CN106884005A

CN106884005A - A kind of preparation method of colorectal cancer T cells with antigenic specificity

Info

Publication number: CN106884005A
Application number: CN201710044530.XA
Authority: CN
Inventors: 樊克兴; 任涛; 刘洋; 王凯玲
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-01-19
Filing date: 2017-01-19
Publication date: 2017-06-23

Abstract

The present invention relates to T cells with antigenic specificity, the particularly preparation method of colorectal cancer T cells with antigenic specificity, and the colorectal cancer T cells with antigenic specificity obtained by methods described.The invention further relates to purposes of the gained T cells with antigenic specificity in the preparation for treating cancer (particularly colorectal cancer) is prepared.

Description

A kind of preparation method of colorectal cancer T cells with antigenic specificity

Technical field

The present invention relates to immunocyte field, relate more specifically to the preparation method of colorectal cancer T cells with antigenic specificity, And the colorectal cancer T cells with antigenic specificity obtained by methods described.

Background technology

Colorectal cancer is the third-largest most common cancer in the whole world, has 1,000,000 or so new cases every year and more than 500,000 people Die from the disease.The treatment of colorectal cancer combines adjuvant radiotherapy, chemotherapy based on performing the operation, although chemoradiation therapy can be certain Extend the life cycle of patient in degree, but also with serious side reaction, and even across positive therapeutic alliance, the disease is still In the presence of recurrence and metastatic rate very high.Patient survival be can effectively extend in the urgent need to developing at present, recurrence and transfer wind reduced The therapeutic strategy of danger.

Transforming growth factor β II receptors (TGF β RII) and O N-Acetyl-D-glucosamine glycosyl transferase (OGT) gene Frameshift produce a kind of neoantigen restricted with human leucocyte antigen A 2 (HLA-A2), be present in 20-40% The restricted colorectal cancer patients of HLA-A2 and it is not present in normal person and other tumours, the Antigenic Peptide after offering is SLYKFSPFPL and RLSSCVPVA.

Increasing evidence shows that immune system can be grown and transfer diffusion with prevention of tumor.Including cell factor, swell Knurl vaccine, immune effector cell, and genetic modification φt cell receptor, monoclonal antibody etc. are as domestic and international immunization therapy research and development Focus.Multinomial colon cancer immunization therapy clinical experimental study is carried out at present, including has promoted the cell factor of immune system, exempts from The blocking of epidemic disease checkpoint, antigen vaccine and adoptive cell therapy etc..Although current genetically modified cell sticks up in turning into immunization therapy Chu, but because preparation technology is complicated, and there is effect of missing the target so that it is hindered in the clinical stage propulsion for the treatment of solid tumor Hinder.

Cytokine-induced killer cell is directed in the past, and NK is treated including the solid tumor including colon cancer Research think that these treatment methods have certain curative effect, but due to lacking antigentic specificity document report clinical therapeutic efficacy It is uneven.

The antigen of tumor cell surface is processed into Antigenic Peptide and is combined with major histocompatibility by antigen presenting cell (APC) Thing (MHC) molecule forming composite is offered and participating in activation is to T cell, forms the cytotoxic T of specific for tumour antigen Cell (CTL).In order to escape identification, MHC molecule is not expressed in downward even to tumour, and Partial tumors patient's immunocyte quantity declines Hypofunction can all cause the tumour antigen cannot to offer, and cause T cell to activate.So aiding in shape using antigen presenting cell It is conventional method into killer T cell, but these methods are unstable and not enough facilitate.

In sum, still need and want more efficient simple immunotherapy and T cells with antigenic specificity simpler and efficient Preparation method.

The content of the invention

In order to solve prior art problems faced, T cells with antigenic specificity is prepared inventor developed one kind, especially It is the method for colorectal cancer T cells with antigenic specificity, it is solved or part solves problem above.

One aspect of the present invention is related to the preparation method of T cells with antigenic specificity, and it includes making T cell and tomour specific Property antigen is incubated altogether.

Further, the tumour specific antigen in methods described can be colorectal cancer specific antigen.

In some embodiments, the colorectal cancer specific antigen is peptide RLSSCVPVA.

