CN113444687A

CN113444687A - DC vaccine and DC-CTL method for transfection of CD40L through cell-penetrating peptide mediated tumor antigen polypeptide sensitization

Info

Publication number: CN113444687A
Application number: CN202110601347.1A
Authority: CN
Inventors: 徐健; 李陶; 张扬
Original assignee: Zhejiang Shengxi'ao Medical Technology Co ltd
Current assignee: Zhejiang Shengxi'ao Medical Technology Co ltd
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2021-09-28

Abstract

The invention belongs to the field of biological medicines and the technical field of cell therapy, and particularly relates to a DC vaccine and a DC-CTL method for transfecting CD40L by sensitization of cell-penetrating peptide mediated tumor antigen polypeptides. Comprises the in vitro culture of DC and CTL; the HLA-A2 restricted tumor antigen peptide is predicted by jointly applying a hyper-motif method, a polynomial scheme and a quantitative motif method for predicting the epitope, and the feasibility of the epitope prediction is verified; constructing an h-CD40L expression system; DC cell transfection of CD40L expression vector; DC cells expressing CD40L are tested for in vitro immune function after being sensitized with tumor antigen peptide through the cell-penetrating peptide mediated CTL; the invention provides a DC vaccine and a DC-CTL method for transfecting CD40L by sensitization of a cell-penetrating peptide mediated tumor antigen polypeptide, which are used for continuously perfecting a tumor antigen peptide library and optimizing DC cell culture, discussing DC cell transfection of a CD40L expression vector and testing the in-vitro immunity function of the DC cell expressing CD40L after sensitization of the cell-penetrating peptide mediated tumor antigen peptide on the basis of synthesizing and screening the tumor antigen peptide with a killing effect on various tumors and a DC induction culture technology.

Description

DC vaccine and DC-CTL method for transfection of CD40L through cell-penetrating peptide mediated tumor antigen polypeptide sensitization

Technical Field

The invention belongs to the field of biological medicines and the technical field of cell therapy, and particularly relates to a DC vaccine and a DC-CTL method for transfecting CD40L by sensitization of cell-penetrating peptide mediated tumor antigen polypeptides.

Background

On the basis of successfully establishing an in vitro DC cell induction culture technology at the early stage, predicting HLA-A2 restrictive tumor antigen peptides, synthesizing, screening and establishing a tumor antigen polypeptide library, deeply researching the influences of processing DC maturation promotion at different time, loading the DC with the antigen peptides at different concentrations, loading the antigen peptides in different modes, transfecting the DC with CD40L and the like on the form, the phenotype and the function of the DC, discussing and optimizing a DC maturation promoting scheme, improving DC loading efficiency, DC antigen presentation efficiency and a DC-mediated specific anti-tumor response mechanism, and providing a new idea for exploring a DC vaccine and DC-CTL cell treatment which have more obvious curative effects, are safer and have more specific immune response.

The basic strategy of tumor vaccines is to activate tumor specific Cytotoxic T Lymphocytes (CTL) by constructing and using the vaccines, so as to effectively induce specific anti-tumor immune response and achieve the purpose of treating tumors. Three criteria determine the tumor killing effect of the tumor vaccine: 1. generating enough immune cells which can highly recognize tumor antigens in vivo, and inducing the generation of long-term memory cells; 2. these immune cells migrate to the tumor site and infiltrate into the tumor stroma, proliferate and activate; 3. immune cells must be activated at tumor sites, and various materials aiming at tumor-associated antigens (TAA) can be used for preparing tumor vaccines, such as tumor cell vaccines, tumor nucleic acid vaccines, tumor genetic engineering vaccines, cytokine tumor vaccines and the like, which are caused by tumor destruction caused by directly dissolving or secreting cytokines. The heteroplasmy protrusion of Dendritic Cell (DC) tumor vaccine appeared in recent years becomes a new hotspot for tumor vaccine research.

In general, mature DCs can be used as vaccines after in vitro loading with tumor antigen peptides, or after maturation of immature DC precursors by receiving tumor proteins or lysates. In order to obtain mature DC and more effectively stimulate the activity of DC, a new stimulating factor needs to be added into a culture system to improve the expression of co-stimulatory molecules and promote the maturation of DC. Compared with immature DC, the mature DC is prone to be used as a tumor vaccine, and the target antigen specificity T cell inhibition can be avoided; ② induction of regulatory T cells.

Through preliminary experiments and reference to domestic and foreign literature reports, a technical system for in vitro induction culture of mature DC cells is established in earlier work, a DC maturation promoting scheme is summarized in the preliminary experiments, and a culture scheme is further optimized.

Studies have shown that in vitro induced DC cells can effectively stimulate allogeneic T cells in MLR, but there is a need for further improvements in loading efficiency and antigen uptake efficiency. How to effectively enter the DC cells, prolong the degradation of the antigen peptide in the cells and delay the half-life period of the antigen peptide so as to enhance the combination of the antigen peptide and the DC cells, effectively present tumor specific antigen through MHCI molecular pathway, and activate T cells so as to generate CTL with specificity to corresponding tumor cells. The method mainly breaks through the improvement of DC function accompanied by in vitro induced maturation DC, obviously improves the reactivity of T cells, relates to DC loading efficiency and tumor antigen polypeptide presenting efficiency, and is related to the concentration of added tumor antigen polypeptide, the selection of antigen peptide carriers and the like.

