CN106645677B - Method, kit and the tumor vaccine of vitro detection tumor neogenetic T cells with antigenic specificity - Google Patents
Method, kit and the tumor vaccine of vitro detection tumor neogenetic T cells with antigenic specificity Download PDFInfo
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Abstract
The present invention relates to method, kit and the tumor vaccines of a kind of vitro detection tumor neogenetic T cells with antigenic specificity.The method of the vitro detection tumor neogenetic T cells with antigenic specificity, comprising: S1, tumor neogenetic antigenic stimulus is added in the cell culture fluid of peripheral blood mononuclear cells and carries out culture incubation；S2, the tumor neogenetic T cells with antigenic specificity that the tumor neogenetic antigen is identified using the detection of IFN γ enzyme-linked immunospot assay.The method and kit for implementing vitro detection tumor neogenetic T cells with antigenic specificity of the invention can accurately detect tumor neogenetic T cells with antigenic specificity by IFN γ enzyme-linked immunospot assay, and detection method is simple, result is clear, low in cost.Implement tumor vaccine of the invention, can effectively expand tumor neogenetic T cells with antigenic specificity, and then effective for the production of immunotherapy of tumors drug.
Description
Technical field
The present invention relates to molecular immunologies and cellular immunology field, more specifically to a kind of vitro detection tumour
Method, kit and the tumor vaccine of neoantigen specific T-cells.
Background technique
T cell passes through the ajor histocompatibility molecule of T cell surface immunity receptor specific recognition tumour cell
The antigen peptide fragment (i.e. epitope) of (Major histocompatibility complex, MHC) submission is killed or is killed
Hurt cancer cell.CD8+ cytotoxic T cell (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.The identification of CD4+ T helper cell
It includes IFN γ, IL2, IL4 and TNF α that cytokine profiles can be secreted after tumour antigen epitope, and activation CD8+CTL is identified and killed
Tumour cell.The peculiar target spot of suitable tumour cell is looked at present, i.e. tumour antigen and specific amplification identifies tumour antigen
Immunocyte be immunotherapy of tumors technology key technology.The target spot of the selection of most cells immunization therapy at present is tumour
Cell is overexpressed 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
The specific target spot of born of the same parents and normal cell are the best targets of tumor vaccine cells treatment.
Detection specific recognition tumor neogenetic antigen T cell can be used MHC- peptide multimer dyeing (such as
The pentamer of ProImmune, the tetramer of Beckman coulter and the dextramer of Immundex company).Peripheral blood
The T cell of middle neoantigen specificity can be in conjunction with the polymer of MHC- tumor neogenetic Antigenic Peptide, the fluorescence being coupled on polymer
Molecule can issue fluorescence under laser active, so combining the special of the tumor neogenetic antigen peptide multimer of MHC- peptide multimer
Property T cell can be detected by Flow cytometry, for analyze in patient's peripheral blood identify tumor neogenetic Antigenic Peptide T cell number
Amount and ratio.
However, being identified using MHC- peptide multimer flow cytometric analysis detection tumor neogenetic T cells with antigenic specificity
Mainly there is following disadvantage:
1) uncertain high: its corresponding MHC molecule of the length and submission of tumor neogenetic antigen all has uncertainty,
Therefore the polymer of stable MHC- nascent peptide can not possibly be generated；
2) susceptibility is low: the specific T-cells ratio that MHC- peptide multimer flow cytometric analysis can be detected effectively is
0.001%, when being lower than this numerical value, will be unable to be detected；
3) afunction: MHC- peptide multimer flow cytometric analysis can only show specific T-cells can be specifically
Whether can be effectively stimulated in conjunction with MHC- peptide complex, but after cannot detecting T cell identification Antigenic Peptide, secreting function cell
The factor or killing tumor cell；
4) costly: each Antigenic Peptide needs are identified by specific MHC molecule submission by T cell, therefore check fee
With height.
Summary of the invention
The technical problem to be solved in the present invention is that in view of the above drawbacks of the prior art, it is strong, quick to provide a kind of certainty
Sensitivity is high, full-featured and low-cost vitro detection tumor neogenetic T cells with antigenic specificity method and kit, and
It can be used for expanding the tumor vaccine of tumor neogenetic T cells with antigenic specificity.