In some embodiments, in suspension, further, the suspension can for the T cell in the inventive method Think PMBC suspension or peripheral blood.

In some embodiments, the T cell in the inventive method is CD8+T cells.

One aspect of the present invention is related to the T cells with antigenic specificity obtained by the inventive method, particularly colorectal cancer T cells with antigenic specificity.

Colorectal cancer T cells with antigenic specificity the invention further relates to be obtained by the inventive method is being prepared for treating knot Purposes in the preparation of the carcinoma of the rectum.

The peptide SLYKFSPFPL (hereafter may be simply referred to as " Antigenic Peptide 1 ", " No. 1 peptide " or " P1 ") and peptide used in the present invention RLSSCVPVA (hereafter may be simply referred to as " Antigenic Peptide 2 ", " No. 2 peptides " or " P2 ") can be chemical synthesis or through bioengineering What technology was produced, it has higher, 99% purity is greater than, with good homogenieity.Directly use peptide The method that RLSSCVPVA treatment obtains T cells with antigenic specificity, step is greatly simplified, and favorable repeatability is grasped with good The property made and practical value.

Brief description of the drawings

Fig. 1 shows the purity of the peptide for preparing；Figure 1A Antigenic Peptides RLSSCVPVA synthesizes the quasi-molecular ions of mass spectral analysis, 12.245 ' occur；Figure 1B Antigenic Peptides RLSSCVPVA synthesizes the main fragment peak of mass spectral analysis, only one, the fragment molecular weight It is 931.76, abundance is 100%, close to the theoretical molecular of RLSSCVPVA.Fig. 1 C Antigenic Peptides SLYKFSPFPL synthesis mass spectrums point Two quasi-molecular ions of analysis, respectively appear in 16.731 ' and 21.317 '.Fig. 1 D OGT Antigenic Peptides SLYKFSPFPL synthesis mass spectrums point The main fragment peak of analysis, there is two, and the molecular weight (abundance) of fragment is respectively 1198.70 (99%) and 1220.81 (1%), the Theoretical molecular of one molecular weight of fragment close to SLYKFSPFPL.

Fig. 2 shown in the case of different effect cell target ration (referred to as " effect target ratio "), uses No. 1 peptide (P1), 2 The equal proportion mixture (P1+2) of number peptide (P2), No. 1 peptide and No. 2 peptides and in the case of being not added with Antigenic Peptide (NP), induction is produced Raw cytotoxic T lymphocyte (hereinafter referred " CTL ") kills the influence of vigor to DLD1.

The CTL of DC Antigens induction directly stimulates with Antigenic Peptide when Fig. 3 shows that effect target ratio is 5/1 (a) and 10/1 (b) Activation CTL kill DLD-1 efficiency ratios compared with.

Fig. 4 show to effect target ratio be two groups of 5/1 (a) and 10/1 (b) CTL activation after IFN-γ secretion amount measure； The CTL (DC+T) that the left side of color obtains for the DC stimulations PBMC of Loading peptides earlier above in every group of contrast, right side is corresponding anti- Former peptide directly stimulates the CTL (PBMC) that PBMC is obtained.

Specific embodiment

First, experiment material

1st, colon carcinoma cell line DLD1 comes from PLA General Hospital tumor center laboratory.

2nd, peripheral blood sample source

2.1 patient's groups：

In November, 2014 in August, 2015 is collected to go to a doctor in the colon carninomatosis made a definite diagnosis of PLA General Hospital tumor center People, diagnosis confirms by through tissue or cell pathology, by including, after exclusion standard screening, and 42 altogether, wherein there is HLA A2 restricted patient 34, is the final patient for including.Histopathology sample is obtained simultaneously by surgery Pathology or enteroscopy Detected through pathology department and determined.All experiments are ratified through PLA General Hospital Ethics Committee, and patient knows and signs informed consent Book.Including for patient is as follows with exclusion standard：

Patient organizes inclusive criteria：

1. the colorectal cancer patients made a definite diagnosis through pathology, histological type only includes gland cancer (high/medium/low differentiation)；

2. without cell therapy before being admitted to hospital；

3. the HLA restricted molecules of A2 are expressed

Patient organizes exclusion standard：

1. the transfer of colon portion or infringement of other position primary tumo(u)rs；

2. the patient of the last radiotherapy of distance/in end of chemotherapy January, previously received cell therapy patient；

3. the restricted molecules of HLA A2 are not expressed

4. with autoimmune disease and disease in the blood system patient；

5. pathology, the incomplete patient of Follow-up Data；

6. with other infection Diseases.

