CN104926944B - Multitarget composite antigen load C D8+The preparation method and its usage of cytotoxic T lymphocyte - Google Patents
Multitarget composite antigen load C D8+The preparation method and its usage of cytotoxic T lymphocyte Download PDFInfo
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Abstract
The present invention provides a kind of multitarget composite antigen, that is, the CTL epitope peptides combined, a kind of polyvaccine to the more target position of tumor cell surface, a kind of multitarget composite antigen load C D8+Cytotoxic T lymphocyte, its preparation method and application in the preparation of medicament for cancer treatment.Present invention also offers a kind of specific CTL effector cell of epitope induction.
Description
Technical field：The present invention relates to biology and medical domain, relates more specifically to multitarget composite antigen load
CD8+The preparation method and applications of cytotoxic T lymphocyte.
Background technology：
Malignant tumour is one of major disease for threatening human health, and in recent years, immune system is in tumor prevention and treatment
In important function accepted extensively, the immunization therapy based on antineoplastic specificity immunologic reconstitution is public in the world at present
That recognizes is most hopeful to eliminate the important means that interior tumor cell is able to effect a radical cure tumour completely after operation, radiation and chemotherapy,
The Main way that will be treated as future tumors.Immunization therapy is mainly intrinsic with activation body by supplementing, inducing in vitro
Biological response regulating system there is cytotoxic activity biologically active cell and cell factor to activate to transfer, raising patient itself
The function of immune system, strengthens the differentiation capability of specific tumor killing cell, on a cellular level killing tumor cell, effectively
Suppress transfer, diffusion and the recurrence of tumour cell, and side reaction is slight, has good clinical therapeutic efficacy, overcomes tumour
Traditional treatment mode " be not thorough, easily transfer, side effect it is big " etc. drawback.With from cell and molecular level to tumour immunity
Research is goed deep into, the tumor-specific cytotoxicity T lymphocytes of Dendritic Cells (DCs) sensitization of load tumour antigen
The pith of (cytotoxic T lymphocyte, CTL) as immunization therapy, can specific recognition tumour, in cloning
Expand and form immunological memory in vivo, mediate lasting antineoplastic specificity immunological effect.And the activation of CTL cells needs tumour
The costimulatory signal that antigen sensibilization and antigen presenting cell (APC) are provided and the three classes signal point provided by cell factor
The collective effect of son.Tumour is related and the specific active immunotherapy that exists for of specific antigen has established theoretical and material base.
The tumour antigen that only immunogenicity is strong, specificity is high could effectively activate the T lymphocytes of tumour-specific.In recent years, pin
The oncopeptide therapeutic vaccine that tumor associated antigen (TAA) or tumour specific antigen (TSA) to tumour expression grow up,
With its high specificity, safety, it is cheap, be easy to preserve and apply the features such as, the shortcomings that being expected to overcome above-mentioned therapy, reach and effectively control
Treat the purpose of tumour.
Tumour antigen must be degraded to small peptide simultaneously in antigen presenting cell (antigen presenting cell, APC)
Forming peptide-MHC-TCR compounds could be identified by T cell, excite corresponding cytotoxic T lymphocyte to react.And polypeptide
The purpose of vaccine is that the major histocompatibility complex for the tumor-antigen peptide of high dose being conveyed to APC surfaces
(major histocompatibility complex, MHC) molecule, therefore suitable and effective tumor-antigen peptide is to tumour
Preparing for vaccine is extremely important.And tumor-antigen peptide is limited by MHC, the only identical patient's ability of MHC I quasi-molecules
Using same peptide, and due to the inhomogeneity of tumour, some tumor antigen peptides inducing immune tolerance rather than may swash
Immune response living.In addition, immunogenicity it is weak be polypeptide vaccine another big weakness, it is (single by being modified epitope polypeptide
The replacement of amino acid, the PMRI modifications of polypeptide and lipopeptid etc.) or use polyvaccine then to effectively improve its immunogenicity, induce
Stronger CTL activity.
The present invention is the follow-up study of the Chinese invention patent based on Application No. 200810037440.9.The present invention is drawn
The document stated is as follows, and following documents are incorporated by reference into present patent application.Patent document 1：Publication number：CN
101302537A；Patent document 2：Publication number：CN 101854945A；Patent document 3：Publication number：CN 102625832A.