The technical solution adopted by the present invention to solve the technical problems is: it is special to construct a kind of vitro detection tumor neogenetic antigen
The method of specific T cell, comprising:
S1, tumor neogenetic antigenic stimulus is added in the cell culture fluid of peripheral blood mononuclear cells and carries out culture and incubates
It educates；
S2, the tumor neogenetic antigen-specific that the tumor neogenetic antigen is identified using the detection of 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, the step S1 includes:
S11, the tumor neogenetic antigen is designed using antigen prediction software netMHC4；
S12, the tumor neogenetic antigen progress is added in the cell culture fluid for suspending the peripheral blood mononuclear cells
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 packet
It includes:
S111, tumor tissue in vitro is taken to do genetic analysis, to find out gene mutation site non-synonymous；
S112, using antigen prediction software netMHC4 based on antigen and phase comprising the gene mutation site non-synonymous
The best antigenic peptide sequence of the binding ability selection binding ability for the ajor histocompatibility molecule answered；
S113, itself and corresponding main group are selected from the antigenic peptide sequence using antigen prediction software netMHC4
The binding ability for knitting biocompatiblity molecules is apparently higher than its corresponding extragenic mutation site and corresponding ajor histocompatibility point
The antigenic peptide sequence of the binding ability of son is 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 is anti-
The Antigenic Peptide that original includes the Antigenic Peptide that sequence is LSKENSLIIQFTSFVAV and sequence is KLVVVGAAGVGKSAL.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, the step S12 packet
It includes:
S121, the peripheral blood mononuclear cells is suspended in serum-free cell culture medium, and it is new that the tumour is added
Raw antigen carries out pre-stimulation；
S122, contain CO in room temperature2Incubator in cultivated.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, the step S2 includes
S21, the peripheral blood mononuclear cells after pre-stimulation is transferred in the container for being coated with IFN γ antibody, is added
Enter the tumor neogenetic antigen to be incubated for, so that special by the tumor neogenetic antigen after the tumor neogenetic antigenic stimulus
Specific T cell secretes IFN γ；
IFN γ described in S22, the IFN γ antibody capture, and newborn macroscopic immune spot is generated by integrated enzyme reaction
Point；
The quantity of S23, the statistics immunodotting, it is anti-to detect the tumor neogenetic based on the quantity of 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, the step S21 is into one
Step includes:
S211, it soaks Elispot plate at normal temperature using ethanol water, the Elispot is then cleaned using PBS
Plate；
S212 plus IFN γ antibody are to be coated with the Elispot plate, and then splice lid low temperature is incubated overnight, and then use PBS
It is sealed up after cleaning and closes fluid-tight and close the unbonded position of the IFN γ antibody, splice lid room temperature is incubated for；
The concentration of the peripheral blood mononuclear cells after pre-stimulation in S213, the adjustment serum-free cell culture medium
Afterwards, the corresponding tumor neogenetic antigen is then added and forms mixing serum-free cell culture medium；
S214, the mixing serum-free cell culture medium is added in the plate hole for the Elispot plate being coated with, is added
Cover board contains CO in room temperature2Incubator in cultivated.
In the method for vitro detection tumor neogenetic T cells with antigenic specificity of the present invention, the step S22 is into one
Step includes:
Then serum-free cell culture medium described in S221, reject is cleaned using PBS, then carried out using cleaning solution
Cleaning；
S222, the IFN γ that biotin coupling is then added identify that antibody, room temperature are incubated for；
S223, it is cleaned using cleaning solution, the horseradish peroxidase of coupling streptavidin, splice lid is then added
Carry out room temperature incubation；
S224, it is cleaned using cleaning solution, the substrate of horseradish peroxidase is then added, room temperature is protected from light, then adopts
It is sufficiently cleaned, is dried with cleaning solution.