The diagnosis of colon cancer gland cancer and histological type are determined according to US National synthesis cancer network (NCCN) hair in 2013 The colon cancer practice guidelines of cloth, patient clinical refers to International Union Against Cancer (UICC) and american cancer joint committee by stages (AJCC) the TNM stagings formulated.According to the RAN and coherence check of including patient, record includes sex, age, pathology Type, clinical stages, the past therapeutic scheme, complication merges other diseases, the letter such as colon cancer correlated inheritance disease or family history Breath.

2.2 control groups：The same period participates in 10 normal healthy controls persons of physical examination in PLA General Hospital MEC and 4 are volunteered The peripheric venous blood of person is as a control group.

Inclusion criteria is：

1. organize the age with patient and sex composition is close

2. the HLA restricted molecules of A2 are expressed

Exclusion standard is：

1. there is acute or chronic inflammation in 1 month

2. by colon cancer or colonic diseases family history

3. the restricted molecules of HLA A2 are not expressed

4. autoimmune disease, hematological system disease and other infect Diseases

2nd, major experimental reagent is as follows, and every other experiment reagent is conventional reagent, and those skilled in the art can be easily It is commercially available

Note：MTS reagent box is using new water soluble compound [3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxylics Carbomethoxy) -2- (4- sulfophenyls) -2H- tetrazoliums (golden father-in-law), inner salt；MTS] fast high-sensitive degree detection cell propagation and cell toxicant The colorimetric detection product of property.

3rd, major experimental instrument

4th, the synthesis of peptide RLSSCVPVA and SLYKFSPFPL, purifying and identification

The synthesis of 4.1 peptides

Peptide RLSSCVPVA and SLYKFSPFPL transfer to BeiJing ZhongKe Yaguang Biology Science Co., Ltd to be commercially synthesized.

The peptide of 4.2 synthesis

BeiJing ZhongKe Yaguang Biology Science Co., Ltd provides the detection obtained on Xevo G2-S QTof mass spectrographs As a result, referring specifically to accompanying drawing 1.The OGT for obtaining the TGF-β RII Antigenic Peptides RLSSCVPVA and 99.05% that purity is 100% resists Former peptide SLYKFSPFPL.

5th, peripheral blood collection

In 20 milliliters of peripheral blood to anticoagulant tube (anticoagulant heparin) is extracted in the case of testing when group objects is admitted to hospital on an empty stomach, blood sampling The front and rear iodophor disinfection of pipe cap blood sampling, it is ensured that aseptic.Blood sample is processed in two hours after collection, with 4 DEG C, 2500 revs/min of centrifugations The pelleted by centrifugation of 15 minutes.Collect upper plasma absorption 1mL to preserve into cryopreservation tube, -80 DEG C of refrigerators are placed in after packing, it is to be checked Survey.In the same way with 14 normal healthy controls persons of collection step, packing 1mL blood plasma is preserved to -80 DEG C of refrigerators, and remaining blood plasma is inhaled Enter 15mL sterile centrifugation tubes, sealing compound sealing is placed in 56 DEG C of water-baths 30 minutes.Water-bath terminates rear blood plasma and puts 23 DEG C, 2500 revs/min The pelleted by centrifugation of centrifugation 15 minutes.Sealing is placed in 4 DEG C of preservations and continues to employ after centrifugation.

6th, the acquisition of PMBC (PBMC) and extracorporeal culturing method

(1) prepare：Experiment starts the previous day, by anti-human CD3 and anti-human CD28 with 1:100 are added in aseptic 1X PBS and mix It is even, so that in 3mL dilutions addition T25 blake bottles, liquid uniform fold bottom of bottle is sealed after masking foil is wrapped up with sealing compound and is put into 4 DEG C overnight.

