency；As a result Such as Fig. 2 --- shown in antigen load Efficiency testing: a is the testing result without loading labeling polypeptide, and b is load biotinyl polypeptide Testing result, the results showed that (FL2-H subset 59.4% indicates that PI stain contaminates to the load efficiency of PBMC by 59.4% To cell account for 59.4%).
3. screening accurate polypeptide
As shown in figure 3, stimulating the FF cell of culture respectively with 12 polypeptides, by detecting the secretion of IFN-γ, detection has The polypeptide of effect, as a result as shown in Figure 2: the burst size of IFN-γ caused by No. 2, No. 6 and No. 12 polypeptides > high baseline+systematic error, Belong to effective accurate polypeptide.
4. flow cytometer detection specific cell ratio
It with No. 12 polypeptides of screening, is repeatedly stimulated on FF cell base, after culture, with flow cytometer detection to precisely more The T cell ratio of peptide specific, as a result as shown in figure 4, being specific T-cells in box.Simultaneously with flow cytometer (FACSCaliburTM (BD Biosciences)) carries out the sorting of CD8+IFN- γ+cell (in box).
5. inhibitive ability of immunity signaling molecule sealing effect
As shown in figure 5, the available closing of 99.2% cell.
Lethal effect of the 6.RFF2 cell to target cell
Killing-efficiency is carried out with the target cell of FF cell, RFF cell and RFF2 cell to mutant antigen epitope source respectively Detection, with flow cytometer detection Apoptosis (TransDetect Annexin V-FITC/PI apoptosis kit), as a result such as It is the cell of apoptosis, lethal effect (31.4%) > RFF cell of RFF2 cells against tumor shown in Fig. 6, in black box (25.8%) > FF cell (23.5%).
The detection of 7.RFF2 cell cytokine release
When with polypeptide stimulating effect cell, due to effector cell, mutant antigen can be identified, therefore, can generate a series of Cell factor, IFN-γ is one of most important cell factor in antitumor action, and Fig. 7 is different training method cells, warp After polypeptide stimulation, the detection of the IFN-γ of release, the results showed that the IFN- generated with effector T cell itself (only effector cell) γ is compared, and RFF cell and RFF2 cell can produce a large amount of IFN-γ, especially RFF2 cell, and effector cell is releasably more More IFN-γ, this result is consistent with killing experiments result, and it is anti-swollen to illustrate that the closing of inhibition target spot can be improved more effectively Tumor ability.
8. tumor-bearing mice survival assay
As a result as shown in figure 8, feeding back the existence improvement of the RFF2 cells against tumor tumor-bearing mice of the embodiment of the present invention has Significantly affect effect.
9. clinical case
Administration process:
First course for the treatment of: monthly RFF2 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year RFF2 cell, quantity 1 × 109A cell, totally 2 times.
Table 2
Number Gender Age Medical diagnosis on disease The Progression free survival time after administration
1 Male 67 Gastric cancer 2017.2- so far
2 Male 61 The cancer of the esophagus 2017.6- so far
3 Male 63 Lung cancer 2017.6- so far
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain Lid is within protection scope of the present invention.Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (9)

1. a kind of RFF2 cell for cellular immunotherapy, which is characterized in that the preparation method of the RFF2 cell include with Lower step:
S1) PBMC cell loading causes the polypeptide of Tumor mutations, then carries out a polypeptide punching to the PBMC cell after load polypeptide It hits；
S2 expand culture after) impacting, to obtain FF cell；
S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；
S4 PBMC cell) is loaded with the accurate polypeptide of step S3 screening, is then cultivated, and during the cultivation process, with accurate polypeptide Carry out repeat impact；
S5 continue to cultivate after) impacting, obtain RFF cell；
S6 the closing of inhibitive ability of immunity signaling molecule) is carried out to RFF cell obtained by step S5 with monoclonal antibody medicine, it is thin to obtain RFF2 Born of the same parents, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD244)。
2. the RFF2 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step s 2, institute Expand culture after stating impact, includes: to obtain FF cell
PBMC cell after polypeptide is impacted is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25；
After cultivating a period of time, it is transferred to and continues to cultivate in the cell culture apparatus containing culture solution OKM-100+12%FBS；
After cultivating a period of time, transfers in the cell culture apparatus containing culture solution OKM-200+5%FBS and continue to cultivate.
3. the RFF2 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step S4, repeat Polypeptide impacts 3~4 times.
4. the RFF2 cell according to claim 4 for cellular immunotherapy, which is characterized in that in step s 4, often A polypeptide impact was carried out every 3~4 days.
5. the RFF2 cell according to claim 1 for cellular immunotherapy, which is characterized in that in polypeptide impact, use The polypeptide solution that concentration is 10 μ of μ g/mL~100 g/mL carries out polypeptide impact.
6. the RFF2 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step S4, will bear PBMC cell after carrying accurate polypeptide is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25, when cultivating one section Between after use the accurate polypeptide of gained to carry out polypeptide impact, 1~4h of attack time；
It is transferred in the cell culture apparatus containing culture solution OKM-100+12%FBS (culture plate or culture bottle) and continues to cultivate, Polypeptide impact, 1~4h of attack time were carried out with identified accurate polypeptide again every 3~4 days.
7. the RFF2 cell according to claim 1 for cellular immunotherapy, which is characterized in that the cause Tumor mutations Polypeptide synthesize to obtain by following methods:
1) exon is sequenced
Full exon sequencing is carried out to tumour cell；
By full exon sequencing result compared with the genome of normal cell, the amino acid sites of mutation are filtered out；
2) Epitope prediction
Centered on the amino acid sites of mutation, to two sides extend 10 amino acid, using the polypeptide of one section of 21 amino acid as Potentially antigenic epitope；
IC50 < 1000nM then thinks that this potentially antigenic epitope is epitope；
3) synthesis polypeptide
Using polypeptide solid-state reaction method synthetic antigen epitope peptide.
8. the RFF2 cell according to claim 7 for cellular immunotherapy, which is characterized in that the tumour cell comes Derived from engineering cell system, the engineering cell system includes H1299, H226, H358, H1563, H2228, A549, Renca, LLC small Mouse Lewis lung carcinoma cell, CRL-6323B16F1, CRL-2539 4T1, U14 mouse carcinoma of uterine cervix cell, the small colloid of BV-2 mouse Oncocyte, G422 mouse glioma cell.
9. the RFF2 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step S6, will walk RFF cell obtained by rapid S5 is resuspended with OKM-200+5%FBS, and the monoclonal antibody medicine of final concentration of 50~200 μ g/mL is then added, The inhibitive ability of immunity signaling molecule of RFF cell is closed.
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