CN104926944A - Preparation method and application of multiple target complex antigen-loaded CD8<+> cytotoxic T lymphocyte - Google Patents
Preparation method and application of multiple target complex antigen-loaded CD8<+> cytotoxic T lymphocyte Download PDFInfo
- Publication number
- CN104926944A CN104926944A CN201510270656.XA CN201510270656A CN104926944A CN 104926944 A CN104926944 A CN 104926944A CN 201510270656 A CN201510270656 A CN 201510270656A CN 104926944 A CN104926944 A CN 104926944A
- Authority
- CN
- China
- Prior art keywords
- lymphocyte
- cell
- cytotoxic
- multitarget
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention provides a multiple target complex antigen, namely a combined cytotoxic T lymphocyte (CTL) antigen epitope peptide, a polyvalent vaccine with multiple target positions for a tumor cell surface, a multiple target complex antigen-loaded CD8<+> CTL, a preparation method of the CTL, and an application of the lymphocyte to preparation of a drug for treating a cancer. The invention also provides an epitope-induced special CTL effect cell.
Description
Technical field: the present invention relates to biology and medical field, relates more specifically to multitarget composite antigen load C D8
+the preparation method of cytotoxic T lymphocyte and application thereof.
Background technology:
Malignant tumour is one of major disease threatening human health, in recent years, the vital role of immunity system in tumor prevention and treatment is extensively admitted, based on the immunotherapy of antineoplastic specificity immunologic reconstitution be generally acknowledge in the world at present be hopeful most to eliminate the important means that interior tumor cell effect a radical cure tumour completely after operation, radiation and chemotherapy, the Main way that future tumors is treated will be become.Immunotherapy mainly through externally to supplement, induction with activate enorganic biological response regulation system and activate transfer there is cytotoxic activity biological activity cell and cytokine, improve the function of patient's self immune system, strengthen the differentiation capability of specific tumor killing cell, killing tumor cell on a cellular level, the transfer of effective inhibition tumor cell, diffusion and recurrence, and side reaction is slight, there is good clinical therapeutic efficacy, overcome the drawbacks such as " not thoroughly, the easily transfer, side effect large " of tumour traditional treatment mode.Along with from going deep into tumour immunity research on cell and molecular level, tumor-specific cytotoxicity T lymphocyte (the cytotoxic T lymphocyte of dendritic cell (DCs) sensitization of load tumour antigen, CTL) as the integral part of immunotherapy, can specific recognition tumour, the amplification in cloning also forms immunological memory in vivo, mediates lasting antineoplastic specificity immunological effect.And the costimulatory signal that the activation of CTL cell needs tumour antigen sensitization and antigen presenting cell (APC) to provide and the acting in conjunction of the 3rd class signaling molecule that provided by cytokine.Tumour is correlated with and the specific active immunotherapy that exists for of specific antigens has established theoretical and basic substance.Only have immunogenicity is strong, specificity is high tumour antigen effectively could activate the T lymphocyte of tumour-specific.In recent years, the oncopeptide therapeutic vaccine that the tumor associated antigen (TAA) of expressing for tumour or tumour specific antigen (TSA) grow up, with its high specificity, safety, cheapness, be easy to the features such as preservation and application, be expected to the shortcoming overcoming above-mentioned therapy, reach the object of effectively treatment tumour.
Tumour antigen must be degraded to small peptide and form peptide-MHC-TCR mixture and could be identified by T cell in antigen presenting cell (antigen presenting cell, APC), excites corresponding cytotoxic T lymphocyte to react.And the object of polypeptide vaccine is just major histocompatibility complex (the major histocompatibility complex tumor-antigen peptide of high dosage being flowed to APC surface, MHC) molecule is therefore suitable and the preparation of effective tumor-antigen peptide to tumor vaccine is extremely important.And tumor-antigen peptide is by MHC restriction, only has MHC I quasi-molecule identical patient could use same peptide, and due to the unhomogeneity of tumour, some tumor antigen peptide may inducing immune tolerance instead of activate immunity be replied.In addition, immunogenicity is weak is another large weakness of polypeptide vaccine, by modifying (replacement of single amino acids, the PMRI modification of polypeptide and lipopeptid etc.) to epitope polypeptide or adopting polyvalent vaccine then effectively can improve its immunogenicity, induce stronger CTL activity.
The present invention is the follow-up study of the Chinese invention patent being 200810037440.9 based on application number.The document that the present invention quotes from is as follows, and following document is attached in present patent application by reference.Patent documentation 1: publication number: CN 101302537A; Patent documentation 2: publication number: CN 101854945A; Patent documentation 3: publication number: CN 102625832A.
Summary of the invention:
The invention provides a kind of multitarget composite antigen (Multiple Target complex antigen, MTCA), namely the mode combined by multiple epi-position, this array mode is according to genetic structure and function difference, at least four kinds of polypeptides in combination matched with human leucocyte antigen are selected from a series of candidate polypeptide, be made into tumor vaccine, thus excite patient's body to the specific immune response of tumour, extend its survival time, MTCA is other tumor vaccines comparatively, there is the unique advantage walking around immunological diversity and tumour unhomogeneity, effectively can induce stronger CTL killing activity.
Therefore, an object of the present invention is to provide a kind of multitarget composite antigen, the CTL epitope peptide namely combined.Another object of the present invention is to provide the polyvalent vaccine to the many target position of tumor cell surface.Another object of the present invention is to provide a kind of multitarget composite antigen load C D8
+cytotoxic T lymphocyte and preparation method thereof.Another object of the present invention is to provide the specific CTL effector cell of a kind of epi-position induction.