(2) PBMC is separated：

Dilution：The haemocyte that blood plasma is removed after centrifugation is diluted with 1X PBS, haemocyte：PBS volume ratio=1:2；Layering： Two 50ml centrifuge tubes are taken, 15ml lymphocyte separation mediums (lower floor) are separately added into, it is thin that inclination is slowly added to about 35ml diluted bloods Born of the same parents (upper strata)；

Centrifugation：Room temperature condition, 2000 revs/min are centrifuged 20 minutes.

(3) wash：Draw the tunica albuginea layer in the middle of layering liquid and be added to new 50ml centrifuge tubes.Note during absorption, rotation is inhaled Take, do not draw lower floor's red blood cell.Blown and beaten with 2 or 3 times of 1X PBS of volume and mixed, be centrifuged 2000 revs/min and be centrifuged 10 minutes. Washing process is carried out twice.If red blood cell layer surface have White Flocculus or centrifugation after find layering it is unclear can will layering it is unclear Liquid washed with PBS after separated with lymphocyte separation medium again.PBMC is derived from, followed by vitro culture.

(4) inoculation is counted：It is resuspended with culture medium after washing for the second time, counted with cell counting count board, by cell concentration It is adjusted to 5 × 10⁶-1×10⁷/ mL, coating buffer is suctioned out and is reclaimed, in cell suspension inoculation to the blake bottle being coated with.

(5) cell factor：Interleukin 1 (IL-1 α), Interleukin-3 recombinant (rIL-3), recombinant interferon Concentration in (rIFN- γ) according to the form below 1 is added in culture medium, and adjusts culture medium total amount for 15mL, in the lump in addition blake bottle.

(6) the 2nd days：Observation of cell situation, recombination leukocyte mesonium-2 (rIL-2) is added by 500U/mL concentration, and will training Support base cumulative volume and add to 10mL.Hereafter expand volume once every 1-2 days and growing state is rolled into a ball according to cell clone and add rIL- 2.According to cell growth status, it is 5% (2.5%-10%) to add autoserum mean concentration.

The cytokine concentrations of table 1. and its addition time

(7) the 4th days：System adds to 20mL, and maintains 5% autoserum and 300U/mL rIL-2 concentration.

(8) the 7th days：Observation of cell growing state, cell is softly blown and beaten, and expands to T75 blake bottles, and total system reaches 40mL, Maintain 5% autoserum and 300U/mL rIL-2 concentration.Thereafter according to the cell growth status replenisher scale of construction.If cell concentration Enough, 1 × 10 is taken out when culture was to the 11st day or 12 days⁷Cell.Lentamente it is well mixed, is made cell mixture.Cell At 4 DEG C, 1800 revs/min are centrifuged 10 minutes, reclaim supernatant and continue as culture medium, and cell presses 5 × 10 with CELL BANKERII⁵ To 5 × 10⁶The slow mixing of cell/mL frozen stock solutions freezes standby to ultra low temperature freezer or liquid nitrogen.

(9) from the 11st day, rIL-2 concentration is adjusted to 100U/mL, the IL-2 of low concentration is found with reference to the past document The CD8 of higher proportion can be obtained⁺T cell.

(10) cell is collected：14th day：Cell is collected in aseptic 50mL centrifuge tubes, is centrifuged 10 minutes by 1800 revs/min, Remove supernatant, cell count.

(11) the cell anti-human CD4 of Bioegend companies streaming antibody PE anti-human CD3, FITC, PE-Cy7 that will be collected are anti- The anti-human CD56 of people CD8, FITC, PE anti human CD 19s are marked to cell.Cell is mixed with 1X PBS, takes 3 × 10⁶Cell is divided equally To 3 streaming pipes, per pipe volume 500uL；According to the form below order adds antibody, antibody to add streaming bottom of the tube, and room temperature is kept away after concussion Light 20 minutes；Shake again, then carry out flow cytometer detection.

The result of flow cytometer detection shown, following change, CD19 in incubation were there occurs from 0 to 14 day⁺B cell disappears, CD8⁺T cell all substantially rises in two groups, and patient's group is culture preceding 35.7%, 74% after culture；Healthy control group is culture Preceding 26.4%, 78.2% after culture, two groups of CD8 after incubation⁺T ratio no significant differences (P>0.05).CD3⁺CD56⁺Phenotype exists Healthy group is expressed as 59.2%, is 44.7% in patient's group, there is obvious significant difference (P between two groups<0.05).