The content of the invention：
The present invention provides a kind of multitarget composite antigen (Multiple Target complex antigen, MTCA), i.e.,
The mode combined by multiple epitopes, the combination are according to genetic 26S Proteasome Structure and Function difference, from a series of candidate polypeptides
In select at least four polypeptides in combination to match with human leukocyte antigen, tumor vaccine is fabricated to, so as to excite patient's machine
Body extends its life span, MTCA has compared with other tumor vaccines and bypasses immunological diversity to the specific immune response of tumour
With the unique advantage of tumour inhomogeneity, stronger CTL killing activities can be effectively induced.
Therefore, it is an object of the present invention to provide a kind of multitarget composite antigen, that is, the CTL epitope peptides combined.
It is a further object to provide the polyvaccine to the more target position of tumor cell surface.Another object of the present invention is to carry
For a kind of multitarget composite antigen load C D8+Cytotoxic T lymphocyte and preparation method thereof.Another object of the present invention
It is to provide a kind of specific CTL effector cell of epitope induction.
Multiple epitope peptides provided by the invention have the ability for the HLA Restricted CTLs for inducing multiple HLA types, so as to reach
To more effective therapeutic effect.Exemplary tumour antigen with induction HLA-A24, HLA-A2 or HLA-A3 supertype at least wraps
Include following：Such as the HLA limitations with induction HLA-1101, HLA-A2402, HLA-A2601, HLA-A3101, HLA-A3303
The ability of property CTL.Plurality of 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, spermatin
17、PSMA、 Survivin。
Preferably, for example, by covering described exemplary oncologic associated antigen polypeptide PRAME, C35-44, Survivin, NY-
ESO-1 (other tumor antigen peptides and combination can be used) etc. can induce the antineoplastic immune for a variety of different epitopes, this
Kind is more more effective than single antigenic stimulus to the polyvaccine of the more target position of tumor cell surface.Preferably, for example, by described in covering
Exemplary oncologic associated antigen polypeptide HER-2/neu, SART and hTERT, CEA can induce for the anti-of a variety of different epitopes
Tumour immunity, this polyvaccine to the more target position of tumor cell surface are more more effective than single antigenic stimulus.
The present invention realizes foregoing invention purpose by following technological means：Selection carries out group no less than four polypeptides below
Close：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, spermatin 17, PSMA, Survivin.Preferably, will
SEQ ID NO：The polypeptides in combination of the amino acid sequence of 1-4 is CTL epitope peptide SEQ ID NO：5.Preferably, by SEQ ID
NO：The polypeptides in combination of the amino acid sequence of 6-9 is CTL epitope peptide SEQ ID NO：10.Prepared using the combination
Polypeptid induction goes out the antineoplastic immune for a variety of different epitopes, this polyvaccine to the more target position of tumor cell surface
It is more more effective than single antigenic stimulus, so as to produce high immunological effect.Corresponding tumour antigen can be chosen, is obtained most preferably with this
Therapeutic effect.
Therefore, on the one hand, the present invention provides a kind of polypeptide, its amino acid sequence is four groups for being selected from following peptide
Close：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, spermatin 17, PSMA, Survivin.Preferably, originally
The amino acid sequence of invention polypeptide is SEQ ID NO：5 and SEQ ID NO：Shown in 10.
On the other hand, the present invention provides a kind of multitarget composite antigen load C D8+Cytotoxic T lymphocyte, its feature
It is that it is prepared by following method：
1) slow virus carrier is built；By pacl restriction endonuclease sites, by hGM-CSF genes directed cloning to slow disease
Recombinant vector is built in poison connection carrier system, recombinant vector and envelope plasmid and packaging structure plasmid are mixed, is formed slow
Viral vector；
2) DC vaccines are prepared：Monocyte is isolated, DC cells are obtained through rhGM-CSF, rhIL-4 and TNF-α induction,
By polypeptide (the preferably SEQ ID NO of the present invention：5 and 10) and step 1) structure slow virus infect DC cells at the same time after obtain DC
Vaccine；
3) Peptide-specific CTL is prepared：The DC vaccines that step 2) obtains are incubated jointly with T lymphocytes, obtain sensitization
Multitarget composite antigen load C D8+Cytotoxic T lymphocyte.