Multiple epitope peptide provided by the invention has the ability of the HLA Restricted CTL of inducing multiple HLA type, thus reaches more effective result for the treatment of.The exemplary tumour antigen with induction HLA-A24, HLA-A2 or HLA-A3 supertype at least comprises following: the ability such as with the HLA Restricted CTL of induction HLA-1101, HLA-A2402, HLA-A2601, HLA-A3101, HLA-A3303.Wherein multiple epitope peptide is selected from PRAME, MAGE A3, HER-2/neu, NY-ESO-1, Ras mutant, p53 mutant, PSA, hTERT, AFP, SART, CEA, CA-125, EGFR-800, C35-44, β-tubuline-309, spermatine 17, PSMA, Survivin.
Preferably, can induce antineoplastic immune for multiple different epitope by such as covering described exemplary oncologic associated antigen polypeptide PRAME, C35-44, Survivin, NY-ESO-1 (can use other tumor antigen peptides and combination) etc., this polyvalent vaccine to the many target position of tumor cell surface is more effective than single antigenic stimulation.Preferably, by such as covering described exemplary oncologic associated antigen polypeptide HER-2/neu, SART and hTERT, CEA can induce antineoplastic immune for multiple different epitope, and this polyvalent vaccine to the many target position of tumor cell surface is more effective than single antigenic stimulation.
The present invention realizes foregoing invention object by following technique means: be no less than four polypeptide below selection and combine: PRAME, MAGE A3, HER-2/neu, NY-ESO-1, Ras mutant, p53 mutant, PSA, hTERT, AFP, SART, CEA, CA-125, EGFR-800, C35-44, β-tubuline-309, spermatine 17, PSMA, Survivin.Preferably, by the polypeptides in combination of the aminoacid sequence of SEQ ID NO:1-4 be CTL epitope peptide SEQ ID NO:5.Preferably, by the polypeptides in combination of the aminoacid sequence of SEQ ID NO:6-9 be CTL epitope peptide SEQ ID NO:10.The polypeptid induction adopting this array mode to prepare goes out the antineoplastic immune for multiple different epitope, and this polyvalent vaccine to the many target position of tumor cell surface is more effective than single antigenic stimulation, thus produces high immunological effect.Corresponding tumour antigen can be chosen, obtain optimum therapeuticing effect with this.
Therefore, on the one hand, the invention provides a peptide species, its aminoacid sequence is four combinations being selected from peptide below: PRAME, MAGE A3, HER-2/neu, NY-ESO-1, Ras mutant, p53 mutant, PSA, hTERT, AFP, SART, CEA, CA-125, EGFR-800, C35-44, β-tubuline-309, spermatine 17, PSMA, Survivin.Preferably, the aminoacid sequence of polypeptide of the present invention is shown in SEQ ID NO:5 and SEQ ID NO:10.
On the other hand, the invention provides a kind of multitarget composite antigen load C D8
+cytotoxic T lymphocyte, is characterized in that it is prepared by method below:
1) lentiviral vectors is built; By pacl restriction endonuclease sites, hGM-CSF gene directed cloning is built recombinant vectors in slow virus connection carrier system, by recombinant vectors and envelope plasmid and the mixing of packaging structure plasmid, form lentiviral vectors;
2) DC vaccine is prepared: isolate monocyte, induce through rhGM-CSF, rhIL-4 and TNF-α and obtain DC cell, by polypeptide of the present invention (preferred SEQ ID NO:5 and 10) and step 1) slow virus that builds obtains DC vaccine after infecting DC cell simultaneously;
3) Peptide-specific CTL is prepared: by step 2) the DC vaccine that obtains and T lymphocyte hatch jointly, obtains the multitarget composite antigen load C D8 of sensitization
+cytotoxic T lymphocyte.
Present invention also offers a kind of polyvalent vaccine to the many target position of tumor cell surface, after it is characterized in that polypeptide of the present invention (preferred SEQ ID NO:5 or SEQ ID NO:10) and slow virus are infected DC cell, obtain DC vaccine simultaneously.Preferably, described slow virus is Lenti-hGM-CSF.
Again on the one hand, the invention provides the specific CTL effector cell of a kind of epi-position induction, it is characterized in that to multitarget composite antigen load C D8 of the present invention
+add GM-CSF, IL-4, IL-2 in the substratum of cytotoxic T lymphocyte to continue to cultivate, collecting cell, obtains the specific CTL effector cell of epi-position induction.
Multitarget composite antigen load C D8 of the present invention
+cytotoxic T lymphocyte for the preparation of the application in the medicine of Therapeutic cancer, and comprises multitarget composite antigen load C D8 of the present invention
+the pharmaceutical composition being used for the treatment of cancer of cytotoxic T lymphocyte is all the scope of protection of present invention.
In embodiments of the present invention, provide the cytotoxic T lymphocyte CTL of specific for tumour antigen, the CTL antigen peptide of described combination is the polypeptide polymer of highly expressing on tumour cell.Preferably, the CTL antigen peptide of combination of the present invention is the polypeptide polymer of highly expressing the polypeptide polymer of PRAME, C35-44, Survivin, NY-ESO-1 and HER-2/neu, SART and hTERT, CEA on tumour cell.
