CN105368778A - Method for preparing human CIK (cytokine-induced killer) cell by combining novel TLR7 agonist GD - Google Patents

Abstract

The invention discloses a method for preparing a high-toxicity CIK (cytokine-induced killer) cell by combining a novel TLR7 agonist GD. The method includes the steps of 1) collecting peripheral venous blood to obtain mononuclear cells; 2) adding the novel TLR7 agonist, a small molecule compound GD to perform CIK cell induced culture; 3) performing CIK cell multiplication culture to obtain a novel heterogeneous cell population, namely a GD-CIK cell. The method has the advantages that compared with a CIK cell cultured by a conventional method, the GD-CIK cell prepared by the method is obviously higher in tumor killing cytotoxic activity and is up to above 95% in tumor killing rate (effector-target ratio: 20:1); the method is low in preparation cost, simple in process, easily controllable in condition, low in requirement on equipment, easy for scale production and the like; the GD-CIK cell prepared by the method is mainly applied to cancer patient treatment or prevention of high-risk groups of cancers.

Description

A kind of method of combining novel TLR7 agonist GD and preparing people's CIK cell

Technical field

The invention belongs to cellular immunology, immunotherapy of tumors field, relate to a kind of method and the application of antitumor adoptive immunotherapy of combining novel TLR7 agonist GD and preparing people's CIK cell.

Background technology

In recent years along with the deterioration of living-pattern preservation and physical environment, add the raising of medical skill, it is in rising trend that tumour detects number, and tumour has become one of important diseases of serious threat human health.Because tumour has higher damaging and higher case fatality rate, the focus that people pay close attention to the investigation and application of antineoplastic treatment means always, immunotherapy of tumors is the clinical the fourth-largest therapy for oncotherapy, wherein immune cell therapy is widely used in China, and domestic at present to carry out maximum be exactly the adoptive feedback therapy of immunocyte.

CIK cell and Cytokine-induced killer cells, be by human peripheral blood single nucleus cell in vitro with cytokine profiles co-cultivation for some time after non-principal histocompatibility complex (MHC) the restrictive cytotoxic T lymphocyte that obtains, have tumour cell and dissolve toxicity efficiently, after DC and CIK cell Dual culture, effectively can improve multiplication capacity and the killing activity of CIK, more being conducive to the antineoplastic immune curative effect improving CIK, is also current domestic application immunotherapy techniques the most widely.

Toll-like receptor (TLRs) is the pattern recognition receptors (patternrecognitionreceptors be present on dissimilar cell, PRRs), the specific molecular pattern that can be identified by these PRRs is classified as a class, be called pathogenic agent associated molecular pattern (pathogen-associatedmolecularpatterns, PAMPs).PAMPs is pathogenic agent conserved structure molecule, and other molecular patterns of host have very large difference, and as pathogen invasion host, body starts for the corresponding immune programme for children of pathogenic agent, thus opposing foreign pathogen is attacked and do not affect host.This pattern recognition mechanisms is the first barrier of body opposing pathogenic bacteria, and Toll-like receptor plays a significant role in this process.

In TLRs family, TLR7 is the Toll-like receptor being first found to be activated by micromolecular compound.The part of TLR7 has the micromolecular compound imidazole quinoline class and guanosine analogue etc. of single stranded RNA (ssRNA), non-viral source thing (as poly dT etc.) and chemosynthesis.The special activation TLR7 of TLR7 part energy, have very strong adjuvant effect, the great majority therefore in these TLR7 parts can use as immunological adjuvant, as produced antiviral and antitumor action by cytokine before induction.The native ligand of TLR7 is single stranded RNA (ssRNA), although it also has good agonism, due to its easy enzymolysis, producing one is class bio-toxicity and its preparation cost high, and its investigation and application is restricted.Although the ssRNA of existing research to synthesis carries out structural modification, namely improve the stability of RNA, also improve immunological effect.But the present invention's synthesized micromolecule TLR7 part used has the advantages such as rock steady structure, hypotoxicity and low cost, the present invention is by the TLR7 agonist GD [Gaoetal.ChemMedChem of independent research, 2015,10 (6): 977-980] use in immunotherapy of tumors as immuno-stimulator, thus prepare more effective CIK cell, be referred to as GD-CIK.Stimulate CIK cell activation through TLR7 agonist GD, stimulate more cell stimulating factor to secrete, produce collaborative hormesis altogether, have and better kill tumor activity.

