CN109957582A - A kind of preparation method of the cytotoxic T lymphocyte of a variety of KRAS mutation epitopes of target tumor - Google Patents
A kind of preparation method of the cytotoxic T lymphocyte of a variety of KRAS mutation epitopes of target tumor Download PDFInfo
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Abstract
The present invention relates to the preparation methods for the cytotoxic T lymphocyte (CTL) for targeting a variety of KRAS mutation epitopes.7 main KRAS mutation epitope sequences are concatenated the new artificial antigen coded sequence being composed by the method for the present invention, it is cloned into Lentiviral, transfect DC, the proliferation and differentiation of stimulation activation CD8+T cell, so that the cytotoxic T lymphocyte (CTL) being finally prepared has the specificity of 7 main KRAS mutation epitopes of targeting.KRAS mutation Antigen-specific cytotoxic T lymphocyte (CTL) prepared by the present invention has many advantages, such as that targeting specific is strong, without side-effects and be not easy to cause immunologic escape, has great application prospect in the treatment of KRAS mutation tumour.
Description
Technical field
The present invention relates to biotechnology development and application research fields, and in particular to targets a variety of KRAS mutation epitopes
Cytotoxic T lymphocyte (CTL) preparation method.
Background technique
KRAS belongs to RAS gene family important member, is " molecular switch " important in Cellular Signaling Transduction Mediated access,
Important regulating and controlling effect is played in MAPK (RAS-RAF-MEK-ERK) and PI3K (PI3K-AKT-mTOR) signal transduction pathway, is participated in
A series of basic bioprocess, including cell Proliferation, existence and differentiation (Jancik et al., 2010).But work as KRAS
When mutation, it cannot be hydrolyzed enzyme hydrolysis inactivation, and be in sustained activation state, cause downstream signal persistently to transduce, to make
At cellular overgrowth, proliferation, and inspire a variety of fatal tumors of the mankind.Even to this day, KRAS mutation is present in about
In 30% human cancer, wherein there is normalization trend in multiple cancers, especially cancer of pancreas, colorectal cancer and lung cancer,
Mutation rate is up to 90%, 45% and 35% respectively.The KRAS most common mode that is activated is point mutation, is mostly occurred the
12,13,61 and No. 146 codons, common mutations site is 12 and No. 13 codons, wherein 7 mutantional hotspots: G12D, G12V,
G13D, G12C, G12A, G12S and G12R, respectively are the highest hot spot of mutation probability, and the 98.5% of Zhan Suoyou KRAS mutation
Above (Prior et al., 2012).
A large amount of clinical effectiveness discoveries, KRAS mutation is unfavorable to Several Kinds of Malignancy patient's prognosis, such as KRAS mutation can be led
Cause lung cancer targeted drug EGFR-TKIs class that initial drug-resistant occurs, while it is western to targeted drug appropriate to also result in colorectal cancer patients
The prognosis of former times monoclonal antibody and Victibix treatment is deteriorated.Compared to the patient of KRAS wild type, the patient survival of KRAS mutation is bright
Aobvious shorten (Jancik et al., 2010).
There are three the RAS gene family being currently known is total there is mutation in gene KRAS, NRAS and HRAS in human tumor,
Wherein KRAS mutation is most commonly seen, accounts for about 85% or so.RAS gene mutation, it has also become current gene most ticklish in the world
Mutation.RAS is an ideal drug target in fact as a very important proto-oncogene.However, being directed at present
There are no effective targeted drugs for the gene.Once multiple RAS targeted drugs all had failed in succession, inhibited compared to EGFR
Forth generation has been developed in agent, up to the present RAS does not see that promising targeting medicine goes through to list also.One of them is most main
It wants reason to be to be mutated the function and RAS of RAS carcinogenic mechanism and does not still illustrate completely at present clear, in addition want to set
It counts and develops one kind to be also nearly impossible for all effective drug of all RAS mutation, therefore, can only find for spy
Surely the targeted drug being mutated, but this will shoulder heavy responsibilities.