In a preferred embodiment in accordance with this invention, the mode of multiple epi-position combination is specially the CTL epitope of described combination, be connected by PRAME, C35-44 and Survivin, NY-ESO-1 tetra-polypeptide, the aminoacid sequence of wherein said PRAME is for shown in SEQ ID NO:1, the aminoacid sequence of described C35-44 is for shown in SEQ ID NO:2, the aminoacid sequence of described Survivin is for shown in SEQ ID NO:3, and the aminoacid sequence of described NY-ESO-1 is for shown in SEQ ID NO:4.The mode connected is prior art, comprises joint connection method, sticky end complementary method, isocaudarner method, series process etc.Preferred mode of connection is series process.
The aminoacid sequence of CTL complex antigen epitope polypeptide of the present invention is as shown in SEQ ID NO:5:
N-ALYVDSLFFL-TLLPAAGPAL-ELTLGEFLKL-SLLMFITQC-C
PRAME：
SEQ ID NO：1
ALYVDSLFFL
C35-44：
SEQ ID NO：2
TLLPAAGPAL
Survivin：
SEQ ID NO：3
ELTLGEFLKL
NY-ESO-1：
SEQ ID NO：4
SLLMFITQC
In another preferred embodiment of the present invention, the CTL antigen peptide of combination is the polypeptide polymer of highly expressing HER-2/neu, SART, hTERT and CEA on tumour cell.The mode of multiple epi-position combination of the present invention is specially the CTL epitope of described combination, be connected by HER-2/neu, SART and hTERT, CEA tetra-polypeptide, the aminoacid sequence of wherein said HER-2/neu is for shown in SEQ ID NO:6, the aminoacid sequence of described SART is for shown in SEQ ID NO:7, the aminoacid sequence of described hTERT is for shown in SEQ ID NO:8, and the aminoacid sequence of described CEA is for shown in SEQ ID NO:9.The mode connected is prior art, comprises joint connection method, sticky end complementary method, isocaudarner method, series process etc.Preferred mode of connection is series process.The aminoacid sequence of CTL complex antigen epitope polypeptide of the present invention is as shown in SEQ ID NO:10: N-YLEEITGYL-DYSARWNEI-YLQVNSLQTV-YLSGADLNL-C
HER-2/neu：
SEQ ID NO：6
YLEEITGYL
SART
SEQ ID NO：7
DYSARWNEI
hTFRT
SEQ ID NO：8
YLQVNSLQTV
CEA
SEQ ID NO：9
YLSGADLNL
In embodiments of the present invention, CTL of kinds of tumors antigen and preparation method thereof is provided for.The present invention can be used for any tumour, include but not limited to: lung cancer, mammary cancer, kidney, cancer of the stomach, colorectal cancer, carcinoma of the pancreas, liver cancer, cervical cancer, bladder cancer, prostate cancer, melanoma and tumor of head and neck etc., also can be applicable to the cancer about blood and marrow.
In the preferred embodiments of the disclosure, the antigen presentation that CTL product of the present invention represents with the described tumour of special correspondence.Therefore, many antigen epitope polypeptide associating slow virus Lenti-hGM-CSF of combination are infected DC cell simultaneously and provides a kind of approach generating single CTL product, described product has specific tumors independent antigen feature.
Significance of the present invention is embodied in: propose a kind of brand-new cellular immunotherapy theory, namely " autoimmune cell broken on tumour immunity tolerance basis is treated " technology, establishes one and is breaking new technology, the novel method that on tumour " immunological tolerance " basis, the efficient specific tumour of enforcement kills and wounds.The bottleneck problems such as this technology solves effector cell in current immunotherapy of tumors, and retention time is short in vivo, and tumor-killing efficiency is low, clinical efficacy difference, significantly improve the clinical efficacy of cellular immunotherapy, have boundless application prospect.
In embodiments of the invention, lentiviral vectors is built: the CDS region sequence for human peripheral hGM-CSF gene (GeneID:NM_000758.3) provides PCR primer by following method, by pcr amplification said gene 5 ' end and 3 ' hold all add restriction enzyme site, with plasmid pORFhGM-CSF for template, PCR method is adopted to amplify hGM-CSF gene fragment; By pacl restriction endonuclease sites, hGM-CSF gene directed cloning is built recombinant vectors in slow virus connection carrier system.By recombinant vectors and envelope plasmid and the mixing of packaging structure plasmid, form slow virus carrier system.Slow virus of the present invention is preferably Lenti-hGM-CSF.
In the preferred embodiment of the invention, described PCR primer is:
GM-CSF gene PCR primer:
F 5′-G
TTAATTAACATGTGGCTGCAGAGCCTGCTGCTCT-′3(SFQ ID NO：6)
R：5′-G
TTAATTAACTCACTCCTGGACTGGCTCCCAGCAG-′3(SEQ ID NO：7)
In above-mentioned primer, underscore part is restriction enzyme site.
In embodiments of the invention, DC vaccine is prepared: isolate monocyte by following method, preferably from peripheral blood, isolate monocyte, induce through rhGM-CSF, rhIL-4 and TNF-α and obtain DC cell, after the antigen epitope polypeptide (preferred SEQ ID NO:5) of combine the present invention and the above-mentioned slow virus of the present invention infect DC cell, obtain DC vaccine simultaneously.