In treatment tumour, conjoint therapy is paid attention to gradually, and the report of TLR7 agonist combined chemotherapy, other immunotherapies also day by day increases, but all belongs to and simply superpose category, and based on common stimulation, the CIK cell of combination therapy theory has no report.The present invention is by TLR7 agonist, and micromolecular compound GD combines conventional cytokine, stimulates altogether, and induction CIK cell, is intended to the associating natural immunity, strengthens tumor-killing effect.Realize conjoint therapy theory in vitro, reduce conjoint therapy risk in body.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, on the basis of the CIK cell optimized by TLR7 small molecule agonist GD, provide a kind of preparation method and application thereof of people's CIK cell of combination therapy theory, i.e. GD-CIK, after solving the cultivation of prior art CIK cell, heterogeneous population somatocyte cytotoxic activity improves the problems such as not remarkable; Obtain may be used for the higher antitumor adoptive immunity active cells of clinical killing-efficiency.

For achieving the above object, the technical solution used in the present invention is:

Combine the method that novel TLR7 agonist GD prepares people's CIK cell, described in its feature concentrates on, comprise step:

1) peripheric venous blood collection, is separated, and obtains mononuclearcell;

2) Day0, adds novel TLR7 agonist GD in conventional CIK cell culture system, follow-uply copies routine, carries out the inducing culture of CIK cell;

3) GD-CIK cell proliferation is cultivated.

As preferably, described step 1): the vein peripheral blood extracting people is in containing in the centrifuge tube of heparin sodium, and centrifugal acquisition serum and complete blood cell, obtain mononuclearcell by complete blood cell through density gradient centrifugation.

Further, described step 1): be specially employing ficoll-general shadow glycosamine density gradient centrifugation separated and collected, and with brine 2 times, after low-speed centrifugal, obtain PBMCs.

Described step 2): be specially and the PBMCs of acquisition is resuspended in GT-T551 ^tMin substratum, add various cytokine, then in 37 DEG C, 5%CO ₂carry out according to CIK cell cultivation conventional scheme with in the incubator of saturated humidity.Day0, is added to aforementioned CIK cell mixed culture 13-15 days by GD, results GD-CIK cell.

Cell form: CD3 positive expression rate more than 90%, CD3 ⁺cD4 ⁺cell is about 10-30%, CD3 ⁺cD8 ⁺cell is at 50-70%, CD3 ⁺cD56 ⁺cell is about 5-10%, CD3 ^-cD56 ⁺cell is about 5-15%.

People GD-CIK cell provided by the present invention, refers to CD3 ⁺cD8 ⁺and CD3 ⁺cD56 ⁺t lymphocyte is main effect cell, within 18 hours, can reach about 98% (imitating target ratio, 20:1) to tumor cytotoxicity activity with K562 co-culture of cells.

" CD3 herein ⁺cD4 ⁺cIK cell " represent that CD3 and CD4 is positive CIK cell." CD3+CD8 ⁺cIK cell " represent that CD3 and CD8 is positive CIK cell." CD3 ⁺cD56 ⁺cIK cell " represent that CD3 and CD56 is positive CIK cell." CD3 ^-cD56 ⁺cell " represent the cell of the negative and CD8 positive of CD3.

Compared with CIK prepared by the GD-CIK of aforesaid method cultivation and cellar culture, killing tumor activity obviously increases, and kills tumor activity and reach more than 98% during experiment in vitro, apparently higher than the CIK cell of ordinary method cultivation.Also have that preparation cost is cheap, operation is simple, condition is easily controlled simultaneously, lower to the requirement of equipment, be easy to the advantages such as large-scale production.The treatment of mainly cancer patient or the prevention of cancer high risk population is applied by the GD-CIK cell prepared by the present invention.

Accompanying drawing explanation

Fig. 1: ripe CIK cell group form.Cultivate the 15th day, the form of ripe CIK under inverted microscope, enlargement ratio is 40 ×;

Fig. 2: cultivate the 15th day flow cytometry and carry out the detection of GD-CIK ripening degree.

Fig. 3: the derived from peripheral blood PBMCs growth curve (n=3) that different training method is cultivated; The PBMCs of derived from peripheral blood is cultivated by different modes, counted with cell counting count board and cell counter respectively at the 0th, 3,5,7,9,11,13,15 day, by the total cellular score of current total cellular score divided by (the 0th day) when starting to cultivate, institute's value is cell proliferation multiple.CIK group: refer to the CIK cell adopting the culture method of Stanford University's application to cultivate; GD-CIK group: refer to training method of the present invention;

Fig. 4: adopt flow cytometer to carry out immunophenotype detection (n=3).The PBMCs of derived from peripheral blood, is cultivated by different modes, gets cell and carries out streaming fluorescence antibody: AntiCD3 McAb-FITC, CD4-PE, CD8-PECY5, CD56-APC carry out padding and detect at the 14th day.CIK group: refer to the CIK cell adopting the culture method of Stanford University's application to cultivate; GD-CIK group: refer to training method of the present invention.