Embodiment one：Prepare the specific cytotoxic T lymphocyte of Antigenic Peptide

1st, 34 adenocarcinoma of colon patients and 14 PBMC of normal healthy controls are obtained according to preceding method；And trained in vitro Support, softly blew and beat the cell of culture at the 7th day, it is 5 × 10 to count adjustment quantity⁶Cells/well is added to 96 orifice plates, each sample Originally three multiple holes are set.

2nd, No. 1 peptide and No. 2 peptides are disposably added by 50ug/mL respectively, stimulates cell to 14 days, collect cell.

3rd, CTL kills efficiency

3.1 tumour cell bed boards：At 13 days of cell culture to using the isometric mixed culture mediums of RPMI-1640/DMEM The DLD-1 of culture, is washed twice with PBS, is digested with pancreatin, and serum-containing media is centrifuged 8 points after terminating digestion with 900 revs/min Clock, the resuspended counting of RPMI-1640/DMEM mixed culture mediums containing 10% hyclone, 1 × 10 is adjusted to by cell concentration³/ 100uL, spreads 96 orifice plates, per hole 100uL, 15 holes of every kind of cell (point four experimental groups and a blank control group, every group 3 Multiple holes), it is put into 37 DEG C, 5%CO₂Adhere-wall culture 6-12 hours in incubator.

3.2CTL is killed：Polypeptide group and control group CTL are pressed 1 × 10 respectively³/100uL、5×10³/ 100uL concentration is added Four groups of tumour cell imitate target ratio respectively 1/1,5/1 per hole 100uL.Respectively after 3 days, MTS measure is carried out after 7 days, comment Estimate killing situations of each treatment group CTL to tumour cell.

3.3MTS：Culture medium in all holes of 96 orifice plates was suctioned out in 3rd day and the 7th day, dispensed to 1.5mL EP by group Pipe is marked, and is frozen into the standby survey cytokine secretion profile of -30 DEG C of refrigerators.According to Promega specifications, by RPMI-1640/DMEM Isometric mixed culture medium:MTS=100uL:20uL/ holes, will add MTS dilutions, 37 DEG C, 5%CO in all holes₂Incubator Middle incubation 2-3 hours, lucifuge on ice after taking-up surveys absorbance (OD) value during 492nm wavelength with ELIASA, and blank control group is only Have culture medium it is acellular (BLANK, the average value that all group resins subtract BLANK is tumour cell Relative Absorbance value, that is, swell Oncocyte relative activity), positive controls only have tumour cell.

3.4 IFN-γ secretions measure fixed：

1) kit heats up 30 minutes to 18-25 DEG C with testing sample；

2) preparation of working solution and standard items carries out that following liquid need to be prepared according to specification：(A. concentrated cleaning solutions are used double Water dilution is steamed, dilution ratio is 1:25；B. standard items 400pg/mL, 200pg/mL, 100pg/mL, 50pg/mL, 25pg/mL and 12.5pg/mL；C. biotinylated antibody working solution；D. enzyme conjugates working solution)；

3) it is loaded：Blank well, standard sample wells and sample well are set.Blank well adds the μ l of sample diluting liquid 100, and remaining hole adds respectively Plus standard items or 100 μ L/ holes samples are measured, sample-adding should impose on ELISA Plate bottom, not touch hole wall, not produce bubble simultaneously light Soft piping and druming is uniform.ELISA Plate film is coated, and 37 DEG C are incubated 90 minutes；

4) liquid is abandoned, buckles dry repeatedly on filter paper, do not washed.The μ L/ holes of addition biotin labelled antibodies working solution 100 ( Film covering is being prepared using first 15 points), overlay film is added on ELISA Plate, 37 DEG C are cultivated 1 hour；

5) liquid in hole is discarded, is dried, board-washing 3 times, every time immersion 1-2 minutes, about 350 μ L/ holes, dry, blotting paper point Boreliquid is hit to pat dry；

6) 100 μ L/ holes of enzyme-added conjugate working solution (preparing for 15 minutes before use), add overlay film, and 37 DEG C are incubated 30 minutes；

7) liquid in hole is discarded, is dried, washed 5 times, the same step 5) of method；

8) each hole adds the μ L of substrate solution (TMB) 90, ELISA Plate to add overlay film, and 37 DEG C are incubated 15 minutes；

9) the μ L/ holes of terminate liquid 50, reaction terminating, the color change of blueness to yellow are added.Terminate liquid addition sequence should be the bottom of with Thing identical order of addition.