In embodiments of the invention, the present invention is combined the DC vaccine obtained after antigen epitope polypeptide (preferred SEQ ID NO:5) and the above-mentioned slow virus of the present invention infect DC cell simultaneously then jointly to hatch with T lymphocyte, obtain the cytotoxic T lymphocyte of sensitization.Preferably, MTCA-DC/APC-CTL immune cell therapy technology expresses CD3
+cD8
+be main cytotoxic T cell be main effects cell.
In embodiments of the invention, prepare Peptide-specific CTL as follows: after being mixed with T lymphocyte by the DC cell of the load tumor associated antigen as above obtained of the present invention, after adding GM-CSF, IL-4, IL-2 continuation cultivation in the medium, namely collecting cell obtains the specific CTL effector cell of epi-position of the present invention induction.
Specific CTL of the present invention has the ability of the secretion of gamma-IFN significantly improved.At MTCA-multitarget composite antigen inducing antigen-specific CTL in the test of the lethal effect of specific tumor cell, the Peptide-specific CTL that MTCA-multitarget composite antigen induction of the present invention produces can form the SL to target cell in effect target cell Dual culture process, namely the CTL of MTCA-multitarget composite antigen induction of the present invention can kill and wound the target cell that load has control peptide simultaneously, expands the scope of application of test polypeptide.
brief Description Of Drawings
Fig. 1 is by pacl restriction endonuclease sites, will build the diagram of recombinant vectors FattbC31UGW-hGM-CSF in hGM-CSF gene directed cloning to slow virus connection carrier system.
Fig. 2 is envelope plasmid VSVG.
Fig. 3 is packaging structure plasmid CMV Δ 8.9-D64N/D116N.
Fig. 4 is that the ability of MTCA-multitarget composite antigen of the present invention (SEQ ID NO:5) inducing antigen-specific CTL generation IFN-γ compares bar graph.
Fig. 5 is that MTCA-multitarget composite antigen of the present invention (the SEQ ID NO:5) lethal effect of inducing antigen-specific CTL to specific tumor cell compares bar graph.
Fig. 6 is that the ability of MTCA-multitarget composite antigen of the present invention (SEQ ID NO:10) inducing antigen-specific CTL generation IFN-γ compares bar graph.
Fig. 7 is that MTCA-multitarget composite antigen of the present invention (the SEQ ID NO:10) lethal effect of inducing antigen-specific CTL to specific tumor cell compares bar graph.
Embodiment
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The structure of embodiment 1 slow virus LentihGM-CSF carrier
CDS region sequence for human peripheral hGM-CSF gene (GeneID:NM_000758.3) provides PCR primer, by pcr amplification said gene 5 ' end and 3 ' hold all add restriction enzyme site, with plasmid pORFhGM-CSF (purchased from Invivogen, article No.: porf-hgmcsf) be template, adopt PCR method to amplify hGM-CSF gene fragment; By pacl restriction endonuclease sites, hGM-CSF gene directed cloning is built recombinant vectors FattbC31UGW-hGM-CSF in slow virus connection carrier system (shown in Fig. 1).By recombinant vectors FattbC31UGW-hGM-CSF and envelope plasmid VSVG (shown in Fig. 2) and packaging structure plasmid CMV Δ 8.9-D64N/D116N (shown in Fig. 3) mixing, blending ratio is 1.5-10: 1-5: 1 formation slow virus carrier system, utilize liposome LipofectamineTM transfection on 293T cell, fluorescence microscopy Microscopic observation again after 24-48h, viral supernatants is collected after there is a large amount of fluorescence after 72h, obtain the lentiviral particle Lenti-hGM-CSF of coding hGM-CSF, the concentrated rear packing of vial supernatant of collection is for subsequent use or use immediately.
Described PCR primer is:
GM-CSF gene PCR primer:
F 5′-G
TTAATTAACATGTGGCTGCAGAGCCTGCTGCTCT-′3
R：5′-G
TTAATTAACTCACTCCTGGACTGGCTCCCAGCAG-′3
In above-mentioned primer, underscore part is restriction enzyme site.
During recombinant slow virus determination of activity, the viral stock after concentrated is done different ratios dilution, carry out fluorescence counting under fluorescent microscope again after cells infected 72h, determine titre.FUGW carrier in contrast.Result shows, and site-specific integration retroviral titre is 7.1X10
-7tU/ml.
Embodiment 2 synthesizes multitarget composite antigen peptide
Polypeptide as shown in SEQ ID NO:5, entrust Shanghai Qiangyao Biotechnology Co., Ltd. to adopt standard Fmoc scheme to synthesize, and adopt high performance liquid chromatography to carry out purifying and purity check, mass spectroscopy carries out identifying and molecular weight determination.Result shows, and the purity of described polypeptide is higher than 95%, and molecular weight conforms to theoretical value.
The pillar that concrete synthesis step: 1.Fmoc protects and monomer must use basic solvent (as piperidine) to remove amino protection group; 2. adopt activator to activate, dissolve the amino acid whose carboxyl terminal needing to be cross-linked, and the monomer of activation and free amino are cross-linked under the effect of linking agent, form peptide bond; 3. by first two steps iterative cycles until whole piece peptide chain synthesis complete.
Embodiment 3 synthesizes multitarget composite antigen peptide
Polypeptide as shown in SEQ ID NO:10, entrust Shanghai Qiangyao Biotechnology Co., Ltd. to adopt standard Fmoc scheme to synthesize, and adopt high performance liquid chromatography to carry out purifying and purity check, mass spectroscopy carries out identifying and molecular weight determination.Result shows, and the purity of described polypeptide is higher than 95%, and molecular weight conforms to theoretical value.The pillar that concrete synthesis step: 1.Fmoc protects and monomer must use basic solvent (as piperidine) to remove amino protection group; 2. adopt activator to activate, dissolve the amino acid whose carboxyl terminal needing to be cross-linked, and the monomer of activation and free amino are cross-linked under the effect of linking agent, form peptide bond; 3. by first two steps iterative cycles until whole piece peptide chain synthesis complete.