Tumour cell immunization therapy has become the hot spot of current oncotherapy research, and be gradually considered as a kind of row it
Effective treatment method.Immunization therapy generates tumor associated antigen (TAAs) and is reacted and attacked by triggering body immune system
Tumour cell is hit, is the most promising treatment method of therapeutic field of tumor, becomes the 4th kind after operation, radiation and chemotherapy
Ideas of cancer therapy.
For the tumour of KRAS mutation, recent multinomial immunotherapy techniques, including immunologic test point inhibitor (PD1 and
PDL1) and TCR-T achieves pleasantly surprised clinical test results.Wherein, III phase clinic examination of the PD1 for NSCLC treatment
Test the result shows that, the patients with lung cancer of KRAS mutation using PD-1 antibody effect it is better than chemotherapy (Borghaei et al.,
2015).In addition, by one of famous immune cell therapy expert Steven A.Rosenberg team development using tumor-infiltrated
The II phase clinic of lymphocyte (tumorinfiltrating lymphocyte, TIL) immunization therapy metastatic patients with bowel cancer is tried
Test as a result, it has been found that, the patients with bowel cancer of a KRAS mutation is in the TIL adoptive immunity for receiving targeting KRAS G12D mutant antigen epitope
After treatment, seven transfer stoves of lung, 6 have diminution (Tran et al., 2016).The research demonstrated for the first time
It can be apparent anti-to the tumour cell generation for expressing such antigen after feeding back the TIL for being directed to autologous patient Tumor mutations neoantigen
Tumor response, this will provide a kind of new effective treatment means for KRAS mutation tumour.But due to the difficulty of preparation technology of TIL
Larger, the problems such as incubation time is longer, is unable to satisfy the demand of future clinical treatment.Even if filtering out driving mutation from TIL
TCR-T is cloned and prepared to T cells with antigenic specificity, and there is also higher technical barriers, including MHC limitation, misses the target and exempts from
The problems such as epidemic disease is escaped.
Dendritic Cells (Dendritic cells, DCs) is the strongest professional antigen presenting cell of function in body
(APC), the ability being proliferated with unique stimulation T cell, is the initiating person of immune response, a kind of natural " immune assistant
Agent " (Steinman and Banchereau, 2007).Just because of pass of the DC in inducing specific anti-tumor immune response
Key effect, a series of researchs about DC vaccine have persistently carried out many decades in global range, and relevant clinical test is also just
It is carrying out or is completing, and presenting preferable curative effect in the tumours such as melanoma, prostate cancer, kidney
(Palucka and Banchereau, 2012).Wherein, 2010, for treating the DC tumor vaccine of prostate cancer
(Sipuleucel-T) become the therapeutic tumor vaccine (Kantoff et al., 2010) of first FDA approval.DC tumour epidemic disease
Seedling is since tolerance is good, few side effects, can produce antitumor immune response, it has also become the research heat in cellular immunotherapy
Point, but from general, their curative effect is still limited.Current clinical research commonly thinks, DC vaccine is to making slow progress
Tumour shows certain curative effect, however to malignant tumour, curative effect is unobvious.
CD8+ cytotoxic T lymphocyte (Cytotoxic T cell, CTL) is the natural enemy of tumour cell most original, tool
There is the effect of tumour specific antigen identification and Efficient killing effect tumour cell, plays an important role in T cell immune response.Cause
This, for malignant tumor patient especially Advanced cancers patient, since tumor load is larger in body and tumour cell increases
It grows rapidly, so expanding a large amount of Peptide-specific CTL in vitro using DC-CTL technology, then feeds back patient's body, can reach
To better therapeutic effect.Meanwhile compared to other immune cell therapy technologies, the CTL based on the activation of DC stimulated in vitro has
The advantages that safely, effectively strong with targeting specific, it will show wide application prospect in the immunization therapy of tumour.
