CN103372205A - Preparation method of tumor dendritic cell vaccine sensitized by glycosylated MUC1 (mucoprotein 1) antigen - Google Patents
Preparation method of tumor dendritic cell vaccine sensitized by glycosylated MUC1 (mucoprotein 1) antigen Download PDFInfo
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Abstract
The invention provides a preparation method of a tumor dendritic cell vaccine sensitized by glycosylated antigen MUC1 (mucoprotein 1). Glycosylation modified tumor antigen peptide is used for sensitizing externally cultured DC (Dendritic Cell) and then the activated DC cells are transfused to a tumor patient. The invention further provides an application of the tumor DC cell vaccine sensitized by the glycosylated MUC1 antigen in treatment of breast cancer and pancreatic cancer. The preparation method provided by the invention has the following advantages (1) the DC cell vaccine amplification efficiency is high; (2) the DC cells have high antigen activity; (3) the DC cells have high maturity degree, and side effects such as immune tolerance are prevented; and (4) small side effects are caused.
Description
Technical field
The present invention relates to a kind of preparation method of antineoplastic immune cell vaccine, particularly a kind of preparation method of antineoplastic immune dendritic cell vaccine.
Background technology
Dendritic cell (Dendritic cell, DC) be the most powerful antigen presenting cell of function in the human body, have picked-up, process antigen, with the form submission antigen of MHCI, II class peptide conjugate and promote the ability of T, bone-marrow-derived lymphocyte propagation, in the natural immunity and acquired immunity, all bringing into play extremely important effect.
Zoopery and the clinical trial in early stage show, tumor antigen with appropriate format, though be DC vaccine that protein, polypeptide or nucleic acid load can both activate can identify, the T cells with antigenic specificity of killing tumor cell, and can produce the immunological memory effect.At present, the DC vaccine mainly contains three major types: the DC that the cellularity tumor antigen is modified; The DC that tumor antigen peptide is modified; The DC of tumor antigen and cytokine gene transfection.
Svane IM etc. has carried out one to be impacted DC with wild type P53 polypeptide thing and prepares vaccine, be used for the treatment of the advanced breast cancer patient's of the HLA-A2 positive I phase clinical research, 6 patients receive treatment, the result confirms all easily tolerances of patient, generation has no side effect, 1 metastatic lymph node occurs and dwindles, and 1 effectively, 2 SD.ELISpot analyzes demonstration: can induce special T lymphocyte reaction (the Svane I.M. for wild type P53 after the immunity, Pedersen A.E., Claesson M.H.Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer:report from a phase I study.Cancer Immunol Immunother.53 (7): 633-41,2004).
Immature DC has extremely strong antigen phagocytic activity, carries out endocytosis, process antigen in endosome in antigen uptaking (comprising external processing), and offer antigen to MHC I and MHC II, thereby start Peptide-specific CTL reaction (Figdor, C.G., van Kooyk, Y.﹠amp; Adema, G.J.C-type lectin receptors on dendritic cells and Langerhans cells.Nature Rev.Immunol.2,77-84,2002).Film at endosome has the receptor of identifying antigen, comprising CLR family (C type agglutinin receptor).There are poly-mannose receptor, CD205 and DC-SIGN in CLR family.Poly-mannose receptor all has expression in various cell types, comprise immature DC cell (iDC) and macrophage.It can be pounced on and catch antigen, is after the processed to pass MHC I and MHC II quasi-molecule (Keler, T., Ramakrishna, V.﹠amp; Fanger, M.W.Mannose receptor-targeted vaccines.Expert Opin.Biol.Ther.4,1953-1962,2004).In an I clinical trial phase, suffers from breast carcinoma, colon cancer, the patient of gastric cancer and rectal cancer uses the MUC1 antigenic peptides vaccine therapy of poly-mannose covalent modification, the result shows that the humoral immune reaction of antigenic specificity appears in half patient, the CTL cell effect appears in the fraction patient, but there is not obvious clinical response (Karanikas, V.et al.Antibody and T cell responses of patients with adenocarcinoma immunized with mannan-MUC1 fusion protein.J.Clin.Invest.100,2783-2792,1997).The III phase clinical research of a tentative MUC1 antigenic peptides treatment breast carcinoma of early stage for the poly-mannose covalent modification of oxidisability shows, the patient of breast carcinoma II phase accepts this test, these patients for the treatment of end post-evaluation survival condition after 5 years, all patients are not all recurred (the matched group relapse rate is 27%) (Apostolopoulos, V.et al.Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1.Breast Cancer Res.8, R27,2006).This test shows that MUC1 antigenic peptides vaccine has very good prospect to oncotherapy and prevention.