The preparation of embodiment 4 DC vaccine
Monocyte is isolated from peripheral blood, induce through rhGM-CSF, rhIL-4 and TNF-α and obtain DC cell, after phase microscope and flow cytometry, jointly hatch with T lymphocyte after the DC vaccine obtained after slow virus Lenti-hGM-CSF prepared by the multitarget composite antigen peptide (SEQ IDNO:5) of synthesis and embodiment 1 is infected DC cell simultaneously, obtain the cytotoxic T lymphocyte of sensitization.MTCA-DC/APC-CTL immune cell therapy technology expresses CD3
+cD8
+be main cytotoxic T cell be main effects cell.
The preparation of embodiment 5 DC vaccine
Monocyte is isolated from peripheral blood, induce through rhGM-CSF, rhIL-4 and TNF-α and obtain DC cell, after phase microscope and flow cytometry, jointly hatch with T lymphocyte after the DC vaccine obtained after slow virus Lenti-hGM-CSF prepared by the multitarget composite antigen peptide (SEQ IDNO:10) of synthesis and embodiment 1 is infected DC cell simultaneously, obtain the cytotoxic T lymphocyte of sensitization.MTCA-DC/APC-CTL immune cell therapy technology expresses CD3
+cD8
+be main cytotoxic T cell be main effects cell.
The preparation of embodiment 6 Peptide-specific CTL
Described CTL cell can derived from periphery blood T lymphocyte, peripheral blood is after lymphocyte separation medium (Ficoll-Hypaque) carries out density gradient centrifugation, collect the Interphase cells being rich in mononuclearcell, be resuspended in RPMI-1640 substratum, put 37 DEG C, 5%CO2 incubator cultivates 2 hours, the suspension cell collecting non-adherent, with after resuspended containing the RPMI-1640 substratum of 3% serum, regulates cell density to be 1 × 10
6/ ml, is rich in T lymphocyte in this suspension.The DC cell of load tumor associated antigen prepared by embodiment 2 and T lymphocyte according to 1: 10 ratio mix after, in RPMI-1640 substratum, add GM-CSF, IL-4, IL-2 continue cultivation 1 week, within every 3 days, half amount changes liquid, and maintenance cell concn is 1X10
6/ ml; Cultivate collecting cell after 1 week and namely obtain the specific CTL effector cell of epi-position induction.
The preparation of embodiment 7 Peptide-specific CTL
Described CTL cell can derived from periphery blood T lymphocyte, peripheral blood is after lymphocyte separation medium (Ficoll-Hypaque) carries out density gradient centrifugation, collect the Interphase cells being rich in mononuclearcell, be resuspended in RPMI-1640 substratum, put 37 DEG C, 5%CO2 incubator cultivates 2 hours, the suspension cell collecting non-adherent, with after resuspended containing the RPMI-1640 substratum of 3% serum, regulates cell density to be 1 × 10
6/ ml, is rich in T lymphocyte in this suspension.The DC cell of load tumor associated antigen prepared by embodiment 3 and T lymphocyte according to 1: 10 ratio mix after, in RPMI-1640 substratum, add GM-CSF, IL-4, IL-2 continue cultivation 1 week, within every 3 days, half amount changes liquid, and maintenance cell concn is 1X10
6/ ml; Cultivate collecting cell after 1 week and namely obtain the specific CTL effector cell of epi-position induction.
The ability that embodiment 8 MTCA-multitarget composite antigen of the present invention inducing antigen-specific CTL produces IFN-γ measures and comparison test
Euzymelinked immunosorbent assay (ELISA) detects the ability of secretion of gamma-IFN: adopt ELISPOT detection kit, add with the IFN-γ capture antibody 100ul of PBS by 1: 100 dilution in 96 orifice plates, be placed in 4 DEG C of overnight incubation, effector cell 100ul is added with after PBS washing, with target cell (human hepatoma cell line HepG2) 100ul, wherein the number ratio (E/T) of effector cell and target cell is 10, at 37 DEG C, and 5%CO
2after hatching 24h under condition, after washing with the PBS containing 0.1% polysorbas20, the biotin labeled anti-IFN-gamma antibodies after the PBS added containing 1%BSA dilutes by 1: 100, at 37 DEG C, 5%CO
2hatch 2h under condition, with the PBS containing 1%BSA by the alkaline phosphatase 100ul of the mark of avidin together after 1: 5000 dilution, hatch 1h, after patting dry culture plate after washing, use distilled water termination reaction, read point after dry.
Experiment one is grouped into: 1, DAGE control group; 2, C35-44 antigen control group; 3, Survivin antigen control group; 4, NY-ESO-1 antigen control group; 5, MTCA-multitarget composite antigen group; 6, blank group.
Result (Fig. 4) shows: the ability of experimental group inducing specific CTL secretion of gamma-IFN is apparently higher than other 5 groups, the ability of its secretion inducing IFN-γ is the strongest, improves more than 3 times relative to the ability of control group single peptide load specific CTL secretion of gamma-IFN.