Currently, DC vaccine in vitro the common strategy of Antigen mainly including the use of section of synthesized peptide, tumor lysis object, wither
The tumour cell and tumor RNA sensitization and induction DC died generate effective and special antitumor reaction (Constantino et
Al., 2017).Although the clinical test at initial stage confirms its safety and validity, but total objective reactivity is very low, fails to
The significant Overall survival for extending patient.Meanwhile these methods are complicated for operation, need a large amount of patient's knurl tissue, due to tumour
The clinical effectiveness that the uncertainty of antigen has been doomed this DC vaccine has uncertainty.Although utilizing tumour-specific or correlation
Antigen polypeptide sensitization DC has good targeting, but since current confirmable tumour antigen epitope is seldom while single
The immune attack of one antigen is not only not enough to effectively kill tumour cell, can induce tumour cell that can generate immune escape instead
Ease.
The gene modification DC vaccine that viral vector infection DC by carrying tumor antigen gene is constructed becomes in recent years
Hot spot in the research of DC tumor vaccine.Wherein, compared to reverse transcription and adenovirus, slow virus carrier, which can both transfect to be in, silk
The cell of active period is divided, and can transfect and divide the slow and cell in telophase, there is metastatic gene segment capacity
Larger, the destination gene expression time is long, stablizes, and is not easy the advantages that inducing immune response, is to be now widely used in laboratory research
With the carrier of clinical test.
Summary of the invention
In consideration of it, the object of the present invention is to provide a kind of specificity of a variety of KRAS mutation epitopes of target tumor
The preparation method of cytotoxic T lymphocyte (CTL).
To achieve the above object, the preparation method of the specific CTL of a variety of KRAS mutation epitopes of a kind of target tumor,
Comprising steps of
A) building of the Lentiviral of 7 series connection KRAS mutation epitope sequences is carried；
B) the virus packaging and concentration of the step a) slow virus carrier；
C) separation and culture of peripheral blood DC cell；
D) DC cell described in slow-virus transfection step c) prepared by step b) and it is induced to differentiate into mature DC cell；
E) the CD8+T cell of paramagnetic particle method sorting derived from peripheral blood；
F) CD8+T cell described in mature DC cell described in step d) and step e) is co-cultured, obtaining has for 7
The specificity cell toxicity T lymphocyte (CTL) of a KRAS mutation epitope.
The main KRAS mutation site (G12A, G12C, G12D, G12R, G12S, G12V and G13D) of described 7 resists
Former epitope sequences are as shown in SEQ ID NO:1-7；The present invention by using Linker sequence as shown in SEQ ID NO:8, by 7
KRAS mutation epitope sequence tandem compound, and starting and terminator codon are added, new artificial antigen coded sequence is created,
As shown in SEQ ID NO:9.This includes h coding's sequence of 7 KRAS mutation epitopes after gene chemical synthesis, clone
To Lentiviral, DC, the proliferation and differentiation of stimulation activation CD8+T cell are transfected, so that being finally prepared into the CTL tool come
There is the specificity of 7 main KRAS mutation epitopes of targeting.
It includes cancer of pancreas, colorectal cancer and lung that above-mentioned Antigen-specific cytotoxic T lymphocyte (CTL), which can be used for manufacturing,
The therapeutic agent of KRAS mutation tumour including cancer.
Compared with the existing technology, the antigen gene based on slow virus carrier building modifies DC vaccine to the present invention, and activate
Peptide-specific CTL is a kind for the treatment of means effective, without side-effects, targeting specific is strong.In addition, due to being based on slow virus
Carrier can generate the Antigenic Peptide of 7 main KRAS mutation epitopes by endogenous expression simultaneously, therefore can prepare generation simultaneously
The Antigen-specific cytotoxic T lymphocyte (CTL) for targeting 7 main KRAS mutation, in actually controlling for KRAS mutation tumour
Have many advantages, such as wide spectrum in treatment and is not easy to cause immunologic escape.
The DC-CTL cell therapy technology of a variety of KRAS mutation epitopes of target tumor of the invention is widely used, and both may be used
To be applied independently in the treatment of KRAS mutation tumor patient, can also in conjunction with operation, chemicotherapy and other immunotherapy techniques into
The treatment of row KRAS mutation tumour.