Present invention also offers a kind of polyvaccine to the more target position of tumor cell surface, it is characterised in that by the present invention's
Polypeptide (preferably SEQ ID NO：5 or SEQ ID NO：10) and slow virus infects acquisition DC vaccines after DC cells at the same time.It is preferred that
Ground, the slow virus are Lenti-hGM-CSF.
Another further aspect, the present invention provide a kind of specific CTL effector cell of epitope induction, it is characterised in that to the present invention
Multitarget composite antigen load C D8+GM-CSF, IL-4, IL-2 is added in the culture medium of cytotoxic T lymphocyte to continue to train
Support, collect cell, obtain the specific CTL effector cell of epitope induction.
The multitarget composite antigen load C D8 of the present invention+Cytotoxic T lymphocyte is preparing the medicine for treating cancer
Application in thing, and include the multitarget composite antigen load C D8 of the present invention+Cytotoxic T lymphocyte is used to treat
The pharmaceutical composition of cancer is all the scope of protection of present invention.
In embodiments of the present invention, there is provided the cytotoxic T lymphocyte CTL of specific for tumour antigen, described group
The CTL Antigenic Peptides of conjunction are the polypeptide polymers that altimeter reaches on tumour cell.Preferably, the CTL of combination of the present invention resists
Former peptide is the polypeptide polymer and HER- that altimeter reaches PRAME, C35-44, Survivin, NY-ESO-1 on tumour cell
The polypeptide polymer of 2/neu, SART and hTERT, CEA.
In a preferred embodiment in accordance with this invention, the mode of multiple epitope combinations is specially the combination
CTL epitopes, are connected by tetra- polypeptides of PRAME, C35-44 and Survivin, NY-ESO-1, wherein the amino of the PRAME
Acid sequence is SEQ ID NO：Shown in 1, the amino acid sequence of the C35-44 is SEQ ID NO：Shown in 2, the Survivin
Amino acid sequence be SEQ ID NO：Shown in 3, the amino acid sequence of the NY-ESO-1 is SEQ ID NO：Shown in 4.Connection
Mode be the prior art, including connector connection method, cohesive end complementary method, isocaudarner method, series process etc..Preferred connection
Mode is series process.
The amino acid sequence of CTL complex antigens epitope polypeptide of the present invention such as SEQ ID NO：Shown in 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 presently preferred embodiment, the CTL Antigenic Peptides of combination are that altimeter reaches on tumour cell
The polypeptide polymer of HER-2/neu, SART, hTERT and CEA.The mode of multiple epitopes combination of the present invention is specially described
The CTL epitopes of combination, are connected by tetra- polypeptides of HER-2/neu, SART and hTERT, CEA, wherein the HER-2/neu
Amino acid sequence is SEQ ID NO：Shown in 6, the amino acid sequence of the SART is SEQ ID NO：Shown in 7, the hTERT
Amino acid sequence be SEQ ID NO：Shown in 8, the amino acid sequence of the CEA is SEQ ID NO：Shown in 9.The side of connection
Formula is the prior art, including connector connection method, cohesive end complementary method, isocaudarner method, series process etc..Preferred connection mode
For series process.The amino acid sequence of CTL complex antigens epitope polypeptide of the present invention such as SEQ ID NO：Shown in 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, there is provided CTL for kinds of tumors antigen and preparation method thereof.The present invention is available
In any tumour, include but not limited to：Lung cancer, breast cancer, kidney, stomach cancer, colorectal cancer, cancer of pancreas, liver cancer, cervical carcinoma, wing
Guang cancer, prostate cancer, melanoma and head and neck neoplasm etc., apply also for the cancer in relation to blood and marrow.
In the preferred embodiments of the disclosure, what CTL products of the present invention were showed with specifically corresponding to the tumour
Antigen presentation.Therefore, more antigen epitope polypeptides joint slow virus Lenti-hGM-CSF of combination is infected DC cells at the same time to provide
A kind of approach for generating list CTL products, the product have a specific tumors independent antigen feature.