Experiment two is grouped into: 1, HER-2/neu antigen control group; 2, SART antigen control group; 3, hTERT antigen control group; 4, CEA antigen control group; 5, MTCA-multitarget composite antigen group; 6, blank group.
Result (Fig. 6) shows: the ability of experimental group inducing specific CTL secretion of gamma-IFN is apparently higher than other 5 groups, the ability of its secretion inducing IFN-γ is the strongest, improves more than 3 times relative to the ability of control group single peptide load specific CTL secretion of gamma-IFN.
Embodiment 9 MTCA-multitarget composite antigen of the present invention inducing antigen-specific CTL is to the lethal effect of specific tumor cell
Time resolved fluorescence cytotoxicity experiment is adopted to analyze.The CTL action effect cell (E) of antigen peptide induction, the T2 cell of Loading peptides is target cell (T), detects the specific killing activity of CTL.At 37 DEG C, 5%CO
2be jointly hatch 4h at 50: 1 by effector cell and target cell according to number ratio under condition, 3 multiple holes established by each sample, average as detected result.
Cell killing activity=(treating gaging hole fluorescence intensity-Spontaneous release hole fluorescence intensity)/(maximum release aperture fluorescence intensity-Spontaneous release hole fluorescence intensity) X100%
Experiment one is grouped into: 1, DAGE control group; 2, C35-44 antigen control group; 3, Survivin antigen control group; 4, NY-ESO-1 antigen control group; 5, MTCA-multitarget composite antigen group.
Result (Fig. 5) shows, the Peptide-specific CTL that the induction of MTCA-multitarget composite antigen produces can form the SL to target cell in effect target cell Dual culture process, and the effector cell that each control group polypeptide is induced only can kill and wound the target cell that load has corresponding peptides, namely the CTL of MTCA-multitarget composite antigen induction can kill and wound the target cell that load has above-mentioned four kinds of control peptides simultaneously, expands the scope of application of test polypeptide.
Experiment two is grouped into: 1, HER-2/neu antigen control group; 2, SART antigen control group; 3, hTERT antigen control group; 4, CEA antigen control group; 5, MTCA-multitarget composite antigen group.
Result (Fig. 7) shows, the Peptide-specific CTL that the induction of MTCA-multitarget composite antigen produces can form the SL to target cell in effect target cell Dual culture process, and the effector cell that each control group polypeptide is induced only can kill and wound the target cell that load has corresponding peptides, namely the CTL of MTCA-multitarget composite antigen induction can kill and wound the target cell that load has above-mentioned four kinds of control peptides simultaneously, expands the scope of application of test polypeptide.
Sequence table
Claims (9)
1. a peptide species, its aminoacid sequence is four combinations being selected from peptide below: PRAME, MAGE A3, HER-2/neu, NY-ESO-1, Ras mutant, p53 mutant, PSA, hTERT, AFP, SART, CEA, CA-125, EGFR-800, C35-44, β-tubuline-309, spermatine 17, PSMA, Survivin.
2. a polypeptide for claim 1, its aminoacid sequence is shown in SEQ ID NO:5 or SEQ ID NO:10.
3. a multitarget composite antigen load C D8
+cytotoxic T lymphocyte, is characterized in that it is prepared by method below:
1) lentiviral vectors is built; By pac1 restriction endonuclease sites, hGM-CSF gene directed cloning is built recombinant vectors in slow virus connection carrier system, by recombinant vectors and envelope plasmid and the mixing of packaging structure plasmid, form lentiviral vectors;
2) prepare DC vaccine: isolate monocyte, induce through rhGM-CSF, rhIL-4 and TNF-α and obtain DC cell, by the polypeptide of claim 1 or 2 and step 1) slow virus that builds obtains DC vaccine after infecting DC cell simultaneously;
3) Peptide-specific CTL is prepared: by step 2) the DC vaccine that obtains and T lymphocyte hatch jointly, obtains the multitarget composite antigen load C D8 of sensitization
+cytotoxic T lymphocyte.
4. the multitarget composite antigen load C D8 of a claim 3
+cytotoxic T lymphocyte, is characterized in that wherein said slow virus is Lenti-hGM-CSF.
5. the multitarget composite antigen load C D8 of claim 3 or 4
+the preparation method of cytotoxic T lymphocyte, comprises the following steps:
1) lentiviral vectors is built; By pac1 restriction endonuclease sites, hGM-CSF gene directed cloning is built recombinant vectors in slow virus connection carrier system, by recombinant vectors and envelope plasmid and the mixing of packaging structure plasmid, form lentiviral vectors;
2) prepare DC vaccine: isolate monocyte, induce through rhGM-CSF, rhIL-4 and TNF-α and obtain DC cell, by the polypeptide of claim 1 or 2 and step 1) slow virus that builds obtains DC vaccine after infecting DC cell simultaneously;
3) Peptide-specific CTL is prepared: by step 2) the DC vaccine that obtains and T lymphocyte hatch jointly, obtains the multitarget composite antigen load C D8 of sensitization
+cytotoxic T lymphocyte.
6., to a polyvalent vaccine for the many target position of tumor cell surface, after it is characterized in that the polypeptide of claim 1 or 2 and slow virus Lenti-hGM-CSF are infected DC cell simultaneously, obtain DC vaccine.
7. a specific CTL effector cell for epi-position induction, is characterized in that the multitarget composite antigen load C D8 to claim 3 or 4
+add GM-CSF, IL-4, IL-2 in the substratum of cytotoxic T lymphocyte to continue to cultivate, collecting cell.