Detailed description of the invention
Fig. 1 show the building schematic diagram for carrying the slow virus carrier of a variety of KRAS mutation antigen encoding sequences.
Fig. 2 show the digestion qualification figure for carrying the slow virus carrier of a variety of KRAS mutation antigen encoding sequences.
Fig. 3 show the flow analysis chart of marker after slow-virus transfection DC cell and induced maturation.
Fig. 4 show the flow analysis chart of the CTL of amplification cultivation.
Fig. 5 show the tumor cytotoxicity detection of Peptide-specific CTL.
Specific embodiment
In order to keep the present invention clearer, the present invention will be further described with reference to the drawings and specific embodiments.
Embodiment 1. carries the building and virus packaging of the Lentiviral of antigen encoding sequences:
1) Lentiviral constructs: the building schematic diagram for carrying the Lentiviral of antigen encoding sequences is for example attached
Shown in Fig. 1, first by using Linker sequence, by 7 KRAS mutation sites (G12A, G12C, G12D, G12R, G12S,
G12V and G13D) epitope sequence (as shown in SEQ ID NO:1-7), tandem compound, and add starting and termination codon
Son creates new artificial antigen coded sequence, as shown in SEQ ID NO:9, sequence size 435bp；Secondly, by creation
The both ends of KRAS mutation antigen encoding sequences are separately added into AscI and XbaI restriction enzyme site sequence and carry out gene chemical synthesis；
Finally, it is connected into Lentiviral (pLVX) by synthetic antigen encoding sequences segment after AscI and XbaI double digestion,
To construct the Lentiviral (pLVX-KRAS7R) for carrying antigen encoding sequences.Carry KRAS mutation antigen encoding sequences
Slow virus carrier digestion qualification figure it is as shown in Fig. 2, Lane 1 and Lane 2 are respectively through AscI and XbaI double digestion
Slow virus empty carrier (pLVX) and the slow virus carrier (pLVX-KRAS7R) for carrying KRAS mutation antigen encoding sequences.This implementation
The Linker sequence of example selection is as shown in SEQ ID NO:8；The tandem compound sequence selected is G12A-G12C-G12D-G12R-
G12S-G12V-G13D。
2) virus packaging: by liposome Lipo2000, by the slow of the carrying KRAS mutation antigen encoding sequences built
Virus expression carrier is transfected into the 293FT (human embryonic kidney cells) of P10-P12 together with package carrier pLP1, pLP2, pLP/VSVG
Slow virus packaging is carried out in cell.To collection contain virulent culture solution, using 20% sucrose cushion Ultracentrifugation Method from
Lentiviral particle is concentrated and purified in supernatant containing virus, dispenses and is saved in -80 DEG C.
The Fiber differentiation of embodiment 2.DC cell and transfection:
Peripheral blood is acquired, separates periphery using Ficoll-Hypaque (Ficoll-Hypaque) density gradient separation
Blood monocyte PBMC, and Fiber differentiation DC cell is stimulated using rhIL-4 (100ng/mL) and GM-CSF (1500U/mL).The
Immature DC cell was transfected with the lentiviral particle packed and be concentrated in three days, it need to be according to the optimal MOI of preliminary experiment
Value calculates the dosage of slow virus.Transfection is changed to fresh DC medium after 24 hours and polyinosinic acid POLY (I: C) (10 is added
μ g/mL) to promote the expression of endogenous gene.Fluid infusion every other day, the 6th day addition TNF (Tumor Necrosis Factor) alpha (10ng/mL), and
7 days maturations by micro- sem observation DC cell, at the same by flow cytomery maturation DC cell surface marker CD83,
CD86 and HLA-DR, as shown in Fig. 3.It should be the results show that comparing immature DC cell, DC cell is through Fiber differentiation and transfection
Afterwards, the expression quantity status enhancing of CD83, CD86 and HLA-DR of the 6th day DC cell surface, further illustrates the DC cell of culture
It is mature, the enhancing of antigen submission ability.