10) ELIASA is used immediately in the OD values in each hole of 450nm wavelength measurements；

11) result of calculation：The IFN-γ concentration of standard is abscissa, and the OD values of corresponding ordinate do standard curve, and Sample concentration is looked into according to standard curve.

Experimental result referring to Fig. 2, the 3rd day of killing, when effect target ratio is 1/1, four patients for the treatment of conditions and control CTL DLD-1 is killed and has no significant difference, also without statistical discrepancy between four treatment in patient's group and in healthy control group：P1, P2, P1 Mean OD value ± the standard deviation that+2, NP treatment conditions patient group are killed is respectively 1.834 ± 0.377,1.924 ± 0.663, 1.629 ± 0.267,1.95 ± 0.769；Healthy control group kill mean OD value ± standard deviation be 1.625 ± 0.584,1.522 ± 0.238,1.546 ± 0.301,1.702 ± 0.559 (P>0.05), red dotted line shows without CTL and exists that only DLD-1's is positive right According to OD values are 2.328, see Fig. 2 a.During 5/1 effect target ratio (Fig. 2 b), patient's group and health group all occur under P2, P1+2 treatment conditions The notable difference of fragmentation effect；The OD values respectively 1.198 that P2 treatment conditions patient group of wherein 5/1 target than under is killed ± 1.134；It is 0.784 ± 0.663 that healthy control group kills OD values.It is in patient's group OD values of P1+2 treatment of the 5/1 effect target than under 0.759±1.526；The OD values of normal healthy controls are：0.456±0.773.Only 10/1 effect target is than display patient under NP treatment conditions The CTL of group kills ability and is better than normal healthy controls (P<0.05), OD values are patient's group：0.465 ± 0.443 healthy group：0.539± 0.092.Illustrating the addition of peptide RLSSCVPVA can effectively improve the ratio of CTL, then the effective vigor for suppressing DLD.

Embodiment two：The comparing of the influence that the Antigenic Peptide and Antigenic Peptide of BMDC (DC) load are individually produced to CTL.

BMDC is the most strong antigen presenting cell of the ability of offering, it is known to those skilled in the art that it is anti-to load DC Former peptide obtains DC vaccines, it is however generally that, DC vaccines will be higher for the efficiency of antigen alone peptide generation CTL.But the present inventor By the result of following experiment, unexpected discovery individually apply Antigenic Peptide obtain it is more more preferable than the DC vaccines of Loading peptides Effect.

1st, the DC of Loading peptides is obtained

(1) peripheral blood sample and 1X PBS are pressed 1:It is slowly added in lymphocyte separation medium after 1 volume dilution, room temperature condition Under, 2000 revs/min are centrifuged 20 minutes；Draw the tunica albuginea layer in the middle of layering liquid and be added to new 50ml centrifuge tubes.With 2 or 3 times of bodies Long-pending 1X PBS piping and druming is mixed, and is centrifuged 2000 revs/min and is centrifuged 10 minutes.Washing process carries out twice, obtaining PBMC.；

(2) PBMC is placed in 6 orifice plates (6 × 10⁶-8×10⁶Cells/well) (5 × 10⁷Cell/6 orifice plate), 37 DEG C of culture 1-3 Hour, gentle aspiration non-adherent cell.

(3) attached cell is cultivated in the RPMI-1640 nutrient solutions containing 10% hyclone, adds the GM- of 800U/ml The rIL-4 of CSF and 500U/ml, half amount is changed liquid and supplements cell factor to original content within the 3rd day, the 5th day non-adherent cell of gained Induce the immature DC for differentiating.

(4) in order to obtain ripe DC, the 5th day LPS to addition 100ng/ml in immature DC stimulates 48 hours.