8. the multitarget composite antigen load C D8 of claim 3 or 4
+cytotoxic T lymphocyte is for the preparation of the application in the medicine of Therapeutic cancer.
9. be used for the treatment of a pharmaceutical composition for cancer, be characterised in that it contains the multitarget composite antigen load C D8 of claim 3 or 4
+cytotoxic T lymphocyte.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510270656.XA CN104926944B (en) | 2015-05-22 | 2015-05-22 | Multitarget composite antigen load C D8+The preparation method and its usage of cytotoxic T lymphocyte |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510270656.XA CN104926944B (en) | 2015-05-22 | 2015-05-22 | Multitarget composite antigen load C D8+The preparation method and its usage of cytotoxic T lymphocyte |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104926944A true CN104926944A (en) | 2015-09-23 |
| CN104926944B CN104926944B (en) | 2018-04-13 |
Family
ID=54114385
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510270656.XA Active CN104926944B (en) | 2015-05-22 | 2015-05-22 | Multitarget composite antigen load C D8+The preparation method and its usage of cytotoxic T lymphocyte |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104926944B (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017059557A1 (en) * | 2015-10-09 | 2017-04-13 | Peking University | Methods for enhancing in vivo persistence and efficacy of exogenously administered t cells, genetically modified t cells and method and method of use |
| CN109294982A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of RFF2 cell |
| CN109294997A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of LRFFT1 cell |
| CN109294983A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of LFF2 cell |
| CN109294996A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of RFFT2 cell |
| CN109679917A (en) * | 2018-09-30 | 2019-04-26 | 北京鼎成肽源生物技术有限公司 | A kind of LRFFT2 cell |
| CN109748974A (en) * | 2019-03-04 | 2019-05-14 | 上海尚泰生物技术有限公司 | A kind of preparation and application of the dendritic cell vaccine of gene modification |
| CN110093316A (en) * | 2018-09-30 | 2019-08-06 | 北京鼎成肽源生物技术有限公司 | A kind of construction method of AFF cell |
| CN110093317A (en) * | 2018-09-30 | 2019-08-06 | 北京鼎成肽源生物技术有限公司 | A kind of construction method of LFF2 cell |
| CN110205341A (en) * | 2018-09-30 | 2019-09-06 | 北京鼎成肽源生物技术有限公司 | A kind of construction method of LRFFT2 cell |
| CN111620955A (en) * | 2020-07-01 | 2020-09-04 | 北京康爱瑞浩细胞技术有限公司 | Multi-target-site composite antigen and application thereof |
| CN112402596A (en) * | 2019-08-22 | 2021-02-26 | 上海细胞治疗集团有限公司 | Polypeptide composition and vaccine |
| CN113461797A (en) * | 2020-03-18 | 2021-10-01 | 北京鼎成肽源生物技术有限公司 | Fallopian tube cancer target antigen, CTL cell cultured by fallopian tube cancer target antigen in stimulation mode and application of CTL cell |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302537A (en) * | 2008-05-15 | 2008-11-12 | 复旦大学 | Site-specific integration retroviral vector system and preparation thereof |
| CN101370513A (en) * | 2005-12-23 | 2009-02-18 | 瓦克松生物技术公司 | Immunogenicity polypeptide composed of optimized concealed peptide derived from tumor antigen |
| CN101765434A (en) * | 2007-07-27 | 2010-06-30 | 伊玛提克斯生物技术有限公司 | Composition of tumour-associated peptides and related anti-cancer vaccine |
| WO2015060235A1 (en) * | 2013-10-21 | 2015-04-30 | 大鵬薬品工業株式会社 | Novel four-ctl epitope-joined peptide |
-
2015
- 2015-05-22 CN CN201510270656.XA patent/CN104926944B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101370513A (en) * | 2005-12-23 | 2009-02-18 | 瓦克松生物技术公司 | Immunogenicity polypeptide composed of optimized concealed peptide derived from tumor antigen |
| CN101765434A (en) * | 2007-07-27 | 2010-06-30 | 伊玛提克斯生物技术有限公司 | Composition of tumour-associated peptides and related anti-cancer vaccine |
| CN101302537A (en) * | 2008-05-15 | 2008-11-12 | 复旦大学 | Site-specific integration retroviral vector system and preparation thereof |
| WO2015060235A1 (en) * | 2013-10-21 | 2015-04-30 | 大鵬薬品工業株式会社 | Novel four-ctl epitope-joined peptide |
Non-Patent Citations (2)
| Title |
|---|
| ZHONG-LI LIAO，ET AL.: "Diepitope multiple antigen peptide of hTERT trigger stronger anti-tumor immune responses in vitro", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
| 张有峰等: "多表位疫苗的构建策略及其在动物疫苗中的应用", 《中国农业科技导报》 * |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017059557A1 (en) * | 2015-10-09 | 2017-04-13 | Peking University | Methods for enhancing in vivo persistence and efficacy of exogenously administered t cells, genetically modified t cells and method and method of use |
| CN108473956A (en) * | 2015-10-09 | 2018-08-31 | 北昊干细胞与再生医学研究院有限公司 | Enhance the method, the T cell of genetic modification and method and application method of the exogenous internal persistence using T cell and effect |