Fig. 5: adopt CCK-8 cytotoxicity assay test kit, cytotoxicity detection (n=3) is carried out to the derived from peripheral blood PBMCs that different training method is cultivated.Cultivated by different modes, got cell at the 14th day and carry out cytotoxicity assay by CCK-8 test kit.CIK group: refer to the CIK cell adopting the culture method of Stanford University's application to cultivate; GD-CIK group: refer to training method of the present invention.X-coordinate: represent effect target cell ratio, represent the ratio of effector cell and target cell be 20: 1, E: T=10: 1 represent the ratio of effector cell and target cell be 10: 1, E: T=5: 1 represent the ratio of effector cell and target cell be 5: 1 at E: T=20: 1.

Fig. 6: GD molecular structural formula, molecular formula, molecular weight, synthetic method to publish thesis compound 9e in [Gaoetal.ChemMedChem, 2015,10 (6): 977-980] in the recent period see inventor.

Embodiment

Illustrate the present invention with example below, but the present invention is not limited.The experimental technique of all unreceipted actual conditionses in example below, the operation instructions that being the method for observing a usual practice and producer provides performs.

Embodiment 1

The present embodiment 1 is separated the PBMCs that obtains and carries out the cultivation of GD-CIK cell.Comprise the following steps:

1. gather detection in peripheral blood of patients underwent 25ml, obtain mononuclearcell through ficoll-general shadow glycosamine density gradient centrifugation.Concrete steps are: 1500 revs/min, centrifugal 10 minutes, draw upper plasma layer, 56 DEG C of deactivations are centrifugal for subsequent use after 30 minutes, and with the hemocyte of physiological saline two-fold dilution precipitation, human lymphocyte parting liquid and dilute blood add in centrifuge tube in the ratio of 1: 2,2000 revs/min, centrifugal 20 minutes, careful draw tunica albuginea layer, with brine 2 times, rotating speed is respectively 1600 revs/min, 1300 revs/min, all centrifugal 7 minutes, namely obtain PBMCs.

The inducing culture of 2.GD-CIK cell.Concrete steps are: the suspension cell collected in embodiment 1 step 1 is resuspended in the GT-T551 containing 10% autologous deactivation blood plasma ^tMdeng in commercialization serum free medium, adjust cell density 2 × 10 ⁶about/ml, and add IFN-γ to final concentration 1000IU/ml, be finally transferred in culturing bottle and cultivate.Cultivate to add after 24 hours anti-CD49d McAb, AntiCD28 McAb, IL-1 α, IL-2,

GD, cultured continuously 13-15 days, wherein often add the fresh culture containing IL-2 for 2-3 days, the cell density after supplemented medium controlled in (1-2) × 10 ⁶/ ml.Wherein, anti-CD49d McAb concentration is 50ng/ml, and the concentration of the concentration of AntiCD28 McAb to be the concentration of 200ng/ml, IL-1 α be 1ng/ml, IL-2 is 1000IU/ml; The concentration adding GD is 8 μMs.

3. counted with cell counting count board and cell counter respectively at the 0th, 3,5,7,9,11,13,15 day, by current total cellular score divided by the total cellular score started when cultivating, institute's value is cell proliferation multiple, reference when dynamically observing cell proliferative conditions using this and add fresh culture as cell, cell proliferation multiple is shown in Fig. 3, table 1.

Table 1: the derived from peripheral blood PBMCs amplification times (n=3) that different training method is cultivated

Result shows, and along with the prolongation of incubation time, two groups of cells are all in growth, and GD-CIK cells expanded is starkly lower than CIK group, significant difference (P<0.05), and two groups of cells all reached climax at 15 days.

Immunophenotype detection is carried out to GD-CIK cell.Step is as follows:

Get the cell of the 14th day, PBS washs, resuspended, and adjustment cell density is 1 × 10 ⁶/ ml, 50 μ l cell suspensions are added in each detector tube, add fluorescent-labeled antibody (AntiCD3 McAb-FITC, CD4-PE, CD8-PECY5.5, CD56-AP3) dyeing, lucifuge hatches 30 minutes, wash with above-mentioned PBS, be resuspendedly diluted to 100 μ l, detect with flow cytometer, data file adopts CFlowPlus software analysis, the results are shown in Figure 4, table 2.

Table 2: different training method human peripheral blood source PBMCs Phenotype (n=3)

Result shows, and cultivates the 14th day, CD3 in three groups of cells ⁺cD4 ⁺and CD3 ^-cD56 ⁺between cell, difference is not remarkable; CD3 ⁺cD56 ⁺in cell, GD-CIK group is apparently higher than CIK group, and difference all significantly (P<0.05); CD3 ⁺cD8 ⁺in cell, GD-CIK group is starkly lower than CIK group, significant difference (P<0.05).

Cytotoxicity assay is carried out to GD-CIK cell.Comprise the following steps:

1. get the cultivation GD-CIK cell of the 14th day, detect the killing activity to K562 tumor cell line.

2. the cell density adjusting K562 is 4 × 10 ⁴/ hole, 96 orifice plates, every hole 100 μ l, ratio in 1: 5,1: 10,1: 20 and GD-CIK cytomixis, each concentration all arranges 3 multiple holes, arranges independent target cell (tumour cell) contrast, individual effect cell (GD-CIK cell and CIK cell) contrast and single culture base blank simultaneously.

3. be placed in 37 DEG C, 5%CO ₂after cultivating 4 hours in the incubator of saturated humidity, every hole adds the CCK-8 solution of 10 μ l, then cultivates 4h, then measures absorbancy (OD) value by microplate reader in 450nm wavelength place.

4., according to formula: kill ratio of outflow (%)=[1-(experimental group OD value-effector cell organizes OD value)]/(effector cell organizes OD value) × 100%, calculate and kill knurl efficiency.Wherein, in formula, each OD value is the value after deducting blank group OD value.The results are shown in Figure 5, table 3.

Table 3: different training method human peripheral blood source PBMCs cytotoxic activity impact (n=3)

Result shows, different effect target ratio, significant difference (P<0.01) between CIK group and GD-CIK groups of cells; .After this illustrates the improved scheme induction of CIK cell, the cytotoxic activity accounting for the lethal cell of most ratio significantly improves, thus CIK cell whole cell cytotoxic activity is also significantly improved, illustrate that GD-CIK cell prepared by Combined culture method of the present invention has better treatment advantage.

Claims (5)

1. combine the method that novel TLR7 agonist GD prepares people's CIK cell, comprise step:

1) peripheric venous blood collection, is separated, and obtains mononuclearcell;

2) in CIK cell culture system, add novel TLR7 agonist GD, carry out the inducing culture of CIK cell;

3) GD-CIK cell proliferation is cultivated.

2. the preparation method of the people GD-CIK cell of high cytotoxic activity as claimed in claim 1, it is characterized in that, described step 1): extract the vein peripheral blood of people in containing in the centrifuge tube of heparin sodium, centrifugal acquisition serum and complete blood cell, by complete blood cell through ficoll-general shadow glycosamine density gradient centrifugation, obtain mononuclearcell.

3. the preparation method of the people GD-CIK cell of high cytotoxic activity as claimed in claim 1, it is characterized in that, described step 2): by step 1) in collect mononuclearcell be resuspended in the serum free medium containing the autologous deactivation blood plasma of 8-10%, adjustment cell density (1-2) × 10 ⁶about/ml, adds TLR7 agonist GD to final concentration 5-15 μM, and adds IFN-γ to final concentration 500-1000IU/ml, be finally transferred in culturing bottle and cultivate; Cultivate and add anti-CD49d McAb, AntiCD28 McAb, IL-1 α, IL-2 after 20-28 hour, continue to cultivate.

4. the preparation method of the people GD-CIK cell of high cytotoxic activity as claimed in claim 1, it is characterized in that, described step 3): by step 2) cell cultured continuously 13-15 days, wherein often within 2-3 days, add the fresh culture containing IL-2, the cell density after supplemented medium is controlled in (1-2) × 10 ⁶/ ml; Cultivate and gather in the crops GD-CIK cell after 13-15 days.

5. the preparation method of the people GD-CIK cell of high cytotoxic activity as claimed in claim 4, it is characterized in that, described anti-CD49d McAb concentration is 50-500ng/ml, and the concentration of AntiCD28 McAb is 50-500ng/ml, the concentration of IL-1 α is the concentration of 0.5-5ng/ml, IL-2 is 50-1000IU/ml; The concentration adding GD is 5-15 μM; Described GD concentration is 8 μMs, and described IL-2 final concentration is 1000IU/ml.