With the identification of more and more different tumor epitope peptides aiming at the same tumor antigen, further research finds that the immune response caused by the stimulation of different antigen epitope peptides is different. Therefore, specific tumor antigen peptides are the key of tumor immunotherapy and are the hot spots of research today. In addition, dozens of different antigen polypeptides of a plurality of same tumor antigens discovered by various methods at present have epitopes presented by HLA-I, II molecules and epitopes presented by HLA-I and II molecules simultaneously, can activate CD4+ and CD8+ T lymphocytes simultaneously, and are beneficial to enhancing the anti-tumor immune response effect and prolonging the duration of specific immunity.

Because the polypeptide vaccine has the limitations of HLA phenotype and the number of antigen peptides, a tumor antigen polypeptide library is mainly established in earlier stage work, and the synthesized antigen epitope peptide presented by the tumor antigen polypeptide is mainly the first type, the epitope presented by HLA-I type molecules is mainly HLA-A2 molecules, and in the population of China, the HLA-A2 positive population accounts for more than half of the population, especially the HLA-A0201 subtype, so the designed and synthesized tumor antigen polypeptide has important application value in the treatment of the industrialized modern immunity technology in China, and the tumor antigen polypeptide library is planned to be further expanded and perfected.

Although numerous studies have indicated that activation of immune cells against tumor antigens can improve tumor immune responses, such as DCs, additional studies have also indicated that the tumor microenvironment determines DC function, and that tumor-suppressive microenvironments can regulate DCs to induce T-cell anergy or suppress tumor-specific responses elicited by DCs. Thus, activation of DCs directed against tumors may not be sufficient to produce an anti-tumor effect. Incubation of activated DCs against TAAs from outside the tumor microenvironment has the potential to improve the anti-tumor immune response. The selection of which tumor antigen and tumor antigen epitope peptide are used and how to combine the tumor antigen peptide with DC in vitro to make up for the functional defect becomes the key to applying DC vaccine for immunotherapy. The search for an effective antigen polypeptide carrier and the improvement of DC presentation efficiency are one of the solutions, and the effect is achieved by adopting the methods of antigen epitope prediction, cell-penetrating peptide mediated tumor antigen polypeptide and CD40L DC infection.

CPPs are polypeptides which directly penetrate cell membranes to enter cells in a receptor-independent mode and a non-classical endocytosis mode, can carry various substances such as DNA, RNA, polypeptides, proteins, small molecular compounds and the like, and have the advantages of wide histocompatibility, stability, low toxicity, low immunogenicity, relatively simple and convenient artificial synthesis and the like. CPPs have been validated in vaccine studies to carry tumor-associated antigens into antigen presenting cells. The report of Wangnrongfu et al shows that the DC cell penetrating efficiency of HLA restrictive polypeptide can be obviously improved by using CPPs, and the half life period is obviously prolonged. A series of researches show that the CTL immune effect caused by the method can highly express CD8+ T lymphocyte expression, and simultaneously, the expression of IFN-gamma and GM-CSF is obviously improved. Indicating that the CPPs are effective.

How to make DC present antigen more specifically and with higher effect is also the focus of research. The CD40L can be combined with the CD40 on the surface of the tumor cell, directly inhibit the proliferation of the tumor cell and increase the sensitivity of the antitumor preparation. More importantly, CD40L can induce DC cell maturation, prolong DC cell survival time, and up-regulate co-stimulatory molecule and endogenous tumor peptide expression; on the other hand, the secretion of some immunostimulating factors is increased. Thereby improving the antigen presentation effect of the DC, promoting the proliferation of T effector cells and enhancing the anti-tumor immunity capability of the host.

The CD40L gene is transfected into DCs to present tumor antigens to naive CD8+ cells by the DCs in a manner that activates CD40 signaling either by itself or by interaction. After the CD40L transfected DC cells are pulsed, the expression of costimulatory molecules, the secretion of IL-12, the proliferation of homologous T cells and the proliferation inhibition of homologous cancer cells are all improved.

In recent years, research shows that a co-culture system consisting of DCs loaded with antigen peptides and CTLs has stronger killing activity on tumor cells expressing related antigens, and the fact that the killing effect has antigen specificity indicates that the system induces and generates Cytotoxic T Lymphocytes (CTLs).

CTLs play an important role in controlling viral infections and tumors. Cells are essential in the differentiation, expansion and induction of memory of tumor-specific CTLs. T cells can recognize and process polypeptide antigens through appropriate MHC molecules, and antigenic determinants identified by CTL cells can be used as vaccines to activate antigenic determinant specific T cells. MHC class I and MHC class II restricted T cell epitopes can be searched from tumor specific proteins for making polypeptide based vaccines.

The DC vaccine and the DC-CTL technology for transfecting CD40L with cell-penetrating peptide mediated tumor antigen polypeptide sensitization aim at solving the key problem (1) the limitation of artificially synthesizing tumor-associated antigen peptide MHC. The solution to be adopted is to predict antigen peptide, apply cell-penetrating peptide mediation and antigen polypeptide combination and the like as important breakthrough points of immunotherapy; (2) the existing antigen peptide library contains antigens which are far from covering gene sites for early cancer screening and the types and the number of tumor markers, expands the antigen library, strengthens the research on the antigens, and provides more suitable antigens for early accurate intervention of tumors, which is also a key problem to be solved urgently; (3) an antigen high-efficiency peptide load DC method and a DC maturation promoting technology; (4) the construction of CD40L involves multiple links, including whether the recombinant cloning vector and the recombinant expression vector meet the assumed results in various screens, and we intend to continue to use the classical basic line to construct the CD40L expression system.