More than test is that glycosylation modified MUC1 is made the tumor antigen peptide vaccine, is injected directly in the tumor patient body, by the activation of sensitization in vivo DC cell, is used for clinical treatment breast carcinoma.But the efficient that this method has DC cellular uptake antigen, offer antigen is not high, easily causes the side effect such as immunologic tolerance.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, a kind of tumor dendritic cell vaccine preparation method of glycosylation MUC1 antigen sensibilization is provided, the DC cell that glycosylation modified tumor antigen peptide is used for sensitization activation In vitro culture, DC cell infusion that again will be activated is to tumor patient, to reach the purpose for the treatment of tumor.Have the following advantages: (1) DC cell vaccine amplification efficiency is high; (2) angtigen presentation of DC cell is active high; (3) Maturity of DC cell is high, avoids causing the side effect such as immunologic tolerance; (4) side effect is less.
The present invention realizes above-mentioned purpose by the following technical solutions:
A kind of tumor DC cell vaccine preparation method of glycosylation MUC1 antigen sensibilization may further comprise the steps:
The VNTR repeated fragment of MUC1 gene is cloned into bacterial expression vector pGEX-3X and expression, and separation and purification obtains to comprise the MUC1 FP fusion rotein of GST and MUC1 VNTR, MUC1 FP is connected with the oxidation of poly-manna sugar chain makes the MUC1-M-FP fusion rotein;
Amplification cultivation DC cell;
Above-mentioned MUC1-M-FP fusion rotein culture fluid is joined DC cell after the cultivation;
The Maturation induction of DC cell;
Obtain the DC cell vaccine of sensitization.
As the further improvement of the technical program, adopt the serum-free medium that contains GM-CSF and IL-4 to carry out amplification cultivation DC cell.
As the further improvement of the technical program, adopt the maturation culture solution that contains CD40 antibody, TNF-α, IL-1 α that the DC cell is carried out Maturation induction.
The present invention also provides a kind of application of tumor DC cell vaccine in breast carcinoma and treatment of pancreatic cancer of glycosylation MUC1 antigen sensibilization.
Compared with prior art, DC cell than not glycosylation modified MUC1 sensitization, the ability of the DC cell educated T cell of glycosylation modified MUC1 sensitization activation is stronger, and the T emiocytosis Cytokines of its domestication is the former about 10 times, and multiplication capacity is the former about 3 times.External contrast experiment by early stage confirms that the DC cell that the method obtains can activate tumor-killing T lymphocyte (CTL cell) effectively.
And than glycosylation modified MUC1 being made the tumor antigen peptide vaccine, be injected directly in the tumor patient body, by the activation of sensitization in vivo DC cell, the present invention directly is used for glycosylation modified tumor antigen peptide the DC cell of sensitization activation In vitro culture, DC cell infusion that again will be activated is to tumor patient, DC cellular uptake and the efficient of offering antigen have been improved, to reach the purpose for the treatment of tumor.
Description of drawings
Fig. 1 is the preparation flow figure of MUC1-M-FP fusion rotein of the present invention;
Fig. 2 is the preparation flow figure of the DC cell vaccine of MUC1-M-FP sensitization of the present invention.
The specific embodiment
Further describe concrete technical scheme of the present invention below in conjunction with drawings and Examples, so that those skilled in the art understands the present invention, and do not consist of its Copyright law.
1.MUC1M-FP preparation (as shown in Figure 1):
1) preparation of MUC1FP: the fragment of 309 bases (pDF9.3, the summary of encoding surpasses the fragment of 60 base motifs in the VNTR zone of 5 MUC1 cDNA) is cloned into the correction frame of bacterial expression vector pGEX-3X and expresses direction.MUC1-FP (comprising GST and MUC1VNTR) induces with IPTG.Centrifugal collecting cell cleans and with ultrasonication cell (in containing the buffer of TritonX-100), the supernatant that comprises soluble protein MUC1-FP is coated with magnetic bead mixing (disulfide bond is connected), then centrifugal collection with glutathion.With fusion rotein MUC1-FP under the buffer solution for cleaning that contains reductive glutathione, with the phosphate buffer dialysis, and SDS-PAGE detects.
2) MUC1 FP connects a manna sugar chain by oxidation at GST.The covalently bound upper manna sugar chain of FP has dual mode: (i) OX-M-FP: in the 0.1M phosphate buffer, with sodium periodate oxidation manna sugar chain.Then, adding ethylene glycol continues to hatch; Mixture is by Sephadex-G25 pillar (with PH6.0-9.0 bicarbonate buffer balance).Then the manna sugar chain vacuum cleaning of oxidation mixes with MUC1 FP, and incubated at room is spent the night, and need not purification can use.(ii) reproducibility (Red-M-FP): the OX-M-FP mixture was processed 3 hours with sodium borohydride, and need not purification can use.