The preparation of 3. Peptide-specific CTL cell of embodiment:
1) CD8+T cell is separately cultured: the PBMC that peripheral blood is separated through Ficoll, sorts CD8+T cell using paramagnetic particle method.
Well-graded CD8+T cell presses 0.5-1 × 106The density of/mL is seeded in T75 culture bottle, and cell factor IFN-γ is added
(1000U/mL), rhIL-2 (1000U/mL) and autologous plasma (10%) Fiber differentiation after culture 3 days, add amplification training in due course
Nutrient solution (1000U/mL rhIL-2).When cell Proliferation to right quantity takes cell progress flow cytometer showed, as shown in Fig. 4, amplification
The CD8+CTL cell purity with higher of culture.
2) preparation of Peptide-specific CTL cell: the mature DC and CTL cell of Antigen is total according to 1: 10 ratio
16h is cultivated, CTL sensitization is made, generates the specificity with 7 main KRAS mutation epitopes of targeting
The tumor cytotoxicity of 4. Peptide-specific CTL of embodiment is tested:
In order to verify Peptide-specific CTL to the fragmentation effect of KRAS mutation tumour cell, the present embodiment has screened 3 plants and has deposited
In the colorectal cancer cell lines SW480 (G12V), LS174T (G12A/G13D) and HCT 116 (G13D), Yi Jiyi of KRAS mutation
The colorectal cancer cell lines HT-29 (WT) of strain KRAS wild type, carries out the killing experiments in vitro of Peptide-specific CTL.First will
Peptide-specific CTL and tumour target cell are incubated for progress specific killing verifying in 6 hours respectively with 1: 1,3: 1 and 10: 1 jointly；
Secondly group of cells OD value is detected using CCK8 detection method；CTL is finally calculated to the killing-efficiency of KRAS mutation tumour cell,
As shown in Fig. 5, the CTL of a variety of KRAS mutation epitopes of targeting prepared as the result is shown has KRAS mutation tumour cell
There are good fragmentation effect and very strong specificity.
In conclusion the cytotoxic T lymphocyte of a variety of KRAS mutation epitopes of target tumor prepared by the present invention
With very strong targeting specific and good fragmentation effect, get a good chance of the clinical treatment for KRAS mutation tumour.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations all should belong to the guarantor of appended claims of the present invention
Protect range.
Claims (6)
1. a kind of preparation method of the cytotoxic T lymphocyte (CTL) of a variety of KRAS mutation epitopes of target tumor, step
It include: the building for a) carrying the Lentiviral of 7 series connection KRAS mutation epitope sequences；B) step a) the slow disease
The virus packaging of poisonous carrier and concentration；C) separation and culture of peripheral blood DC cell；D) slow-virus transfection prepared by step b)
DC cell described in step c) is simultaneously induced to differentiate into mature DC cell；E) the CD8+T cell of paramagnetic particle method sorting derived from peripheral blood；f)
CD8+T cell described in mature DC cell described in step d) and step e) is co-cultured, obtaining has for 7 main KRAS
The specificity cell toxicity T lymphocyte (CTL) of mutant antigen epitope.
2. the cytotoxic T lymphocyte (CTL) of a variety of KRAS mutation epitopes of target tumor as described in claim 1
Preparation method, it is characterized in that: mutant antigen epitope sequences described in the step a) refer to 7 KRAS through Linker sequence
Mutant antigen epitope sequences tandem compound, and starting and terminator codon are added, the new artificial antigen coded sequence of creation, such as
Shown in SEQ ID NO:9.
3. expression vector described in step a) according to claim 1, it is characterised in that the expression vector is slow virus
Carrier.
4. expression vector described in step a) according to claim 1, it is characterised in that the antigen encoding that carries and can express
Sequence is shown in SEQ ID NO:9.
5. a kind of cytotoxic T lymphocyte (CTL) of antigentic specificity made from preparation method as described in claim 1.
6. a kind of controlled by Antigen-specific cytotoxic T lymphocyte described in claim 5 (CTL) for KRAS mutation tumour
Treat the application of drug.
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