Disclosure of Invention

The invention provides a DC vaccine and a DC-CTL method for transfecting CD40L by inducing the cell-penetrating peptide mediated tumor antigen polypeptide, which aims to continuously perfect a tumor antigen peptide library and optimize DC cell culture, discuss the DC cell transfected by a CD40L expression vector and test the in-vitro immune function of the DC cell expressing CD40L after the DC cell is sensitized by the cell-penetrating peptide mediated tumor antigen peptide on the basis of synthesizing and screening the tumor antigen peptide with stronger killing effect on various tumors and a DC induction culture technology.

The technical scheme adopted by the invention is as follows: the DC-CTL method for transfecting CD40L by mediating tumor antigen polypeptide sensitization through the cell-penetrating peptide is characterized in that: the method comprises the following steps:

s1: culturing DC and CTL in vitro; observing the surface morphology and the internal structure change of the DC by using an inverted microscope, a scanning electron microscope and a transmission electron microscope; detecting the expression of DC surface molecules CD1a, CD83, CD80, CD86 and HLA-DR by flow cytometry; detecting the proliferation promoting effect of DC cells on T cells in a mixed lymphocyte assay (MLR) by using a thiazole blue (MTT) method; detecting the specific killing activity of CTL to tumor cells by using an LDH method (lactate dehydrogenase killing test); the DC and CTL in-vitro culture comprises a standard process of in-vitro induction DC culture, a standard operation procedure of in-vitro CTL efficient amplification culture technology, identification of dendritic cells, functional identification of DC, culture of tumor antigen polypeptide loaded dendritic cells, effector cell CTL and killing capacity of CTL loaded with tumor antigen polypeptide DC to tumor cells;

s2: the HLA-A2 restricted tumor antigen peptide is predicted by jointly applying a hyper-motif method, a polynomial scheme and a quantitative motif method for predicting the epitope, and the feasibility of the epitope prediction is verified; separating, culturing and identifying peripheral blood DC, inducing CTL activation and amplification after the DC is loaded with tumor antigen peptide, further researching the killing capability to various tumor cells, and screening the tumor antigen peptide with strong killing effect to various tumors.

S3: constructing an h-CD40L expression system;

s4: DC cell transfection of CD40L expression vector;

s5: DC cells expressing CD40L are tested for in vitro immune function after being sensitized with tumor antigen peptide through the cell-penetrating peptide mediated CTL; comprises the secretion of cell factors, the detection of the killing effect of the sensitized and modified DC-CTL cell on tumor cells by a CCK-8 method, the adoption of Si-RNA and the killing of the sensitized and modified DC-CTL cell on in-vivo tumor cells, the screening of the change of immune related genes and tumor markers in tumor tissues by a chip, and the detection of the content of specific CTL cells in the tumor tissues by immunohistochemistry.

The standard process of in vitro induction DC culture comprises the following steps:

s1: the first day: cell preparation

A: 75ml of anticoagulated peripheral blood is extracted, 800g is extracted, and centrifugation is carried out at room temperature for 15min (without breaking);

b: preparation of autologous plasma: collecting supernatant plasma, and placing in water bath at 56 deg.C for 30 min; then standing for 10min at-20 ℃; finally, centrifuging for 15min at 4 ℃ and 1100g, and storing at 4 ℃ for later use;

c: taking the centrifuged lower cell component, adding D-PBS to 50ml, mixing, adding into 3 50ml centrifuge tubes containing 20ml medical grade lymphocyte separation liquid, centrifuging at room temperature for 15min (without breaking) at 800 g;

d: taking the cell layer, adding the culture medium to 50ml, 600g for 10min, and removing the supernatant;

e: preparing 50ml of separated PBMC into cell suspension, adding the cell suspension into a T75 culture flask, and culturing in a 5.0% CO2 incubator at the saturated humidity of 37 ℃ for 2 h;

f: transferring the suspension cells to a CIK bottle for CIK culture, and performing DC culture on adherent cells;

g: adding 5ml of blood plasma into a DC culture bottle, and adding a proper amount of cell factors;

s2: the next day, adding the residual blood plasma and proper amount of cell factors into the DC culture bottle;

s3: on the fourth day, a proper amount of cytokine is added into the DC culture bottle;

s4: on the fifth day, a proper amount of cytokines is added into the DC culture bottle;

s5: on the sixth day, a proper amount of cell factors and a proper amount of cell-penetrating peptide-mediated tumor antigen polypeptide are added into the DC culture bottle;

s6: on day seven, DCs were collected.

The standard operation procedures of the in vitro CTL high-efficiency amplification culture technology are as follows:

s1: coating: adding a proper amount of cell factors into 20ml of D-PBS, keeping the mixture at 4 ℃, keeping out of the sun, and coating a T175 culture bottle overnight; or placing in 37 deg.C incubator, and coating for at least 2 hr;

s2: the first day: cell preparation

A: extracting 50ml of anticoagulated peripheral blood, 800g, and centrifuging at room temperature for 15min (without breaking);

c: taking the centrifuged lower cell component, adding D-PBS to 50ml, mixing, adding into 250ml centrifuge tubes containing 20ml medical grade lymphocyte separation liquid, centrifuging at room temperature for 15min (without breaking) at 800 g;

e: adding 40ml of CIK culture solution into the separated PBMC to prepare cell suspension, adding 5ml of plasma, adding into a T175 culture flask from which the coating solution is removed, simultaneously adding a proper amount of cell factors, and culturing in a 5.0% CO2 incubator at the saturated humidity of 37 ℃;