2.MUC1 the DC vaccine (as described in Figure 2) of sensitization:
1) under the room temperature, uses blood cell separator to separate patient P BMC, from Medical device box, take out carefully the sack that patient's hemocyte is housed, after the surface sterilization, move into Biohazard Safety Equipment;
2) cell that gathers joins two pipes and contains on the heparin solution, mixing, and room temperature is centrifugal, sucks supernatant;
3) add the normal saline re-suspended cell, mixing, other gets a centrifuge tube, puts filter, filtration cell liquid;
4) Ficoll liquid gradient centrifugation separates mononuclearcell, get two centrifuge tubes, carefully cell suspension is added in above the lymphocyte separation medium in each centrifuge tube, room temperature is centrifugal, collect the middle mononuclear cell confluent monolayer cells of plasma layer and lymphocyte separation medium layer, whole sucking-off PBMC try one's best;
5) wash PBMC 2 times with normal saline, centrifugal under the room temperature, supernatant discarded is used the serum-free medium re-suspended cell;
6) cell counting is got 10 μ l and is mixed at 1: 10 with the Turk working solution behind the mixing, add the blue 10 times of dilution countings of 10 μ l Placenta Hominiss behind the lucifuge reaction 15min again.All the other cells are resuspended in the 54ml serum-free medium, get 6 six well culture plates, and every hole adds 1.5ml cell suspension, and 37 ℃, the 5%CO2 incubator leaves standstill cultivates 30min;
7) behind the 30min, adherent cell is used for the cultivation of DC in six orifice plates, and not adherent cell is lymphocyte, is used for follow-up detection;
8) cultivation of immature DC cell: the 0th day, the PBMC adhere-wall culture, not adherent cell suspension in sucking-off six orifice plates, and wash once with serum-free medium, adherent cell in six orifice plates, add DC complete culture solution (serum-free medium that contains rhGM-CSF, rhIL-4 and L-glutaminate, human albumin) in every hole, the DC complete culture solution was mended in every hole in the 2nd, 4 day, place 37 ℃, the incubator of 5%CO2 to cultivate six orifice plates, added the culture fluid that contains the MUC1-M-FP fused antigen in the 4th day.
9) the 6th day, the DC complete culture solution that contains rhTNF-and CD40 antibody was added in the every hole of six orifice plates, and culture plate places 37 ℃, cultivates in the 5%CO2 incubator;
10) maturation of DC cell: the 7th day, results DC.With cell scraper cell is swept, sucking-off cell suspension, every hole is washed 1 time with normal saline, collects in the lump, and room temperature is centrifugal, removes supernatant, uses normal saline washed cell 3 times, and cell counting is used for the 1st patients undergoing subcutaneous injecting of DC; Frozen 3 untransfecteds, six orifice plate cells, frozen 16 hole transfection DC use normal saline washed cell 2 times, frozen 5 respectively; Get 106 transfection DC and be used for the flow cytometry Phenotypic examination.
3.DC learning, the fluidic cell of cell detects
1) the DC cell of 106 cultivations of collection is put into graduated centrifuge tube, and 4 ℃, the centrifugal 5min of 1000r/min abandon supernatant.
2) adding 1ml PBS is resuspended, and counting is distributed into about 1 * 106 cell of every pipe in the EP pipe.4 ℃, the centrifugal 5min of 1000r/min.
3) abandon supernatant, wash 3 times with PBS again, use at last 200ml PBS resuspended.
4) then press the reagent description and add fluorescent-labeled antibody: PE-anti-human CD80 (1ul), FITC-anti-human CD86 (1ul), FITC-anti-human MHC II (1ul) and PE-anti-ratOX62 (10ul), 4 ℃ of lucifuges are hatched 30min.
5) Staining buffer washing is 3 times, and flow cytometer detects.
Detect lymphocytic emiocytosis cytokine situation 4.DC be total to culture method with lymphocyte
1) get the lymphocyte that above-mentioned separation obtains, spread 24 orifice plates and cultivate, every hole 0.5ml adds and contains serum RPMI-1640;
2) each hole adds the DC cell for preparing, and each 0.5ml is divided into the blank group, cultivate basis set, DC cell+MUC1 group and DC cell+MUC1-M-FP group, each is organized and establishes 6 again holes.Blow and beat gently mixing, put 37 ℃, the 5%CO2 cell culture incubator is cultivated;
3) respectively at behind the bed board 48,72h collects supernatant, relevant cell factor test to be done.