The significance of the present invention is embodied in：A kind of brand-new cellular immunotherapy theory is proposed, i.e., " breaks tumour to exempt from
Autoimmune cell treatment on the basis of epidemic disease tolerance " technology, establishes one kind and implements on the basis of tumour " immune tolerance " is broken
New technology, the new method of efficient specific tumour killing.This technology solves in current immunotherapy of tumors effector cell in body
Interior retention time is short, and tumor-killing efficiency is low, and the bottleneck problem such as clinical efficacy difference, greatly improved the clinic of cellular immunotherapy
Curative effect, has boundless application prospect.
In embodiments of the invention, slow virus carrier is built with following methods：For human peripheral hGM-CSF bases
Because of (GeneID：NM_000758.3 CDS region sequences) provide PCR primer, by PCR amplifications at 5 ' ends of said gene and 3 '
Restriction enzyme site is added at end, and using plasmid pORFhGM-CSF as template, hGM-CSF genetic fragments are amplified using PCR method；It is logical
Pacl restriction endonuclease sites are crossed, hGM-CSF genes directed cloning to slow virus is connected structure restructuring in carrier system carries
Body.Recombinant vector and envelope plasmid and packaging structure plasmid are mixed, form slow virus carrier system.Slow disease of the present invention
Poison is preferably Lenti-hGM-CSF.
In the preferred embodiment of the invention, the PCR primer is：
GM-CSF gene PCR primer：
F 5′-GTTAATTAACATGTGGCTGCAGAGCCTGCTGCTCT-′3(SFQ ID NO：6)
R：5′-GTTAATTAACTCACTCCTGGACTGGCTCCCAGCAG-′3(SEQ ID NO：7)
In above-mentioned primer, underscore part is restriction enzyme site.
In embodiments of the invention, DC vaccines are prepared by following methods：Monocyte is isolated, preferably from periphery
Monocyte is isolated in blood, DC cells are obtained through rhGM-CSF, rhIL-4 and TNF-α induction, by the united antigen of the present invention
Epitope polypeptide (preferably SEQ ID NO：5) and the above-mentioned slow virus of the present invention infects acquisition DC vaccines after DC cells at the same time.
In embodiments of the invention, by present invention joint antigen epitope polypeptide (preferably SEQ ID NO：And this hair 5)
Bright above-mentioned slow virus infects the DC vaccines obtained after DC cells and then is incubated jointly with T lymphocytes at the same time, obtains the thin of sensitization
Cytotoxic T Lymphocytes.Preferably, MTCA-DC/APC-CTL immune cell therapies technology is to express CD3+CD8+Based on it is thin
Cytotoxic T cells are main effects cell.
In embodiments of the invention, Peptide-specific CTL is prepared as follows：The load being obtained as described above of the present invention is swollen
After the DC cells of knurl related antigen are mixed with T lymphocytes, GM-CSF, IL-4, IL-2 are added in the medium and continues to cultivate
Afterwards, specific CTL effector cell of the cell up to epitope of the present invention induction is collected.
Specific CTL of the present invention has the ability of the secretion of gamma-IFN significantly improved.Lured in MTCA- multitarget composite antigens
Lead in experiment of the Peptide-specific CTL to the lethal effect of specific tumor cell, MTCA- multitarget composite antigens of the present invention
The Peptide-specific CTL produced is induced to form the specific dissolution to target cell during effect target cell co-cultures, i.e.,
The CTL of MTCA- multitarget composite antigens induction of the present invention can kill the target cell for being loaded with control peptide at the same time, and it is more to expand experiment
The scope of application of peptide.
Brief Description Of Drawings
Fig. 1 is by pacl restriction endonuclease sites, by hGM-CSF genes directed cloning to slow virus connection carrier system
The diagram of recombinant vector FattbC31UGW-hGM-CSF is built in system.
Fig. 2 is envelope plasmid VSVG.
Fig. 3 is packaging structure plasmid CMV Δs 8.9-D64N/D116N.
Fig. 4 is MTCA- multitarget composite antigens of the present invention (SEQ ID NO：5) inducing antigen-specific CTL produces IFN-
The ability of γ compares bar chart.
Fig. 5 is MTCA- multitarget composite antigens of the present invention (SEQ ID NO：5) inducing antigen-specific CTL is to specificity
The lethal effect of tumour cell compares bar chart.
Fig. 6 is MTCA- multitarget composite antigens of the present invention (SEQ ID NO：10) inducing antigen-specific CTL produces IFN-
The ability of γ compares bar chart.
Fig. 7 is MTCA- multitarget composite antigens of the present invention (SEQ ID NO：10) inducing antigen-specific CTL is to special
The lethal effect of property tumour cell compares bar chart.
Embodiment
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
The structure of 1 slow virus LentihGM-CSF carriers of embodiment
For human peripheral hGM-CSF genes (GeneID：NM_000758.3 CDS region sequences) provide PCR primers, lead to
Cross PCR amplification and add restriction enzyme site at 5 ' ends of said gene and 3 ' ends, (be purchased from plasmid pORFhGM-CSF
Invivogen, article No.：Porf-hgmcsf it is) template, hGM-CSF genetic fragments is amplified using PCR method；Limited by pacl
Property restriction enzyme site processed, by hGM-CSF genes directed cloning, into slow virus connection carrier system (shown in Fig. 1), structure restructuring carries
Body FattbC31UGW-hGM-CSF.By recombinant vector FattbC31UGW-hGM-CSF and envelope plasmid VSVG (shown in Fig. 2) with
And packaging structure plasmid CMV Δs 8.9-D64N/D116N (shown in Fig. 3) mixing, mixed proportion 1.5-10: 1-5: 1 form slow
Virus carrier system, is transfected using liposome LipofectamineTM on 293T cells, fluorescence microscope again after 24-48h
Lower observation, collects viral supernatants after occurring a large amount of fluorescence after 72h, obtains the lentiviral particle Lenti-hGM- of coding hGM-CSF
CSF, dispenses spare or uses immediately after the vial supernatant concentration of collection.
The PCR primer is：
GM-CSF gene PCR primer：
F 5′-GTTAATTAACATGTGGCTGCAGAGCCTGCTGCTCT-′3
R：5′-GTTAATTAACTCACTCCTGGACTGGCTCCCAGCAG-′3
In above-mentioned primer, underscore part is restriction enzyme site.
During recombinant slow virus determination of activity, the viral stock after concentration is made into different proportion dilution, after infection cell 72h
Fluorescence counting is carried out under fluorescence microscope again, determines titre.FUGW carriers are as control.The results show that site-specific conformability
Slow virus titre is 7.1X10-7TU/ml。
Embodiment 2 synthesizes multitarget composite antigen peptide
Such as SEQ ID NO：Polypeptide shown in 5, commission Shanghai Qiangyao Biotechnology Co., Ltd. use standard Fmoc schemes
Synthesized, and use high performance liquid chromatography is purified and purity analysis, mass spectrography is identified and molecular weight determination.Knot
Fruit shows that the purity of the polypeptide is higher than 95%, and molecular weight is consistent with theoretical value.
Specific synthesis step：The pillar and monomer of 1.Fmoc protections must use basic solvent (such as piperidine) to remove ammonia
The protection group of base；2. need the c-terminus of crosslinked amino acid using activator activation, dissolving, and by the monomer of activation and trip
From amino be crosslinked under the action of crosslinking agent, formed peptide bond；3. by first two steps iterative cycles until whole peptide chain synthesizes
Finish.
Embodiment 3 synthesizes multitarget composite antigen peptide
Such as SEQ ID NO：Polypeptide shown in 10, commission Shanghai Qiangyao Biotechnology Co., Ltd. use standard Fmoc side
Case is synthesized, and use high performance liquid chromatography is purified and purity analysis, and mass spectrography is identified and molecular weight determination.
The results show that the purity of the polypeptide is higher than 95%, molecular weight is consistent with theoretical value.Specific synthesis step：1.Fmoc protections
Pillar and monomer must use the protection group of basic solvent (such as piperidine) removal amino；2. activated using activator, is molten
Solution needs the c-terminus of crosslinked amino acid, and the monomer of activation and free amino are handed under the action of crosslinking agent
Connection, forms peptide bond；3. by first two steps iterative cycles until the synthesis of whole peptide chain finishes.
The preparation of 4 DC vaccines of embodiment
Monocyte is isolated from peripheral blood, DC cells is obtained through rhGM-CSF, rhIL-4 and TNF-α induction, passes through
After phase contrast microscope and flow cytometry, by multitarget composite antigen peptide (the SEQ ID NO of synthesis：5) made with embodiment 1
Standby slow virus Lenti-hGM-CSF is incubated jointly after infecting the DC vaccines obtained after DC cells at the same time with T lymphocytes, is obtained
To the cytotoxic T lymphocyte of sensitization.MTCA-DC/APC-CTL immune cell therapy technologies are to express CD3+CD8+Based on
Cytotoxic T cell be main effects cell.
The preparation of 5 DC vaccines of embodiment
Monocyte is isolated from peripheral blood, DC cells is obtained through rhGM-CSF, rhIL-4 and TNF-α induction, passes through
After phase contrast microscope and flow cytometry, by multitarget composite antigen peptide (the SEQ ID NO of synthesis：And embodiment 1 10)
The slow virus Lenti-hGM-CSF of preparation is incubated jointly after infecting the DC vaccines obtained after DC cells at the same time with T lymphocytes,
Obtain the cytotoxic T lymphocyte of sensitization.MTCA-DC/APC-CTL immune cell therapy technologies are to express CD3+CD8+For
Main cytotoxic T cell is main effects cell.
The preparation of 6 Peptide-specific CTL of embodiment
The CTL cells can be derived from periphery blood T lymphocyte, and peripheral blood is through lymphocyte separation medium (Ficoll-
Hypaque after) carrying out density gradient centrifugation, the Interphase cells rich in mononuclearcell is collected, are resuspended in RPMI-1640 culture mediums
In, put 37 DEG C, 5%CO2 incubators culture 2 it is small when, collect non-adherent suspension cell use containing 3% serum RPMI-1640 cultivate
After base weight is hanged, it is 1 × 10 to adjust cell density6/ ml, is rich in T lymphocytes in this suspension.Load prepared by embodiment 2 is swollen
After the DC cells and T lymphocytes of knurl related antigen are according to 1: 10 ratio mixing, GM- is added in RPMI-1640 culture mediums
CSF, IL-4, IL-2 continue culture 1 week, and every 3 days half amounts change liquid, and maintenance cell concentration is 1X106/ml；Collected after cultivating 1 week
The specific CTL effector cell that cell induces up to epitope.
The preparation of 7 Peptide-specific CTL of embodiment
The CTL cells can be derived from periphery blood T lymphocyte, and peripheral blood is through lymphocyte separation medium (Ficoll-
Hypaque after) carrying out density gradient centrifugation, the Interphase cells rich in mononuclearcell is collected, are resuspended in RPMI-1640 culture mediums
In, put 37 DEG C, 5%CO2 incubators culture 2 it is small when, collect non-adherent suspension cell use containing 3% serum RPMI-1640 cultivate
After base weight is hanged, it is 1 × 10 to adjust cell density6/ ml, is rich in T lymphocytes in this suspension.Load prepared by embodiment 3 is swollen
After the DC cells and T lymphocytes of knurl related antigen are according to 1: 10 ratio mixing, GM- is added in RPMI-1640 culture mediums
CSF, IL-4, IL-2 continue culture 1 week, and every 3 days half amounts change liquid, and maintenance cell concentration is 1X106/ml；Collected after cultivating 1 week
The specific CTL effector cell that cell induces up to epitope.
The ability that the MTCA- multitarget composite antigens inducing antigen-specific CTL of the present invention of embodiment 8 produces IFN-γ is surveyed
Fixed and comparative test
Enzyme-linked immunization detects the ability of secretion of gamma-IFN：Using ELISPOT detection kits, add and use in 96 orifice plates
PBS is placed in 4 DEG C of overnight incubations, effector cell is added after being washed with PBS by 1: 100 diluted IFN-γ capture antibody 100ul
100ul, and target cell (human hepatoma cell line HepG2) 100ul, wherein the quantity ratio (E/T) of effector cell and target cell are 10,
At 37 DEG C, 5%CO2Under the conditions of be incubated 24h after, after being washed with the PBS containing 0.1% polysorbas20, add the PBS containing 1%BSA
By the anti-IFN-γ antibody of the biotin labeling after 1: 100 dilution, at 37 DEG C, 5%CO2Under the conditions of be incubated 2h, with containing 1%BSA
PBS by 1: 5000 dilution after Avidin together mark alkaline phosphatase 100ul, be incubated 1h, pat dry culture plate after washing
Afterwards, terminated and reacted with distilled water, read point after drying.