| CN108473956B (en) * | 2015-10-09 | 2022-04-05 | 北昊干细胞与再生医学研究院有限公司 | Methods of enhancing in vivo persistence and efficacy of exogenously administered T cells, genetically modified T cells and methods of use |
| CN109294996A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of RFFT2 cell |
| CN109679917B (en) * | 2018-09-30 | 2020-09-25 | 北京鼎成肽源生物技术有限公司 | LRFFT2 cell |
| CN109294997A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of LRFFT1 cell |
| CN109679917A (en) * | 2018-09-30 | 2019-04-26 | 北京鼎成肽源生物技术有限公司 | A kind of LRFFT2 cell |
| US11718827B2 (en) | 2018-09-30 | 2023-08-08 | Beijing Dcty Biotech Co., Ltd. | LRFFT2 cell |
| CN110093316A (en) * | 2018-09-30 | 2019-08-06 | 北京鼎成肽源生物技术有限公司 | A kind of construction method of AFF cell |
| CN110093317A (en) * | 2018-09-30 | 2019-08-06 | 北京鼎成肽源生物技术有限公司 | A kind of construction method of LFF2 cell |
| CN110205341A (en) * | 2018-09-30 | 2019-09-06 | 北京鼎成肽源生物技术有限公司 | A kind of construction method of LRFFT2 cell |
| CN109294982A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of RFF2 cell |
| CN109294983A (en) * | 2018-09-30 | 2019-02-01 | 北京鼎成肽源生物技术有限公司 | A kind of LFF2 cell |
| CN109748974B (en) * | 2019-03-04 | 2022-07-08 | 上海尚泰生物技术有限公司 | Preparation and application of gene modified dendritic cell vaccine |
| CN109748974A (en) * | 2019-03-04 | 2019-05-14 | 上海尚泰生物技术有限公司 | A kind of preparation and application of the dendritic cell vaccine of gene modification |
| CN112402596A (en) * | 2019-08-22 | 2021-02-26 | 上海细胞治疗集团有限公司 | Polypeptide composition and vaccine |
| CN112402596B (en) * | 2019-08-22 | 2023-12-08 | 上海细胞治疗集团有限公司 | Polypeptide composition and vaccine |
| CN113461797A (en) * | 2020-03-18 | 2021-10-01 | 北京鼎成肽源生物技术有限公司 | Fallopian tube cancer target antigen, CTL cell cultured by fallopian tube cancer target antigen in stimulation mode and application of CTL cell |
| CN111620955A (en) * | 2020-07-01 | 2020-09-04 | 北京康爱瑞浩细胞技术有限公司 | Multi-target-site composite antigen and application thereof |
| CN111620955B (en) * | 2020-07-01 | 2022-11-08 | 北京康爱瑞浩细胞技术有限公司 | Multi-target-site composite antigen and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104926944B (en) | 2018-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104926944B (en) | Multitarget composite antigen load C D8+The preparation method and its usage of cytotoxic T lymphocyte | |
| ES2317379T3 (en) | TUMOR ASSOCIATED PEPTIDES UNITED TO MHC MOLECULES. | |
| Speidel et al. | Priming of cytotoxic T lymphocytes by five heat‐aggregated antigens in vivo: conditions, efficiency, and relation to antibody responses | |
| Calinescu et al. | Overview of current immunotherapeutic strategies for glioma | |
| RU2020113032A (en) | NEW PEPTIDES AND PEPTID COMBINATIONS FOR USE IN LUNG CANCER IMMUNOTHERAPY, INCLUDING NON-SMALL CELL LUNG CANCER (NSCLC) AND OTHER CANCER SPECIES | |
| US20230250151A1 (en) | Artificial antigen presenting cells for genetic engineering of immune cells | |
| WO2014004385A2 (en) | Anti-cancer vaccines | |
| Oertli et al. | Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo | |
| CN106279392B (en) | Tumor associated antigen XAGE-1b small peptides and its application | |
| Moran et al. | Alphaviral vector-transduced dendritic cells are successful therapeutic vaccines against neu-overexpressing tumors in wild-type mice | |
| JP2021536479A (en) | HERV-K-derived antigen as a shared tumor antigen for anti-cancer vaccines | |
| CN108456247A (en) | Target the T cell receptor and application thereof of NY-ESO-1 | |
| CN117487000A (en) | Tumor T cell epitope peptide, pMHC, and preparation and application thereof | |
| CN110526970A (en) | Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of CD133 | |
| RU2012147590A (en) | CDCA5 PEPTIDES AND THEIR VACCINES CONTAINING THEM | |
| CN105950662B (en) | A kind of replication defective recombinant slow virus CAR-T transgene carrier targeting CD22 and its construction method and application | |
| CN106589106B (en) | HLA-A24-restricted ECM 1-specific CTL epitope peptide and application thereof | |
| CN109371005B (en) | HLA-0201 restrictive PADI4 epitope polypeptide and application thereof | |
| JP2022501046A (en) | Arginase 1 polypeptide | |
| CN110157674A (en) | A kind of targeting T lymphocyte and its preparation method and application | |
| AU2018316253A1 (en) | LMP1-expressing cells and methods of use thereof | |
| CN109790224A (en) | Tumor-antigen peptide and its application derived from CACNA1H | |
| Liu et al. | Immunotherapy of Epstein-Barr virus associated malignancies using mycobacterial HSP70 and LMP2A356–364 epitope fusion protein | |
| CN111620955B (en) | Multi-target-site composite antigen and application thereof | |
| RU2283129C1 (en) | Protein-peptide antitumor composition, cell preparation activated therewith and method for tumor prophylaxis or treatment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |