CN106645677A - Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine - Google Patents
Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine Download PDFInfo
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Abstract
The invention relates to a method and kit for in vitro detection of tumor neoantigen specificity T cells and a tumor vaccine. The method for in vitro detection of the tumor neoantigen specificity T cells comprises a step S1 of adding tumor neoantigen stimulation in cell culture fluid of peripheral blood mononuclear cells and performing culture incubation; a step S2 of adopting an IFN gamma enzyme linked immunospot assay method to detect and identify the tumor neoantigen specificity T cells of tumor neoantigens. By implementing the method and kit for in vitro detection of the tumor neoantigen specificity T cells, the tumor neoantigen specificity T cells can be detected in high precision through the IFN gamma enzyme linked immunospot assay method, the detection method is simple, results are clear, and the cost is low. By implementing the tumor vaccine, the tumor neoantigen specificity T cells can be effectively amplified and are effectively used for manufacturing tumor immunotherapy medicines.
Description
Technical field
The present invention relates to molecular immunology and cellular immunology field, more particularly, it relates to a kind of vitro detection tumour
The method of neoantigen specific T-cells, kit, and tumor vaccine.
Background technology
The ajor histocompatibility molecule that T cell passes through T cell surface immunity receptor specific recognition tumour cell
The antigen peptide fragment (i.e. epitope) of (Major histocompatibility complex, MHC) submission is killing or kill
Hinder cancer cell.CD8+ cytotoxic T cells (Cytotoxic T lymphocytes, CTL) can the secretion of specific recognition target cell
Important cytokines interferon-gamma (Interferon γ, IFN γ), and direct killing cancer cell.CD4+ helper cell is recognized
Cytokine profiles can be secreted after tumour antigen epi-position including IFN γ, IL2, IL4 and TNF α, activation CD8+CTL is recognized and killed
Tumour cell.The peculiar target spot of suitable tumour cell, i.e. tumour antigen, and specific amplification identification tumour antigen are looked at present
Immunocyte be immunotherapy of tumors technology key technology.The target spot that at present most cells immunization therapy is selected is tumour
Cell overexpression antigen, i.e., the expression in tumour cell is significantly larger than normal cell.It is thin that tumor neogenetic antigen is to discriminate between tumour
Born of the same parents and the specific target spot of normal cell, are the best targets of tumor vaccine cells treatment.
The T cell of detection specific recognition tumor neogenetic antigen can be dyeed (such as using MHC- peptide multimers
The pentamer of ProImmune, the tetramer of Beckman coulter and the dextramer of Immundex companies).Peripheral blood
The T cell of middle neoantigen specificity can be with reference to the polymer of MHC- tumor neogenetic Antigenic Peptides, the fluorescence being coupled on polymer
Molecule can send fluorescence under laser active, so the tumor neogenetic Antigenic Peptide for combining MHC- peptide multimers is polymeric special
Property T cell can be detected by Flow cytometry, for analyzing patient's peripheral blood in recognize tumor neogenetic Antigenic Peptide T cell number
Amount and ratio.
However, using the detection tumor neogenetic T cells with antigenic specificity identification of MHC- peptide multimers flow cytometric analysis
Mainly there is following shortcoming：
1) it is uncertain high：The length and submission of tumor neogenetic antigen its corresponding MHC molecule all has uncertainty,
Therefore the polymer of stable MHC- nascent peptides cannot possibly be produced；
2) susceptibility is low：The specific T-cells ratio of MHC- peptide multimer flow cytometric analysis energy effective detections is
0.001%, when less than this numerical value, will be unable to be detected；
3) afunction：MHC- peptide multimers flow cytometric analysis can only represent specific T-cells can be specifically
With reference to MHC- peptide complexs, but can not detect T cell identification Antigenic Peptide after whether can effectively be stimulated, secreting function cell
The factor or killing tumor cell；
4) it is costly：Each Antigenic Peptide needs to be recognized by T cell by specific MHC molecule submission, therefore check fee
With height.
The content of the invention
The technical problem to be solved in the present invention is, for the drawbacks described above of prior art, there is provided a kind of certainty is strong, quick
The method and kit of sensitivity height, complete function and the vitro detection tumor neogenetic T cells with antigenic specificity of low cost, and
Can be used for expanding the tumor vaccine of tumor neogenetic T cells with antigenic specificity.
The technical solution adopted for the present invention to solve the technical problems is：Construct a kind of vitro detection tumor neogenetic antigen special
The method of specific T cell, including：
S1, add in the cell culture fluid of PMNC tumor neogenetic antigenic stimulus and carry out culture and incubate
Educate；
S2, the tumor neogenetic antigen-specific that the identification tumor neogenetic antigen is detected using IFN γ enzyme-linked immunospot assay
Property T cell.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, step S1 includes：
S11, the tumor neogenetic antigen is designed using antigen prediction software netMHC4；
S12, in the cell culture fluid for suspending the PMNC tumor neogenetic antigen is added to carry out
Pre-stimulation is simultaneously cultivated.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, the step S11 bag
Include：
S111, take tumor tissue in vitro and do genetic analysis, to find out non-synonymous gene mutation site；
S112, using antigen prediction software netMHC4 based on comprising the non-synonymous gene mutation site antigen and phase
The binding ability of the ajor histocompatibility molecule answered selects the best antigenic peptide sequence of binding ability；
S113, it is selected with corresponding main group from the antigenic peptide sequence using antigen prediction software netMHC4
The binding ability of biocompatiblity molecules is knitted apparently higher than its corresponding extragenic mutation site and corresponding ajor histocompatibility point
The antigenic peptide sequence of the binding ability of son is used as the tumor neogenetic antigen.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, the tumor neogenetic resists
Original includes the Antigenic Peptide that the Antigenic Peptide and sequence that sequence is LSKENSLIIQFTSFVAV is KLVVVGAAGVGKSAL.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, the step S12 bag
Include：
S121, the PMNC is suspended in serum-free cell culture medium, and add the tumour new
Raw antigen carries out pre-stimulation；
S122, normal temperature contain CO2Incubator in cultivated.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, step S2 includes
S21, by pre-stimulation after the PMNC be transferred to and be coated with the container of IFN γ antibody, plus
Enter the tumor neogenetic antigen to be incubated, so that special by the tumor neogenetic antigen after the tumor neogenetic antigenic stimulus
Specific T cell secretes IFN γ；
S22, IFN γ described in the IFN γ antibody capture, and newborn macroscopic immune spot is produced by integrated enzyme reaction
Point；
S23, the quantity for counting the immunodotting, detect that the tumor neogenetic resists with the quantity based on the immunodotting
The quantity of former specific T-cells.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, step S21 enters one
Step includes：
S211, Elispot plates are soaked at normal temperatures using ethanol water, subsequently adopt Elispot described in PBS
Plate；
S212, plus IFN γ antibody to be coated with the Elispot plates, then splice lid low temperature overnight incubation, subsequently uses PBS
Closing fluid-tight is added to close the unconjugated position of the IFN γ antibody, the incubation of splice lid normal temperature after cleaning；
The concentration of the PMNC after pre-stimulation in S213, the adjustment serum-free cell culture medium
Afterwards, it is subsequently added the corresponding tumor neogenetic antigen and forms mixing serum-free cell culture medium；
S214, by it is described mixing serum-free cell culture medium be added in the plate hole of the Elispot plates being coated with, plus
Cover plate contains CO in normal temperature2Incubator in cultivated.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, step S22 enters one
Step includes：
Serum-free cell culture medium described in S221, reject, is then cleaned using PBS, is subsequently carried out using cleaning fluid
Cleaning；
S222, the IFN γ identification antibody for being subsequently added biotin coupling, normal temperature incubation；
S223, cleaned using cleaning fluid, be subsequently adding the horseradish peroxidase for being coupled streptavidin, splice lid
Carry out normal temperature incubation；
S224, cleaned using cleaning fluid, be subsequently adding the substrate of horseradish peroxidase, then room temperature lucifuge is adopted
Fully cleaned with cleaning fluid, dried.
The present invention solves another technical scheme for being adopted of its technical problem：Construct a kind of vitro detection tumor neogenetic to resist
The kit of former specific T-cells, including：Tumor neogenetic antigen, Elispot plates for being coated with IFN γ antibody and enzyme-linked anti-
Answer reagent；The tumor neogenetic antigen includes that Antigenic Peptide and sequence that sequence is LSKENSLIIQFTSFVAV are
The Antigenic Peptide of KLVVVGAAGVGKSAL, the integrated enzyme reaction reagent includes IFN γ identification antibody, the coupling chain that biotin is coupled
The horseradish peroxidase of enzyme Avidin, the substrate of horseradish peroxidase, and auxiliary agent.
In the kit of vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, the auxiliary agent includes
Cleaning fluid, PBS, confining liquid, serum-free cell culture medium.
The invention further relates to a kind of tumor vaccine, including new for the tumour that expands tumor neogenetic T cells with antigenic specificity
Raw antigen, the tumor neogenetic antigen includes that Antigenic Peptide and sequence that sequence is LSKENSLIIQFTSFVAV are
The Antigenic Peptide of KLVVVGAAGVGKSAL.
Implement the method and kit of the vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, by IFN γ enzyme
Connection immune spot-ing can accurately detect tumor neogenetic T cells with antigenic specificity, and detection method is simple, result is bright
Really, it is with low cost.Implement the tumor vaccine of the present invention, can effectively expand tumor neogenetic T cells with antigenic specificity, and then effectively
For the making of immunotherapy of tumors medicine.
Description of the drawings
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing：
Fig. 1 is the described vitro detection tumor neogenetic T cells with antigenic specificity of first embodiment of the invention
The flow chart of method；
Fig. 2 is described vitro detection tumor neogenetic T cells with antigenic specificity according to the second embodiment of the present invention
The flow chart of method；
Fig. 3 is the Elispot figures of the immune response of tumor neogenetic T cells with antigenic specificity；
Fig. 4 is the immunodotting schematic diagram of tumor neogenetic T cells with antigenic specificity.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only to explain the present invention, and
It is not used in the restriction present invention.
Those skilled in the art know that in the following embodiments, in addition to special instruction, each reagent, instrument are city
Commercially available conventional products on face.Unreceipted particular technique or condition person in the following embodiments, according to text in the art
Offer described technology or condition or carry out according to product description.For the reagent concentration in following embodiments, process temperature
Degree and process time, can be adjusted according to actual conditions by those skilled in the art or be changed.
Fig. 1 is the described vitro detection tumor neogenetic T cells with antigenic specificity of first embodiment of the invention
The flow chart of method.As shown in figure 1, in step sl, add tumour new in the cell culture fluid of PMNC
Raw antigenic stimulus simultaneously carries out culture incubation.In one embodiment of the invention, can be set using antigen prediction software netMHC4
Count the tumor neogenetic antigen.In another embodiment of the present invention, can using it is well known in the prior art it is any can quilt
The tumor neogenetic antigen of tumor neogenetic T cells with antigenic specificity identification.In a preferred embodiment of the invention, the tumour
Neoantigen has the long peptide or whole antigen protein of non-synonymous gene mutation.In the present invention, can adopt in this area
Any technology known and method obtain the PMNC, and using tumor neogenetic antigenic stimulus and carry out culture and incubate
Educate.
In step s 2, the tumor neogenetic of the identification tumor neogenetic antigen is detected using IFN γ enzyme-linked immunospot assay
T cells with antigenic specificity.After by the tumor neogenetic antigenic stimulus, the tumor neogenetic T cells with antigenic specificity point
The IFN γ secreted can produce newborn macroscopic immune spot by pericellular IFN γ antibody capture by integrated enzyme reaction
Point, by counting the quantity of immunodotting the quantity of the tumor neogenetic T cells with antigenic specificity can be just detected.
Therefore first, the peptide fragment of the tumor neogenetic antigen in the present invention is the common incubation of PMNC
During, cut into naturally and itself by the antigen presenting cell in PMNC such as dendritic cells and B cell
The antigen fragment that ajor histocompatibility molecule is consistent, submission gives the tumor neogenetic T cells with antigenic specificity.Therefore, examined
The peptide fragment of the tumor neogenetic antigen of the tumor neogenetic T cells with antigenic specificity identification for measuring is that nature is presented, and
And include by the unpredictable epitope for arriving of Bioinformatic methods, so can ensure that the tumor neogenetic antigentic specificity
The natural sex of epitope of T cell identification and comprehensive.
Secondly, 3x10 can be detected using IFN γ enzyme-linked immunospot assay5A specific T-cells in PBMC, because
This its sensitiveness is high, is conducive to detecting the tumor neogenetic T cells with antigenic specificity of low ratio.And IFN γ enzyme linked immunological
Spotting method detection is to recognize that the tumor neogenetic antigen specific T of simultaneously energy secreting function cell factor IFN γ after tumour antigen is thin
Born of the same parents, are that feature is detected, the immune response of direct reaction tumor neogenetic T cells with antigenic specificity.
Additionally, the good tumor neogenetic antigen of immunogenicity is patient's cancer cell is different from the distinctive target spot of normal cell,
Can be applied in immunotherapy of tumors as tumor vaccine or for expanding tumor specific T cells.
Fig. 2 is described vitro detection tumor neogenetic T cells with antigenic specificity according to the second embodiment of the present invention
The flow chart of method.As shown in Fig. 2 in step sl, tumor neogenetic antigen is designed using antigen prediction software netMHC4.
In a preferred embodiment of the present invention, the tumor tissue in vitro that can take tumour patient does genetic analysis, finds out non-synonymous
Gene mutation site (including due to the point mutation of nucleic acid, insertion or the amino acid sequence for lacking and being formed), using antigen
Forecasting software netMHC4 whole candidate tumor neoantigens of the prediction comprising gene mutation site.It is subsequently soft using antigen prediction
Part netMHC4 is designed and synthesized comprising 15-30 amino acid lengths based on it with the binding ability of ajor histocompatibility molecule
Long peptide is used as tumor neogenetic antigen.After tumor neogenetic antigen is designed, those skilled in the art can adopt prior art
In any known technology and method synthesis purity>95% related neoplasms neoantigen.
In step s 2, the tumor neogenetic is added to resist in the cell culture fluid for suspending the PMNC
Original carries out pre-stimulation and is cultivated.In one embodiment of the invention, PMNC is isolated from peripheral blood
(Peripheral blood mononuclear cells, PBMC) is suspended with serum-free cell culture medium, adds tumor neogenetic
Antigenic Peptide (the 15-30 amino acid long peptides comprising mutational site) pre-stimulation, at 37 DEG C, 5%CO2Incubator is co-cultured 2 days.At this
In embodiment, incubation time is stimulated to be adjusted according to actual conditions.
In step s3, by pre-stimulation after the PMNC be transferred to the appearance for being coated with IFN γ antibody
In device, the tumor neogenetic antigen is added to carry out incubation 1 day, so that by the tumour after the tumor neogenetic antigenic stimulus
Neoantigen specific T-cells secrete IFN γ.Incubation time can be adjusted according to actual conditions.
In step s 4, the IFN γ of the tumor specific T cells secretion is led to by pericellular IFN γ antibody capture
Cross integrated enzyme reaction and produce macroscopic immunodotting.
In step s 5, the quantity of the immunodotting is counted, it is described swollen with the quantity detection based on the immunodotting
The quantity of knurl neoantigen specific T-cells.Quantity reflection patient's peripheral blood pair of the tumor neogenetic T cells with antigenic specificity
The immune response of nascent tumor is strong and weak.
Therefore first, the peptide fragment of the tumor neogenetic antigen in the present invention is the common incubation of PMNC
During, cut into naturally and itself by the antigen presenting cell in PMNC such as dendritic cells and B cell
The antigen fragment that ajor histocompatibility molecule is consistent, submission gives the tumor neogenetic T cells with antigenic specificity.Therefore, examined
The peptide fragment of the tumor neogenetic antigen of the tumor neogenetic T cells with antigenic specificity identification for measuring is that nature is presented, and
And include by the unpredictable epitope for arriving of Bioinformatic methods, so can ensure that the tumor neogenetic antigentic specificity
The natural sex of epitope of T cell identification and comprehensive.
Secondly, 3x10 can be detected using IFN γ enzyme-linked immunospot assay5A specific T-cells in PBMC, because
This its sensitiveness is high, is conducive to detecting the tumor neogenetic T cells with antigenic specificity of low ratio.And IFN γ enzyme linked immunological
Spotting method detection is to recognize that the tumor neogenetic antigen specific T of simultaneously energy secreting function cell factor IFN γ after tumour antigen is thin
Born of the same parents, are that feature is detected, the immune response of direct reaction tumor neogenetic T cells with antigenic specificity.Tumor neogenetic antigen-specific
The immune response of property T cell can be represented by macroscopic immunodotting, quickly and easily quantitative determination patient peripheral blood
For the immune response of tumor neogenetic antigen in the immunogenicity and tumour patient peripheral blood of middle tumor neogenetic antigen.
Additionally, the good tumor neogenetic antigen of immunogenicity is patient's cancer cell is different from the distinctive target spot of normal cell,
Can be applied in immunotherapy of tumors as tumor vaccine or for expanding tumor specific T cells.
Also, tumor neogenetic antigen predicts and synthesizes that method is simple using Bioinformatic methods.Tumor neogenetic antigen is cut
Cut and submission is passed in confluent monolayer cells in antigen and is naturally done, it is not necessary to spend high cost to synthesize the polymer of MHC- Antigenic Peptides.
Below by by way of example in detail to the vitro detection tumour described according to the third embodiment of the invention
The method of neoantigen specific T-cells is described further.
Embodiment 1, the prediction of tumor neogenetic antigen and synthesis
Take tumor tissue in vitro and do genome analysis, find out non-synonymous gene mutation site (including due to the point of nucleic acid
Mutation, insertion or the amino acid sequence for lacking and being formed), using antigen prediction software netMHC4 based on comprising described non-
The antigen in equivalent gene mutational site selects binding ability best with the binding ability of corresponding ajor histocompatibility molecule
Tumor neogenetic Antigenic Peptide of the antigenic peptide sequence as candidate.The tumor neogenetic Antigenic Peptide of candidate is logical with the binding ability of corresponding MHC
Cross and reach stable MHC- Antigenic Peptides and represent with reference to required Antigenic Peptide least concentration.Concentration is lower, represents Antigenic Peptide with MHC point
The combination of son is more stable, and its antigen originality is stronger, can preferably stimulate the propagation of specific T-cells.
Then it is selected with corresponding Main Tissues from the antigenic peptide sequence using antigen prediction software netMHC4
The binding ability of biocompatiblity molecules is apparently higher than its corresponding extragenic mutation site and corresponding ajor histocompatibility molecule
Binding ability antigenic peptide sequence as the tumor neogenetic antigen.Screening and phase i.e. from the tumor neogenetic Antigenic Peptide of candidate
The adhesion of MHC molecule is answered apparently higher than corresponding not mutated peptide (i.e. primary peptide) and the Antigenic Peptide sequence of the adhesion of MHC molecule
Row are used as the tumor neogenetic antigen.The tumor neogenetic Antigenic Peptide for determining candidate is the distinctive target spot of tumour cell, can be stimulated
The specific immune response of tumour cell targeting is produced, without the normal cell of the primary peptide of recognition expression.
Subordinate list 1 is using netMHC4 software screening methods two tumor neogenetic Antigenic Peptides out and their primary peptides of correspondence
Peptide sequence.As shown in subordinate list 2, the two tumor neogenetic Antigenic Peptides are with the adhesion of corresponding MHC molecule all higher than corresponding primary
Peptide and the adhesion of MHC molecule, represent that the two mutation generate required tumor neogenetic antigen.
The tumor neogenetic antigen of the prediction of table 1 and its amino acid sequence of the primary peptide of correspondence
Note：Runic simultaneously adds the amino acid of lower setting-out for the mutational site of tumor neogenetic antigen
The tumor neogenetic antigen of the prediction of table 2 and the binding ability (Kd, nM*) of corresponding MHC
*：Potential tumor neoantigen peptide is combined with the binding ability of corresponding MHC by reaching stable MHC- Antigenic Peptides
Required Antigenic Peptide least concentration represents that concentration is lower, and the combination of expression peptide and MHC molecule is more stable, potentially antigenic originality
It is stronger.
In tumor neogenetic antigen of the prediction comprising mutational site, design as shown in table 1 comprising tumor neogenetic Antigenic Peptide
The long peptide of 15-30 amino acid lengths, the tumor neogenetic Antigenic Peptide for delivering biotech firm's synthesis purity ＞ 95% of peptide symthesis is standby.
The tumor neogenetic Antigenic Peptide pre-stimulation of embodiment 2
Same example tumour patient extracts patient peripheric venous blood 10mL in the case of in the know informing with liquaemin anticoagulant tube.
With lymphocyte separation medium (Fresenius Kabi Norge AS, LymphoprepTM) isolated by density-gradient centrifugation method
After PMNC PBMC, suspended with fresh serum-free medium (AIM-V of Life Technology companies),
Cell density is in 1-3x106/mL.In serum-free medium, above-mentioned tumor neogenetic Antigenic Peptide or corresponding primary peptide are added
(1-10 μ g/mL), at 37 DEG C, 5%CO236-48h is cultivated in incubator.Detect that immune response of patient P BMC to primary peptide is
In order to check whether it is targeting mutant nucleotide sequence to the immune response of tumor neogenetic antigen, to determine tumor neogenetic antigen specific T
Only selectively targeted identification mutant cell is cancer cell and does not kill normal cell cell.One be it is possible to additionally incorporate without related peptide (ratio
As inhibition of HIV envelope protein a peptide fragment) as negative control, be not added with peptide as blank, for assessing PBMC in
The background level of immune response.
Embodiment 3 detects the tumor neogenetic antigen of the identification tumor neogenetic antigen using IFN γ enzyme-linked immunospot assay
Specific T-cells (U-CyTech BV, CT230-PR5)
Operating process reference reagent box service manual.Specific experiment flow process is as follows：
With 70% ethanol water in normal temperature wetting Elispot plates 1 hour, subsequently using PBS 2 times after, plus IFN γ
Coated antibody is coated with Elispot plates, and 4 DEG C of overnight incubations of splice lid are closed the IFN γ and resisted with PBS 3 times, plus closing fluid-tight
The unconjugated position of body, 37 DEG C of splice lid is incubated 1 hour；
The PBMC concentration that adjustment pre-stimulation is crossed is 1-3x106/ ml, is separately added into corresponding 1-10ug/mL tumor neogenetics and resists
Former (nascent peptide 1, nascent peptide 2 in table 1), tumour native antigen (primary peptide 1 and primary peptide 2 in table 1), control peptide fragment (ratio
As inhibition of HIV envelope protein a peptide fragment) serum-free cell culture medium and blank serum-free cell culture medium；Often
Hole adds 100ul serum-free cell culture mediums to the Elispot plates being coated with (i.e. per hole 1-3x105Cell, each Antigenic Peptide
Do 2-3 parallel hole), 37 DEG C of splice lid, 5%CO2Culture 16-24h；
Serum-free cell culture medium described in reject, first with PBS 2 times, then is washed 5 times with cleaning fluid, adds biotin idol
The IFN γ identification antibody of connection, is incubated 1 hour at 37 DEG C；
Washed 5 times with cleaning fluid, add the horseradish peroxidase for being coupled streptavidin, added a cover 37 DEG C and be incubated 1 hour；
Washed 5 times with cleaning fluid, add the substrate of horseradish peroxidase, it is room temperature lucifuge 25-50 minute, fully clear with clear water
The positive and negative of board-washing, dries.
With immunodotting number (as shown in Figure 3) in Elispot plate analysis device statistics hole, with reference to the cell of PBMC in every hole
Sum, calculates the tumour of specific recognition tumor neogenetic antigen and secreting function cell factor IFN γ in certain amount PBMC
The number of neoantigen specific T-cells, that is, recognize the ratio of tumor neogenetic T cells with antigenic specificity, outside the higher explanation of ratio
Immune response in all blood for tumor neogenetic antigen is stronger, and the immunogenicity of tumor neogenetic antigen is also stronger.
As shown in accompanying drawing 3-4, nonmutationed two parent tumor peptides can be recognized in the tumour patient peripheral blood in present case
And secrete the immunocyte number of IFN γ and there is no notable difference with negative control, so primary peptide does not have effective stimulus tumour special
The ability of specific T cell, i.e., without immunogenicity.And two tumor neogenetic Antigenic Peptides and secreting function cell factor can be recognized
The immunocyte number of IFN γ apparently higher than for their corresponding not mutated primary peptides or negative control, especially for
The immunodotting of nascent peptide 1 clearly (accompanying drawing 3), shows that the two potential tumor neoantigens have immunogenicity, can pierce
The T cell of specificity, can be used to prepare tumor specific T cells or tumor vaccine is applied to tumour and exempts from sharp patient's peripheral blood
Epidemic disease is treated.
In sum, the invention discloses using swollen in IFN γ elispot assay detection tumour patient peripheral blood
Knurl neoantigen specific T-cells react, and determine the immunogenicity of tumor neogenetic antigen, are that the immunotherapy of tumors of patient is looked for
Effectively target spot, is applied to the making of clinical immunotherapy medicine.
The tumor neogenetic antigen of further open candidate of the invention predicts and synthesizes that method is simple using Bioinformatic methods
It is single.The cutting of neoantigen and submission are passed in confluent monolayer cells in antigen and are naturally done, it is not necessary to spend high cost to synthesize MHC-
The polymer of Antigenic Peptide.The immune response of neoantigen specific T-cells can be represented by macroscopic immunodotting, very
Intuitively reflect that the immunity in the immunogenicity and tumour patient peripheral blood of tumor neogenetic antigen for tumor neogenetic antigen is anti-
Should.The good tumor neogenetic antigen of immunogenicity is that patient's cancer cell is different from the distinctive target spot of normal cell, can be used as swollen
Knurl vaccine is applied in the making of immunotherapy of tumors medicine for expanding tumor specific T cells.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of method of vitro detection tumor neogenetic T cells with antigenic specificity, it is characterised in that include：
S1, add in the cell culture fluid of PMNC and tumor neogenetic antigenic stimulus and carry out culture incubation；
S2, detect that the tumor neogenetic antigen specific T of the identification tumor neogenetic antigen is thin using IFN γ enzyme-linked immunospot assay
Born of the same parents.
2. the method for vitro detection tumor neogenetic T cells with antigenic specificity according to claim 1, it is characterised in that institute
Stating step S1 includes：
S11, the tumor neogenetic antigen is designed using antigen prediction software netMHC4；
S12, in the cell culture fluid for suspending the PMNC tumor neogenetic antigen is added to carry out pre- thorn
Swash and cultivated.
3. the method for vitro detection tumor neogenetic T cells with antigenic specificity according to claim 2, it is characterised in that institute
Stating step S11 includes：
S111, take tumor tissue in vitro and do genetic analysis, to find out non-synonymous gene mutation site；
S112, using antigen prediction software netMHC4 based on comprising the non-synonymous gene mutation site antigen with it is corresponding
The binding ability of ajor histocompatibility molecule selects the best antigenic peptide sequence of binding ability；
S113, it is selected with corresponding Main Tissues phase from the antigenic peptide sequence using antigen prediction software netMHC4
The binding ability of capacitive molecule is apparently higher than its corresponding extragenic mutation site and corresponding ajor histocompatibility molecule
The antigenic peptide sequence of binding ability is used as the tumor neogenetic antigen.