Experiment one is grouped into：1st, PRAME antigen control group；2nd, C35-44 antigen controls group；3rd, Survivin antigen controls
Group；4th, NY-ESO-1 antigen controls group；5th, MTCA- multitarget composite antigens group；6th, blank control group.
As a result (Fig. 4) shows：The ability of experimental group inducing specific CTL secretion of gamma-IFN apparently higher than other 5 groups, its
The ability of secretion inducing IFN-γ is most strong, is improved relative to the ability of control group single peptide load specific CTL secretion of gamma-IFN
More than 3 times.
Experiment two is grouped into：1st, HER-2/neu antigen controls group；2nd, SART antigen controls group；3rd, hTERT antigen controls
Group；4th, CEA antigen controls group；5th, MTCA- multitarget composite antigens group；6th, blank control group.
As a result (Fig. 6) shows：The ability of experimental group inducing specific CTL secretion of gamma-IFN apparently higher than other 5 groups, its
The ability of secretion inducing IFN-γ is most strong, is improved relative to the ability of control group single peptide load specific CTL secretion of gamma-IFN
More than 3 times.
The MTCA- multitarget composite antigens inducing antigen-specific CTL of the present invention of embodiment 9 is to specific tumor cell
Lethal effect
Analyzed using time-resolved fluorescence cytotoxicity experiment.The CTL of antigen inducing peptide as effector cell (E),
The T2 cells of Loading peptides are target cell (T), detect the specific killing activity of CTL.At 37 DEG C, 5%CO2Under the conditions of will
Effector cell is 50: 1 common incubation 4h according to quantity ratio with target cell, and each sample sets 3 multiple holes, is averaged as detection
As a 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：1st, PRAME antigen control group；2nd, C35-44 antigen controls group；3rd, Survivin antigen controls
Group；4th, NY-ESO-1 antigen controls group；5th, MTCA- multitarget composite antigens group.
As a result (Fig. 5) shows, the Peptide-specific CTL that the induction of MTCA- multitarget composite antigens produces is total in effect target cell
The specific dissolution to target cell can be formed in incubation, and the effector cell that each control group polypeptide is induced is only capable of killing
The target cell of corresponding peptides is loaded with, i.e. the CTL of MTCA- multitarget composite antigens induction can be killed at the same time is loaded with above-mentioned four kinds
The target cell of control peptide, expands the scope of application of test polypeptide.
Experiment two is grouped into：1st, HER-2/neu antigen controls group；2nd, SART antigen controls group；3rd, hTERT antigen controls
Group；4th, CEA antigen controls group；5th, MTCA- multitarget composite antigens group.
As a result (Fig. 7) shows, the Peptide-specific CTL that the induction of MTCA- multitarget composite antigens produces is total in effect target cell
The specific dissolution to target cell can be formed in incubation, and the effector cell that each control group polypeptide is induced is only capable of killing
The target cell of corresponding peptides is loaded with, i.e. the CTL of MTCA- multitarget composite antigens induction can be killed at the same time is loaded with above-mentioned four kinds
The target cell of control peptide, expands the scope of application of test polypeptide.
Claims (9)
1. a kind of polypeptide, is connected in sequence from N-terminal to C-terminal by tetra- polypeptides of HER-2/neu, SART, hTERT and CEA, wherein
The amino acid sequence of the HER-2/neu is SEQ ID NO：Shown in 6, the amino acid sequence of the SART is SEQ ID NO：7
Shown, the amino acid sequence of the hTERT is SEQ ID NO：Shown in 8, the amino acid sequence of the CEA is SEQ ID NO：9
It is shown.
2. polypeptide according to claim 1, its amino acid sequence as or SEQ ID NO：Shown in 10.
A kind of 3. multitarget composite antigen load C D8+Cytotoxic T lymphocyte, it is characterised in that：It is by following method
Prepare：
1) slow virus is built；By pacl restriction endonuclease sites, hGM-CSF genes directed cloning to slow virus is connected and is carried
Recombinant vector is built in system system, recombinant vector and envelope plasmid and packaging structure plasmid are mixed, forms slow virus；
2) DC vaccines are prepared：Monocyte is isolated, DC cells are obtained through rhGM-CSF, rhIL-4 and TNF-α induction, by right
It is required that the slow virus of 1 or 2 polypeptide and step 1) structure obtains DC vaccines after infecting DC cells at the same time；
3) Peptide-specific CTL is prepared：The DC vaccines that step 2) obtains are incubated jointly with T lymphocytes, obtain the more of sensitization
Target spot complex antigen load C D8+Cytotoxic T lymphocyte.
4. multitarget composite antigen load C D8 according to claim 3+Cytotoxic T lymphocyte, it is characterised in that：Wherein
The 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, including with
Lower step：
1) slow virus is built；By pacl restriction endonuclease sites, hGM-CSF genes directed cloning to slow virus is connected and is carried
Recombinant vector is built in system system, recombinant vector and envelope plasmid and packaging structure plasmid are mixed, forms slow virus；
2) DC vaccines are prepared：Monocyte is isolated, DC cells are obtained through rhGM-CSF, rhIL-4 and TNF-α induction, by right
It is required that the slow virus of 1 or 2 polypeptides and step 1) structure obtains DC vaccines after infecting DC cells at the same time；
3) Peptide-specific CTL is prepared：The DC vaccines that step 2) obtains are incubated jointly with T lymphocytes, obtain the more of sensitization
Target spot complex antigen load C D8+Cytotoxic T lymphocyte.
A kind of 6. polyvaccine to the more target position of tumor cell surface, it is characterised in that：By the polypeptide of claim 1 or 2 with
Slow virus Lenti-hGM-CSF obtains DC vaccines after infecting DC cells at the same time.
A kind of 7. specific CTL effector cell of epitope induction, it is characterised in that：It is compound to the Mutiple Targets of claim 3 or 4
Antigen load CD8+GM-CSF, IL-4, IL-2 are added in the culture medium of cytotoxic T lymphocyte to continue to cultivate, and collect cell.
8. the multitarget composite antigen load C D8 of claim 3 or 4+Cytotoxic T lymphocyte is being prepared for treating cancer
Application in the medicine of disease.
A kind of 9. pharmaceutical composition for treating cancer, it is characterised in that：It is compound containing the Mutiple Targets of claim 3 or 4
Antigen load CD8+Cytotoxic T lymphocyte.
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| 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 |
| CN109136277A (en) * | 2018-09-30 | 2019-01-04 | 北京鼎成肽源生物技术有限公司 | A kind of construction method of 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 |
| CN109294997B (en) * | 2018-09-30 | 2020-09-25 | 北京鼎成肽源生物技术有限公司 | LRFFT1 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 |
| 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 |
| CN109679917B (en) | 2018-09-30 | 2020-09-25 | 北京鼎成肽源生物技术有限公司 | LRFFT2 cell |
| CN109294996B (en) * | 2018-09-30 | 2020-11-06 | 北京鼎成肽源生物技术有限公司 | RFFT2 cell |
| CN109294982B (en) * | 2018-09-30 | 2020-11-06 | 北京鼎成肽源生物技术有限公司 | RFF2 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 |
| CN112402596B (en) * | 2019-08-22 | 2023-12-08 | 上海细胞治疗集团有限公司 | Polypeptide composition and vaccine |
| CN114315964B (en) * | 2020-03-18 | 2023-06-13 | 北京鼎成肽源生物技术有限公司 | Oviduct cancer target antigen combination, CTL cells stimulated and cultured by oviduct cancer target antigen and application of CTL cells |
| 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 |
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| US7994276B2 (en) * | 2007-07-27 | 2011-08-09 | Immatics Biotechnologies Gmbh | Composition of tumour-associated peptides and related anti-cancer vaccine |
| CN101302537B (en) * | 2008-05-15 | 2010-09-15 | 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 |
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