s3: supplementing 60ml of CTL culture solution the next day, adding 5ml of blood plasma, and adding a proper amount of cell factors;

s4: supplementing 50ml of CTL culture solution in the fourth day, adding all the remaining plasma, and adding a proper amount of cell factors;

s5: on the fifth day, the cells in the culture bottle are completely blown down and transferred to culture bags, and the culture bags are divided into 2 culture bags, and each culture bag is recommended to be supplemented to 400 ml; s6: on the seventh day, according to the growth state of cells, each culture bag is recommended to be supplemented with 300ml of CTL culture solution;

s7: on the tenth day, according to the growth state of cells, each culture bag is recommended to be supplemented with 600ml of CTL culture solution, and a third party is sent to be tested to perform detection on bacteria, fungi, mycoplasma and endotoxin and simultaneously perform self-test;

s8: the thirteenth day: 1400ml of CTL cell suspension is collected, supernatant is discarded after centrifugation at 1500rpm multiplied by 8min, 200ml of physiological saline in each tube of a 250ml centrifuge tube is washed (1500rpm multiplied by 8min) for 1 time, 50ml of physiological saline in a 50ml centrifuge tube is washed (1200rpm multiplied by 8min) for 1 time, cells are resuspended according to 100ml of physiological saline and 5ml of 20% serum albumin, and the cells are sent to a ward after being packaged, and meanwhile, samples are reserved and sealed for later detection; supplementing 200 ml/bag of CTL culture solution, and sending to a third party for detection to perform self-detection on bacteria, fungi, mycoplasma and endotoxin;

s9: on the fifteenth day: 1600ml of CTL cell suspension is collected, centrifuged at 1500rpm multiplied by 8min and then the supernatant is discarded, 200ml of physiological saline is washed (1500rpm multiplied by 8min) for 1 time per tube by 2 tubes of a 250ml centrifuge tube, then 50ml of physiological saline of a 50ml centrifuge tube is washed (1200rpm multiplied by 8min) for 1 time, cells are resuspended according to 100ml of physiological saline and 5ml of 20% serum albumin, and the cells are sent to a ward after being packaged, and meanwhile, the cells are preserved and sealed for later detection.

The identification of the dendritic cells comprises morphological observation of the DC and phenotypic identification of the DC, and the DC surface molecules HLA-DR, CD83 and CD86 are detected by flow cytometry, wherein the morphological observation step of the DC comprises the following steps:

s1: and (4) inverted microscope observation: observing the morphological change of the cells by an inverted microscope every day during the culture period;

s2: and (3) observation by an optical microscope: collecting cultured mDC for 8d, centrifuging at 1000rmp for 5min to obtain cell precipitate, fixing with formalin, dehydrating with gradient ethanol, clearing with xylene, soaking in paraffin and embedding, conventionally making paraffin section, staining with hematoxylin-eosin (HE), observing under optical microscope, and taking picture;

s3: and (3) observing by a transmission electron microscope: collecting cultured mDC of 8d, centrifuging at 1000rmp for 5min to obtain cell precipitate, performing double fixation with 2.5% glutaraldehyde and osmic acid, gradient acetone dehydration, epoxy resin soaking and embedding, conventionally making ultrathin section, staining with lead and uranium, and observing under a transmission electron microscope and taking a picture;

the phenotype identification of the DC, the expression steps of detecting DC surface molecules HLA-DR, CD83 and CD86 by flow cytometry are as follows:

s1: collecting im DC cultured for 4d and m DC cultured for 8d, centrifuging at 1000rpm for 5min, counting, and adjusting cell concentration to 1 × 106/tube;

s2: PBS washing 2 times, abandoning the supernatant, 100u l PBS re-suspension cell;

s3: adding flow antibody (2 μ g/106 cells), and incubating at room temperature for 30min in the dark;

s4: supplementing PBS to 500 μ l, and mixing well;

s5: and (4) detecting by using a flow cytometer.

The functional identification steps of the DC are as follows:

s1: preparation of stimulated cells: collecting im DC cultured for 4d and m DC cultured for 8d, respectively, and adjusting cell concentration to 1 × 105/m;

s2: preparation of reaction cells: collecting lymphocytes separately cultured from umbilical cord blood of different batches of the stimulated cells im DC and mdC, and adjusting the cell concentration to be 5 multiplied by 106/ml;

s3: the test is carried out on a 96-well plate, and the test is divided into 6 groups, each group is provided with 3 multiple wells, and the specific groups are as follows: blank control group: cell culture medium 100. mu.l im DCs; stimulation of cell groups: im DC 50. mu.l + cell culture broth 50. mu.l m DC; stimulation of cell groups: m DC 50 μ l + cell culture fluid 50 μ l; reaction cell group: lymphocytes 50. mu.l + cell culture broth 50. mu.l im DCs; mixed reaction cell group: im DCs 50. mu.l + lymphocytes 50. mu.l m DCs; mixed reaction cell group: mDC 50. mu.l + lymphocytes 50. mu.l; (3) culturing in a 5% CO2 incubator at 37 deg.C for 72h, and observing cell growth with an inverted microscope;

s4: adding 15 mul of MTT (methyl thiazolyl tetrazolium) with the concentration of 5mg/ml into each hole, and continuously culturing for 4 hours;

s5: adding equivalent volume of acidified isopropanol into each hole, and horizontally oscillating for 15min to fully dissolve crystals;

s6: measuring the OD value of each hole at 570nm on a microplate reader, and calculating the average value of 3 multiple holes in each group;

s7: calculating the proliferation level of lymphocytes: the Stimulation Index (SI) reflects the level of lymphocyte proliferation, and is (OD value of mixed response cell group-OD value of Stimulation cell group)/OD value of response cell group, and the Stimulation index of im DC and mdc on lymphocytes is compared.

The tumor antigen polypeptide loads dendritic cells and comprises the following steps:

s1: collecting cultured im DC, centrifuging at 1000rpm for 5min, counting, adjusting cell concentration to 1 × 106/ml, and replanting back to 6-well plate;

s2: the experiment is divided into 4 groups, including a control group and A, B, C reaction groups;

adding polypeptides with final concentrations of 25. mu.g/ml, 50. mu.g/ml and 100. mu.g/ml into the three reaction groups at the 5 th day of DC culture, continuously culturing for 48h, adding no polypeptide into the control group, adding 20% fetal calf serum RPMI-1640 culture solution containing 200u/ml rh TNF-alpha into the 4 th day of DC culture, and continuously culturing for 24 h;

control group: no polypeptide is added; reaction group: group A: 25 μ g/ml polypeptide; group B: 50 μ g/ml polypeptide; group C: 100 μ g/ml polypeptide;

the culture steps of the effector cells CTL are as follows:

s1: culturing the control group and the reaction group DC until the 8 th day is mature, collecting cells, centrifuging at 1000rpm for 5min, counting, and adjusting the cell concentration to 2 × 105/ml;

s2: collecting the lymphocytes of the same batch of isolated culture, centrifuging at 1000rpm for 5min, counting, and adjusting the cell concentration to 1 × 107/ml;

s3: mixing lymphocyte and DC at a ratio of 50:1, adding 20% fetal bovine serum RPMI-1640 culture solution containing rh IL-21000 u/ml and 1% PHA into a disposable plastic culture bottle, and culturing at 37 deg.C in a 5% CO2 incubator;

s4: culturing for 72h, centrifuging at 1000rpm for 5min, collecting effector cells CTL, counting, and adjusting cell concentration to 1 × 108/ml.

The killing capacity of CTL induced by the loaded tumor antigen polypeptide DC to tumor cells is detected by an LDH (layered double hydroxide) release method, the killing capacity of CTL induced by the loaded tumor antigen polypeptide DC to tumor cells is divided into the following groups:

s1: experiments are carried out on 96-well culture plates in batches according to the sequence of the polypeptide and the types of tumor cells, CTL is effector cells, and the tumor cells are target cells;

s2: each batch of experiments are divided into 5 groups, namely a reaction group, a target cell natural release group, a target cell maximum release group, an effector cell natural release group and a blank control group, wherein each group is provided with 3 multiple holes;

s3: the reaction components are divided into A, B, C, D four groups, which are respectively: target cells (induced by 5-Aza-Cd R) + effector cells (induced by polypeptide-loaded DC), target cells + effector cells (induced by polypeptide-unloaded DC), and target cells + effector cells (not induced by DC);

the experimental steps of the killing capacity of CTL induced by the tumor antigen loaded polypeptide DC to tumor cells are as follows:

s1: adding 50 mul of effector cells and target cells into each group of the reaction group according to the effective target ratio of 50: 1;

s2: adding 100 mu l of target cells into the target cell natural release group;

s3: target cell maximum release group added target cells 100. mu.l (without lysis buffer);

s4: the effector cell natural release group is added with 100 mu l of effector cells;

s5: only 100 mul of cell culture solution is added into the blank control group;

s6: continuously culturing for 6h in a 5% CO2 incubator at 37 ℃;

s7: adding 5 μ l of lysis solution into each well of the maximum release group, and continuously incubating for 15 min;

s8: centrifuging at low speed of 250g/min for 10min, carefully sucking 100 μ l of supernatant of each group, transferring into another 96-well plate, adding 100 μ l of freshly prepared LDH reaction solution into each well, and reacting for 15min at room temperature in a dark place;

s9: adding 50 mul of stop solution into each hole, and slightly shaking for 10s to stop the reaction;

s10: on an enzyme labeling detector, after zero setting is carried out by using a blank control hole, the OD value of each hole at 490nm is measured, and the average value of 3 multiple holes is taken for each group to calculate the result;

s11: calculating the killing rate of CTL to tumor cells: the killing rate is (E-S1-S2)/(M-S1) × 100%, where E is the experimental well OD average, S1 is the target cell natural release pore OD average, S2 is the effector cell natural release pore OD average, and M is the maximum release pore OD average, and the killing rate reflects the killing ability of CTL to tumor cells;

s12: the above experiment was repeated and all data were statistically analyzed.

The h-CD40L expression system:

s1: separating T lymphocytes from blood by using a cell separation and culture technology, and extracting total RNA of the T cells after activation;

s2: reversing and amplifying the CD40L gene segment by an RT-PCR method;

s3: connecting the purified CD40L gene segment with a pMD-18-T vector to form a CD40L recombinant cloning vector, transforming the recombinant cloning vector into DH5 alpha allelopathy, screening ampicillin, blue and white spots, carrying out PCR and DNA sequencing on bacterial liquid plasmids, and identifying the recombinant cloning vector;

s4: the recombinant cloning vector pMD-18-T-CD40L and the eukaryotic expression vector pcDNA3.1(+) plasmid are respectively extracted from the transformed bacteria by using a plasmid extraction kit, and the recombinant eukaryotic expression vector pcDNA3.1-CD40L is obtained by enzyme digestion, purification and connection and is transformed into TOP10 competent bacteria. Then identifying the recombinant eukaryotic expression vector pcDNA3.1-CD40L by ampicillin screening, PCR of bacterial liquid plasmid, double enzyme digestion of Nhe I and Knp I and DNA sequencing;

the DC cell transfection of the CD40L expression vector comprises the steps of culturing a DC cell, and transfecting the cell pcDNA3.1-CD40L by a liposome method; and (3) detecting the CD40LmRNA transcription of the DC transfected cells and the CD40L cell membrane expression rate after the PE marker anti-CD 40L staining by using RT-PCR and flow cytometry.