4) the ELISA method detects the generation of cytokine: be taken at the supernatant of respectively organizing co-culture media, detect T lymphocytic emiocytosis IFN-γ, the TNF-α ability respectively organized with the ELISA test kit.
5.MTT method detects the Proliferation of lymphocytes situation
1) gets 96 porocyte culture plates.Every hole adds each 100ul of lymphocyte that the above-mentioned separation of people obtains, and then adds and contains serum RPMI-1640,5%CO2,37 ℃ of cultivation 4h.
2) in cultured cell, add: contain serum RPMI-1640 200ul (matched group); The DC cell of cultivating+MUC1 200ul (DC+MUC1 group); DC cell+MUC1-M-FP 200ul (DC+MUC1-M-FP group); All establish 6 multiple holes.Establish simultaneously blank.
3) continue to cultivate 48h.
4) the every hole of 4h adds MTT (5mg/ml) 20ul before experiment.
5) suck gently supernatant, every hole adds 150ul DMSO, and 10min gently vibrates.
6) photometry absorption value on enzyme mark photometer.The mensuration wavelength is 490nm, and reference wavelength is 630nm, and the gained absorbance value represents the relative populations of respective aperture cell.
The present invention is by directly being used for glycosylation modified tumor antigen peptide the DC cell of sensitization activation In vitro culture, and DC cell infusion that again will be activated is to tumor patient.The result shows, DC cell than not glycosylation modified MUC1 sensitization, the ability of the DC cell educated T cell of glycosylation modified MUC1 sensitization activation is stronger, and the T emiocytosis Cytokines of its domestication is the former about 10 times, and multiplication capacity is the former about 3 times.External contrast experiment by early stage confirms that the DC cell that the method obtains can activate tumor-killing T lymphocyte (CTL cell) effectively.
Above-described embodiment only is explanation technological thought of the present invention and characteristics; its purpose is to make those skilled in the art can understand content of the present invention and implements according to this; can not limit according to this protection scope of the present invention; the equalization of namely being done with disclosed spirit changes or derives, and must be encompassed in protection scope of the present invention.
Claims (4)
1. the tumor DC cell vaccine preparation method of a glycosylation MUC1 antigen sensibilization may further comprise the steps:
The VNTR repeated fragment of MUC1 gene is cloned into bacterial expression vector pGEX-3X and expression, and separation and purification obtains to comprise the MUC1 FP fusion rotein of GST and MUC1 VNTR, MUC1 FP is connected with the oxidation of poly-manna sugar chain makes the MUC1-M-FP fusion rotein;
Amplification cultivation DC cell;
Above-mentioned MUC1-M-FP fusion rotein culture fluid is joined DC cell after the cultivation;
The Maturation induction of DC cell;
Obtain the DC cell vaccine of sensitization.
2. the tumor DC cell vaccine preparation method of a kind of glycosylation MUC1 antigen sensibilization according to claim 1 is characterized in that, adopts the serum-free medium that contains GM-CSF and IL-4 to carry out amplification cultivation DC cell.
3. the tumor DC cell vaccine preparation method of a kind of glycosylation MUC1 antigen sensibilization according to claim 1 is characterized in that, adopts the maturation culture solution that contains CD40 antibody, TNF-α, IL-1 α that the DC cell is carried out Maturation induction.
4. the application of tumor DC cell vaccine in breast carcinoma and treatment of pancreatic cancer of a kind of glycosylation MUC1 antigen sensibilization claimed in claim 1.
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| CN104800838A (en) * | 2015-04-14 | 2015-07-29 | 苏静 | MUC1-Fc polypeptide vaccine as well as preparation method and application thereof |
| CN105734014A (en) * | 2016-02-23 | 2016-07-06 | 南京融卓生物科技有限公司 | Dendritic cell loaded with broad spectrum antigens and preparation method |
| CN105734014B (en) * | 2016-02-23 | 2019-03-26 | 南京融卓生物科技有限公司 | A kind of Dendritic Cells and preparation method loading wide spectrum antigen |
| CN112458058A (en) * | 2020-11-24 | 2021-03-09 | 康九生物科技(长春)有限公司 | DC cell over-expressing TRAF6, DC cell vaccine, construction method and application |
| CN112458058B (en) * | 2020-11-24 | 2023-09-01 | 康九生物科技(长春)有限公司 | TRAF6 over-expression DC cell, DC cell vaccine, construction method and application |
| CN113249223A (en) * | 2021-05-13 | 2021-08-13 | 深圳罗兹曼国际转化医学研究院 | Application of leishmania transformed with expression plasmid in promotion of DC maturation, method for promoting DC maturation, and expression plasmid |
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