Add No. 1 peptide and No. 2 peptides within (5) the 6th days, respectively by 10ug/mL, 20ug/mL, 50ug/mL, 100ug/mL, 200ug/mL is disposably added, and stimulates cell by the 8th day, obtains the DC of Loading peptides.

(6) the 8th days, collecting cell carried out flow cytometer showed detection cell phenotype, and 5 pipe lists dye matches somebody with somebody Isotype control：FITC is anti-human The anti-human HLA-DR of CD80, PE anti-human CD86, PE anti-human CD83, FITC anti-human CD11c, PE.

(7) the 8th days, DC points three groups (P1, P2, P1+2) of Antigen body stimulated autologous PBMC, and the 4th group is not load The DC and PBMC of antigen are co-cultured, and culture medium is replaced by LONZA X-VIVO 15, add 5% autoserum and 300U/mL IL-2.Antigenic Peptide (P1, P2, P1+2) and PBS (negative control) are carried out according to 1 couple of corresponding volunteer PBMC of embodiment simultaneously Directly stimulate.Cultivate by the 14th day and collect all cells.

2nd, CTL kills efficiency

(1) tumour cell bed board：The 13rd day of cell culture in step 1 is to isometric using RPMI-1640/DMEM The DLD-1 of mixed culture medium culture, is washed twice with PBS, with pancreatin digest, serum-containing media terminate digestion after with 900 turns/ Separate the heart 8 minutes, the RPMI-1640/DMEM resuspended countings of isometric mixed culture medium containing 10% hyclone, by cell concentration It is adjusted to 1 × 10³/ 100uL, spreads 96 orifice plates, per hole 100uL, 15 holes of every kind of cell (point four experimental groups and a blank pair According to group, every group of 3 multiple holes), it is put into 37 DEG C, 5%CO₂It is adherent 6-12 hours in incubator.

(2) CTL is killed：At the 14th day, by P1, CTL the and DC Antigens stimulation of tetra- groups of Healthy Peoples of P2, P1+2, NP Healthy human T-cell, the individuality according to cell derived is corresponded, respectively by 1 × 10³/100uL、5×10³/100uL、1×10⁴/ 100uL、2×10⁴/ 100uL concentration adds four groups of tumour cell per hole 100uL, effect target ratio respectively 1/1,5/1,10/1, 20/1.Respectively after 3 days, MTS measure is carried out after 7 days, killing situations of each treatment group CTL to tumour cell is assessed.

(3) MTS killing experiments and IFN-γ secretion situation：Culture medium in all holes of 96 orifice plates was inhaled in 3rd day and the 7th day Go out, dispensed into 1.5mL EP by group and manage and mark, freeze into the standby survey cytokine secretion profile of -30 DEG C of refrigerators.According to Promega specifications, by the isometric mixed culture mediums of RPMI-1640/DMEM:MTS=100uL:20uL/ holes, by all holes Add MTS dilutions, 37 DEG C, 5%CO₂It is incubated 2-3 hour in incubator, lucifuge on ice after taking-up, with ELIASA survey 492nm ripples Absorbance (OD) value when long, blank control group only has culture medium, and acellular (BLANK (blank), all group resins are subtracted The average value of BLANK is tumour cell Relative Absorbance value, i.e. tumour cell relative activity), it is thin that positive controls only have tumour Born of the same parents.IFN-γ secretion amount is determined using ELISA method.

, referring to Fig. 3, the CTL of DC Antigens induction is straight with antigen when effect target ratio is 5/1 (A) and 10/1 (B) for experimental result Connect stimulation activation CTL kill DLD-1 efficiency ratios compared with.P1DC+T, P2DC+T, P1+2DC+T, P0DC+T represent DLD-1 and divide respectively The CTL that PBMC is obtained is not stimulated to be incubated altogether by the DC of Loading peptides 1, Antigenic Peptide 2, Antigenic Peptide 1+2 and unsupported Antigenic Peptide. PBMC+P1, PBMC+P2, PBMC+P12, T cell represent DLD-1 and respectively by the direct stimulation of Antigenic Peptide 1,2,1+2 respectively The CTL and nonreactive former peptide that PBMC is obtained stimulate the CTL that PBMC is obtained.

The CTL of DC Antigens induction directly stimulates activation CTL to DLD-1 fragmentation effect killing activity formula with antigen For:

BLANK OD values are 3.523 for the OD values of 0.0403, DLD-1, when it is 5/1 to imitate target ratio, the DC of Loading peptides 1 The CTL that PBMC is obtained directly is stimulated to be respectively 26.7% and 65.8% (P=0.054) to the killing rate of DLD-1 with Antigenic Peptide 1, The DC of Loading peptides 2 directly stimulate with Antigenic Peptide 2 CTL that PBMC is obtained the killing rate of DLD-1 is respectively 20.7% and The DC and Antigenic Peptide 1+2 of 81.3% (P=0.039), Loading peptides 1+2 directly stimulate the CTL that PBMC is obtained to kill DLD-1 The rate of wound is respectively 39.9% and 69.2% (P=0.048).When it is 10/1 to imitate target ratio, DC and the Antigenic Peptide 1 of Loading peptides 1 The CTL that PBMC is obtained directly is stimulated to be respectively 36.3% and 88.8% (P=0.0384), Antigen to the killing rate of DLD-1 The PBMC that the DC of peptide 2 directly stimulates with Antigenic Peptide 2 obtains CTL and is respectively 37.5% and 89.7% (P=to the killing rate of DLD-1 0.0224), the DC of Loading peptides 1+2 and Antigenic Peptide 1+2 directly stimulates the CTL that PBMC is obtained to distinguish the killing rate of DLD-1 It is 45.6% and 86.6% (P=0.043).

CTL activation to imitating two groups when target ratio is 5/1,10/1 does the measure of IFN-γ secretion amount, such as Fig. 4, manage bar at two It is shown in receiving the CTL of PBMC activation of Healthy People when P1 Antigenic Peptides stimulate under part advantageously.

Experimental result according to embodiment 2 can be seen that and directly stimulated PBMC using Antigenic Peptide and particularly receive Antigenic Peptide 1 In the case of stimulation, while cell factor is present, the CTL of acquisition uses DC Antigens to the killing ability ratio of DLD-1 Stimulate the killing ability of the CTL in the same volunteer PBMC sources of activation strong afterwards.PBMC is directly stimulated to simulate body using Antigenic Peptide Interior periphery blood component receives the situation of Antigenic Peptide stimulation, and outside DC is activated, Antigenic Peptide has equally activated other immune responses Link, different from full tumour cell or tumor cell debris activation T cell, tumor neogenetic Antigenic Peptide optimizes identification process, makes Only possess specific antigen TCR T cell it is amplifying activated, can more accurate targets neoplastic cells, therefore using directly making Identical effect can also be realized with antigenic stimulus peripheral blood.

Sequence table

<110>Fan Kexing

<120>A kind of preparation method of colorectal cancer T cells with antigenic specificity

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>The neoantigen peptide that No. 1 peptide, i.e. transforming growth factor β II receptors frameshift are produced

<400> 1

Ser Leu Tyr Lys Phe Ser Pro Phe Pro Leu

1 5 10

<210> 2

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>No. 2 peptides, i.e. the neoantigen peptide that O N-Acetyl-D-glucosamine glycosyl transferase frameshift is produced

<400> 2

Arg Leu Ser Ser Cys Val Pro Val Ala

1 5

Claims

1. the preparation method of T cells with antigenic specificity, it includes making T cell be incubated altogether with tumour specific antigen.

2. the method described in claim 1, wherein the tumour specific antigen is colorectal cancer specific antigen.

3. the method described in claim 2, wherein the colorectal cancer specific antigen is peptide RLSSCVPVA.

4. the method any one of claims 1 to 3, wherein the T cell is in suspension.

5. the method described in claim 4, wherein the suspension is PMBC suspension or peripheral blood.

6. the method any one of claims 1 to 3, wherein the T cell is through in vitro culture.

7. the method any one of claims 1 to 3, wherein T cell are CD8+T cells.

8. the T cells with antigenic specificity that method according to any one of claim 1 to 7 is obtained.

9. the T cells with antigenic specificity described in claim 8, it is colorectal cancer T cells with antigenic specificity.

10. the colorectal cancer T cells with antigenic specificity described in claim 9 is in the preparation for treating colorectal cancer is prepared Purposes.