The secretion of the cell factors is that a cancer cell line in a logarithmic growth phase is paved on a 12-hole plate by 105 cells/hole, sensitized and modified DC-CTL cells are added in the next day, the cells are cultured for 4 hours together at the concentration of an effective target ratio of 5:1, supernate is collected, and the secretion levels of the cells IFN-gamma and TNF-alpha are detected by ELISA Kit Human IFN-gamma and ELISA Kit Human TNF-alpha; the CCK-8 method is used for detecting the killing effect of the sensitized and modified DC-CTL cells on the tumor cells:

s1: taking tumor cells in logarithmic growth phase, carrying out trypsinization, counting the cells, adjusting the density to 1X105/ml by RPMI 1640, and inoculating 10000 cells/hole in a 96-hole plate, wherein each hole is 100 ul;

s2: after 24 hours of incubation, 25ul of each chemotherapeutic was added to bring the final concentration to the set molarity. The treatment is carried out for 48 hours, each treatment is provided with three multiple wells, and blank wells are replaced by 125ul of culture solution; the experiment was repeated 3 times;

s3: adding CCK-810 ul into each well, and continuously incubating for 2-4 h;

s4: adding 10ul of 1N HCl into each hole, and stopping the reaction;

s5: detecting a light absorption value at 450nm by using an enzyme-labeling instrument;

s6: and (4) performing statistical analysis, and calculating the mean soil standard deviation for 3 times.

The invention has the beneficial effects that:

the HLA-A2 restricted epitope possibly existing in tumor specific antigen is predicted by combining a hyper-sequence method, a polynomial scheme and a quantitative motif method for predicting the epitope, and the epitope is identified by T cells which cover tumors most widely according to the fact that an epitope peptide library of Chinese immune characteristics (HLA types) contains mutation sites and is optimized.

Secondly, the tumor specific antigen polypeptide is easy to prepare and purify, chemically synthesized and not limited by material sources; the chemical property and the thermodynamic property are stable, so that the transportation and the storage are convenient; no toxic or infectious factor pollution, no potential carcinogenicity, small side effect and easy control.

Thirdly, loading DC cells by adopting a combination of various antigen peptides: the tumor basal antigen and the specific antigen are loaded with DC cells together.

And fourthly, increasing antigen intake by applying the cell-penetrating peptide, and obviously improving the DC cell loading efficiency.

Fifthly, by utilizing a gene engineering method, cloning the NKG2D full-length gene, constructing and identifying an expression vector to obtain a recombinant expression vector of NKG2D, and activating and modifying the NK cell through NKG2D, so that the immunosuppression effect caused by the tumor cell and a microenvironment thereof, including inhibitory cells, can be effectively avoided, the tumor killing effect of the NK cell is improved, the survival time of the NK cell can be prolonged, the NK cell can survive in vivo for a long time, and the capability of thoroughly eliminating the tumor cell is realized.

And sixthly, an effective DC (dendritic cell) maturation promoting scheme ensures the antigen presenting function of the DC cells and efficiently induces antigen peptide specific CTL.

And seventhly, on the basis of a classical DC maturation promoting scheme, the DC maturation is promoted by using CD40L transfected DC and the like, the antigen presentation function of DC cells is ensured, and antigen-specific CTL is induced efficiently.

Drawings

FIG. 1 is a technical scheme diagram of a DC vaccine and a DC-CTL method for transfection of CD40L by the mediation of tumor antigen polypeptide of cell-penetrating peptide.

Detailed Description

The DC-CTL method for transfecting CD40L by mediating tumor antigen polypeptide sensitization through the cell-penetrating peptide is characterized in that: the method comprises the following steps:

S3: constructing an h-CD40L expression system;

s4: DC cell transfection of CD40L expression vector;

Adopting an Si-RNA interference technology and a gene transfection technology to adjust the antigen expression condition of tumor cells, adopting the SiRNA interference technology to reduce the surface antigen expression quantity of the tumor cells, simultaneously adopting the gene transfection technology to up-regulate the same surface antigen expression quantity of another tumor cell, detecting the killing effect of the sensitized and modified DC-CTL cell on the adjusted tumor cells by using a CCK-8 method, calculating the statistical significance of the change difference of the killing rate of two tumor cells expressing different surface antigens, and guessing that the killing effect of the sensitized and modified DC-CTL cell on the tumor cells expressing different antigen expression quantities can be achieved by adjusting the target spot of the antigen;

killing of sensitized modified DC-CTL cells on in vivo tumor cells 30 female nude mice of 6 weeks old were taken, and 1X 107 tumor cell lines were subcutaneously injected into the right underarm of each nude mouse, and randomly divided into 3 groups: a normal saline control group, a DC-CTL cell group and a sensitized and modified DC-CTL cell group. Sensitized and modified DC-CTL cells and DC-CTL cells are used as effector cells, and the effector cells are injected into tail vein for 1 × 107 cells/time, the total injection volume is 200ul, and the injection is performed 2 times per week. The control group was replaced with the same volume of saline. Tumor volume was calculated by measuring the long and short diameters of the tumor with a vernier caliper before each treatment (length a mm, width b mm, tumor volume calculation formula: 1/2ab 2). Counting the death number every day, and drawing a survival rate curve of the transplanted tumor model mouse;

the chip screens the change of immune related genes and tumor markers in tumor tissues, and after one month of treatment, a transplanted tumor model mouse is dislocated and killed in cervical vertebra and tumor tissues are collected. Grinding tumor tissue to extract RNA, carrying out reverse transcription to obtain cDNA, and sending the cDNA as a template to a professional biological company for detecting expression changes of immune related genes and tumor markers by a TaqMan chip method;

immunohistochemical detection of specific CTL cell content in tumor tissue transplanted tumor model mice were sacrificed by dislocation of cervical vertebrae and tumor tissue was collected one month after treatment. The tumor tissue was sectioned into tissue pieces with a thickness of 4 μm and then immunohistochemically stained. Formalin-fixed paraffin-embedded tumor tissue was sequentially soaked in alcohol and xylene at different concentration gradients, placed in citrate buffer PH10 and heated for 25 minutes for antigen retrieval, 3% hydrogen peroxide solution was reacted at room temperature for 5 minutes, 1: 50 diluted monoclonal CD3 antibody was reacted at room temperature for 60 minutes, the secondary antibody was reacted at room temperature for 30 minutes, reacted with DAB solution at room temperature for 5 minutes, stained with hematoxylin solution for 5 minutes, and the sections were placed on an Olympus microscope after being coverslipped for observation and evaluation.

s1: the first day: cell preparation

s6: on day seven, DCs were collected.

s2: the first day: cell preparation

s4: supplementing PBS to 500 μ l, and mixing well;

s5: and (4) detecting by using a flow cytometer.

The functional identification steps of the DC are as follows:

Mixed Lymphocytes Reaction (MLR): since DCs can effectively induce the proliferation of allogeneic lymphocytes in vitro, the functions of imdcs and mdcs can be determined by performing mixed culture using imdcs and mdcs as stimulating cells and allogeneic lymphocytes as reacting cells, respectively, and detecting the proliferation ability of lymphocytes by the MTT method.

the culture steps of the effector cells CTL are as follows:

s2: adding 100 mu l of target cells into the target cell natural release group;

s6: continuously culturing for 6h in a 5% CO2 incubator at 37 ℃;

The h-CD40L expression system:

s2: reversing and amplifying the CD40L gene segment by an RT-PCR method;

s3: adding CCK-810 ul into each well, and continuously incubating for 2-4 h;

s4: adding 10ul of 1N HCl into each hole, and stopping the reaction;

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. The DC-CTL method for transfecting CD40L by mediating tumor antigen polypeptide sensitization through the cell-penetrating peptide is characterized in that: the method comprises the following steps:

s1: culturing DC and CTL in vitro; observing the surface morphology and the internal structure change of the DC by using an inverted microscope, a scanning electron microscope and a transmission electron microscope; detecting the expression of DC surface molecules CD1a, CD83, CD80, CD86 and HLA-DR by flow cytometry; detecting the proliferation promoting effect of DC cells on T cells in a mixed lymphocyte test MLR by using a thiazole blue MTT method; detecting the specific killing activity of CTL to the tumor cells by using LDH method lactate dehydrogenase killing test; the DC and CTL in-vitro culture comprises a standard process of in-vitro induction DC culture, a standard operation procedure of in-vitro CTL efficient amplification culture technology, identification of dendritic cells, functional identification of DC, culture of tumor antigen polypeptide loaded dendritic cells, effector cell CTL and killing capacity of CTL loaded with tumor antigen polypeptide DC to tumor cells;

s2: the HLA-A2 restricted tumor antigen peptide is predicted by jointly applying a hyper-motif method, a polynomial scheme and a quantitative motif method for predicting the epitope, and the feasibility of the epitope prediction is verified; separating, culturing and identifying peripheral blood DC, inducing CTL activation and amplification after the DC is loaded with tumor antigen peptide, further researching killing capacity to various tumor cells, and screening out the tumor antigen peptide with strong killing effect to various tumors;

s3: constructing an h-CD40L expression system;

s4: DC cell transfection of CD40L expression vector;

2. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the standard process of in vitro induction DC culture comprises the following steps:

s1: the first day: cell preparation

A: 75ml of anticoagulated peripheral blood is extracted, 800g is extracted, and centrifugation is carried out for 15min at room temperature with within the range of about one hundred twenty minutes;

c: taking the centrifuged lower cell component, adding D-PBS to 50ml, mixing uniformly, adding into 3 50ml centrifuge tubes filled with 20ml medical grade lymphocyte separation liquid, centrifuging at room temperature for 15min at 800g, and centrifuging with out breaking;

s6: on day seven, DCs were collected.

3. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the standard operation procedures of the in vitro CTL high-efficiency amplification culture technology are as follows:

s2: the first day: cell preparation

A: extracting 50ml of anticoagulated peripheral blood, 800g, and centrifuging at room temperature for 15min with without breaking;

c: taking the centrifuged lower cell component, adding D-PBS to 50ml, mixing uniformly, adding into 250ml centrifuge tubes filled with 20ml medical grade lymphocyte separation liquid, centrifuging at room temperature for 15min at 800g, and centrifuging with out breaking;

s8: the thirteenth day: 1400ml of CTL cell suspension is collected, supernatant is discarded after centrifugation at 1500rpm multiplied by 8min, 200ml of physiological saline of each tube of a 250ml centrifuge tube is washed at 1500rpm multiplied by 8min1 times, 50ml of physiological saline of a 50ml centrifuge tube is washed at 1200rpm multiplied by 8min1 times, cells are resuspended according to 100ml of physiological saline and 5ml of 20% serum albumin, the cells are packaged and then sent to a ward, and meanwhile, samples are reserved and sealed for later detection; supplementing 200 ml/bag of CTL culture solution, and sending to a third party for detection to perform self-detection on bacteria, fungi, mycoplasma and endotoxin;

s9: on the fifteenth day: collecting 1600ml of CTL cell suspension, centrifuging at 1500rpm multiplied by 8min, removing supernatant, washing 200ml of physiological saline per tube by a 250ml centrifuge tube set 2 tubes at 1500rpm multiplied by 8min1 times, washing 50ml of physiological saline per tube by a 50ml centrifuge tube at 1200rpm multiplied by 8min1 times, re-suspending cells according to 100ml of physiological saline and 5ml of 20% serum albumin, packaging, delivering to a ward, and simultaneously preserving samples for later detection.

4. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the identification of the dendritic cells comprises morphological observation of the DC and phenotypic identification of the DC, and the DC surface molecules HLA-DR, CD83 and CD86 are detected by flow cytometry, wherein the morphological observation step of the DC comprises the following steps:

s3: adding flow antibody 2 μ g/106 cells, incubating at room temperature for 30min in dark;

s4: supplementing PBS to 500 μ l, and mixing well;

s5: and (4) detecting by using a flow cytometer.

5. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the functional identification steps of the DC are as follows:

s3: the test is carried out on a 96-well plate, and the test is divided into 6 groups, each group is provided with 3 multiple wells, and the specific groups are as follows: blank control group: cell culture medium 100. mu.l im DCs; stimulation of cell groups: im DC 50. mu.l + cell culture broth 50. mu.l m DC; stimulation of cell groups: m DC 50 μ l + cell culture fluid 50 μ l; reaction cell group: lymphocytes 50. mu.l + cell culture broth 50. mu.l im DCs; mixed reaction cell group: im DCs 50. mu.l + lymphocytes 50. mu.l m DCs; mixed reaction cell group: mDC 50. mu.l + lymphocytes 50. mu.l; 3, culturing for 72 hours in a 5% CO2 incubator at 37 ℃, and observing the growth condition of the cells by an inverted microscope;

6. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the tumor antigen polypeptide loads dendritic cells and comprises the following steps:

the culture steps of the effector cells CTL are as follows:

7. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the killing capacity of CTL induced by the loaded tumor antigen polypeptide DC to tumor cells is detected by an LDH (layered double hydroxide) release method, the killing capacity of CTL induced by the loaded tumor antigen polypeptide DC to tumor cells is divided into the following groups:

s3: the reaction components are divided into A, B, C, D four groups, which are respectively: the method comprises the following steps that after the induction of target cells by 5-Aza-Cd R, the + effect cells are subjected to DC induction group loaded with polypeptide, before the induction of the target cells by 5-Aza-Cd R, the + effect cells are subjected to DC induction group loaded with polypeptide, the + effect cells of the target cells are subjected to DC induction group not loaded with polypeptide, and the + effect cells of the target cells are not subjected to DC induction group;

s2: adding 100 mu l of target cells into the target cell natural release group;

s3: adding 100 mu l of target cells into the target cell maximum release group without adding lysate;

s6: continuously culturing for 6h in a 5% CO2 incubator at 37 ℃;

8. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the h-CD40L expression system:

s2: reversing and amplifying the CD40L gene segment by an RT-PCR method;

s4: respectively extracting recombinant cloning vector pMD-18-T-CD40L and eukaryotic expression vector pcDNA3.1(+) plasmid from the transformed bacteria by using a plasmid extraction kit, carrying out enzyme digestion, purification and connection to obtain a recombinant eukaryotic expression vector pcDNA3.1-CD40L, and transforming the recombinant eukaryotic expression vector pcDNA3.1-CD40L into TOP10 competent bacteria; then identifying the recombinant eukaryotic expression vector pcDNA3.1-CD40L by ampicillin screening, PCR of bacterial liquid plasmid, double enzyme digestion of Nhe I and Knp I and DNA sequencing;

9. The method for DC-CTL transfection of CD40L through mediation of tumor antigen polypeptide by the cell-penetrating peptide according to claim 1, wherein: the secretion of the cell factors is that a cancer cell line in a logarithmic growth phase is paved on a 12-hole plate by 105 cells/hole, sensitized and modified DC-CTL cells are added in the next day, the cells are cultured for 4 hours together at the concentration of an effective target ratio of 5:1, supernate is collected, and the secretion levels of the cells IFN-gamma and TNF-alpha are detected by ELISA Kit Human IFN-gamma and ELISA Kit Human TNF-alpha;

the CCK-8 method is used for detecting the killing effect of the sensitized and modified DC-CTL cells on the tumor cells:

s2: after 24 hours of culture, 25ul of chemotherapeutic drugs are respectively added to make the final concentration reach the set molarity; the treatment is carried out for 48 hours, each treatment is provided with three multiple wells, and blank wells are replaced by 125ul of culture solution; the experiment was repeated 3 times;

s3: adding CCK-810 ul into each well, and continuously incubating for 2-4 h;

s4: adding 10ul of 1N HCl into each hole, and stopping the reaction;