4. the method for vitro detection tumor neogenetic T cells with antigenic specificity according to claim 3, it is characterised in that institute
It is the anti-of KLVVVGAAGVGKSAL for the Antigenic Peptide and sequence of LSKENSLIIQFTSFVAV that tumor neogenetic antigen is stated including sequence
Former peptide.
5. the method for vitro detection tumor neogenetic T cells with antigenic specificity according to claim 2, it is characterised in that institute
Stating step S12 includes：
S121, the PMNC is suspended in serum-free cell culture medium, and add the tumor neogenetic to resist
Original carries out pre-stimulation；
S122, normal temperature contain CO2Incubator in cultivated.
6. the side of the vitro detection tumor neogenetic T cells with antigenic specificity according to any claim in claim 1-5
Method, it is characterised in that step S2 includes
S21, by pre-stimulation after the PMNC be transferred to and be coated with the container of IFN γ antibody, add institute
State tumor neogenetic antigen to be incubated, so that by the tumor neogenetic antigentic specificity after the tumor neogenetic antigenic stimulus
T cell secretes IFN γ；
S22, IFN γ described in the IFN γ antibody capture, and newborn macroscopic immunodotting is produced by integrated enzyme reaction；
S23, the quantity for counting the immunodotting, detect that the tumor neogenetic antigen is special with the quantity based on the immunodotting
The quantity of specific T cell.
7. the method for vitro detection tumor neogenetic T cells with antigenic specificity according to claim 6, it is characterised in that institute
State step S21 to further include：
S211, Elispot plates are soaked at normal temperatures using ethanol water, subsequently adopt Elispot plates described in PBS；
S212, plus IFN γ antibody to be coated with the Elispot plates, then splice lid low temperature overnight incubation, subsequently uses PBS
Afterwards plus the unconjugated position of the IFN γ antibody is closed in closing fluid-tight, the incubation of splice lid normal temperature；
After the concentration of the PMNC after pre-stimulation in S213, the adjustment serum-free cell culture medium,
It is subsequently added the corresponding tumor neogenetic antigen and forms mixing serum-free cell culture medium；
S214, the mixing serum-free cell culture medium is added in the plate hole of the Elispot plates being coated with, plus cover plate
Contain CO in normal temperature2Incubator in cultivated.
8. the method for vitro detection tumor neogenetic T cells with antigenic specificity according to claim 7, it is characterised in that institute
State step S22 to further include：
Serum-free cell culture medium described in S221, reject, is then cleaned using PBS, is subsequently cleaned using cleaning fluid；
S222, the IFN γ identification antibody for being subsequently added biotin coupling, normal temperature incubation；
S223, cleaned using cleaning fluid, be subsequently adding the horseradish peroxidase for being coupled streptavidin, splice lid is carried out
Normal temperature is incubated；
S224, cleaned using cleaning fluid, be subsequently adding the substrate of horseradish peroxidase, room temperature lucifuge, then using clear
Washing lotion is fully cleaned, and is dried.
9. a kind of kit of vitro detection tumor neogenetic T cells with antigenic specificity, it is characterised in that include：Tumor neogenetic resists
Elispot plates and integrated enzyme reaction reagent former, that be coated with IFN γ antibody；The tumor neogenetic antigen is including sequence
The Antigenic Peptide and sequence of LSKENSLIIQFTSFVAV is the Antigenic Peptide of KLVVVGAAGVGKSAL, and the integrated enzyme reaction reagent includes
IFN γ identification antibody, horseradish peroxidase, the bottom of horseradish peroxidase of coupling streptavidin that biotin is coupled
Thing, and auxiliary agent.
10. a kind of tumor vaccine, it is characterised in that include the tumor neogenetic for expanding tumor neogenetic T cells with antigenic specificity
Antigen, the tumor neogenetic antigen includes that Antigenic Peptide and sequence that sequence is LSKENSLIIQFTSFVAV are
The Antigenic Peptide of KLVVVGAAGVGKSAL.
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