Another technical solution adopted by the present invention to solve the technical problem thereof is that: it is anti-to construct a kind of vitro detection tumor neogenetic
The kit of former specific T-cells, comprising: tumor neogenetic antigen is coated with the Elispot plate of IFN γ antibody and enzyme-linked anti-
Answer reagent；The tumor neogenetic antigen includes the Antigenic Peptide that sequence is LSKENSLIIQFTSFVAV and sequence is
The Antigenic Peptide of KLVVVGAAGVGKSAL, the integrated enzyme reaction reagent include IFN γ identification antibody, the coupling chain of biotin coupling
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 solution, PBS, confining liquid, serum-free cell culture medium.
The invention further relates to a kind of tumor vaccines, new including the tumour for expanding tumor neogenetic T cells with antigenic specificity
Raw antigen, the tumor neogenetic antigen includes the Antigenic Peptide that sequence is LSKENSLIIQFTSFVAV and sequence is
The Antigenic Peptide of KLVVVGAAGVGKSAL.
The method and kit for implementing vitro detection tumor neogenetic T cells with antigenic specificity of the invention, pass through 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, low in cost.Implement tumor vaccine of the invention, can effectively expand tumor neogenetic T cells with antigenic specificity, and then effectively
Production for immunotherapy of tumors drug.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples, in attached drawing:
Fig. 1 is the vitro detection tumor neogenetic T cells with antigenic specificity of first embodiment according to the present invention
The flow chart of method；
Fig. 2 is the vitro detection tumor neogenetic T cells with antigenic specificity of second embodiment according to the present invention
The flow chart of method；
Fig. 3 is the Elispot figure 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 to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of 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.Particular technique or condition person are not specified in the following embodiments, according to text in the art
It offers described technology or conditions or is carried out according to product description.For the reagent concentration in following embodiments, processing temperature
Degree and processing time, it can be adjusted or be changed according to the actual situation by those skilled in the art.
Fig. 1 is the vitro detection tumor neogenetic T cells with antigenic specificity of first embodiment according to the present invention
The flow chart of method.As shown in Figure 1, in step sl, it is new that tumour is added in the cell culture fluid of peripheral blood mononuclear cells
Raw antigenic stimulus simultaneously carries out culture incubation.In one embodiment of the invention, it 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 entire antigen protein of gene mutation non-synonymous.In the present invention, it can use in this field
Any technology and methods known obtain the peripheral blood mononuclear cells, and use tumor neogenetic antigenic stimulus and carry out culture and incubate
It educates.
In step s 2, the tumor neogenetic of the tumor neogenetic antigen is identified using the detection of 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 generate newborn macroscopic immune spot by integrated enzyme reaction by the IFN γ antibody capture of cell peripheral
Point, the quantity by counting immunodotting can detect the quantity of the tumor neogenetic T cells with antigenic specificity.
Therefore firstly, the peptide fragment of the tumor neogenetic antigen in the present invention is the total incubation of peripheral blood mononuclear cells
In the process, by the antigen presenting cell in peripheral blood mononuclear cells such as dendritic cells and B cell be cut into naturally and itself
The antigen fragment that ajor histocompatibility molecule is consistent, submission give the tumor neogenetic T cells with antigenic specificity.Therefore, it is examined
The peptide fragment of the tumor neogenetic antigen of the tumor neogenetic T cells with antigenic specificity identification measured is nature presentation, and
It and include that the tumor neogenetic antigentic specificity can ensure that by the unpredictable epitope arrived of Bioinformatic methods in this way
The natural sex of epitope of T cell identification and comprehensive.
Secondly, can detecte 3x10 using IFN γ enzyme-linked immunospot assay5A specific T-cells in PBMC, because
This its sensibility is high, is conducive to the tumor neogenetic T cells with antigenic specificity for detecting low ratio.And IFN γ enzyme linked immunological
What spotting method detected is after identifying tumour antigen and the tumor neogenetic antigen specific T of energy secreting function cell factor IFN γ is thin
Born of the same parents are functional detection, directly react the immune response of tumor neogenetic T cells with antigenic specificity.
In addition, the good tumor neogenetic antigen of immunogenicity is that patient's cancer cell is different from the distinctive target spot of normal cell,
It can be used as tumor vaccine or for expanding tumor specific T cells applied in immunotherapy of tumors.
Fig. 2 is the vitro detection tumor neogenetic T cells with antigenic specificity of second embodiment according to the present invention
The flow chart of method.As shown in Fig. 2, in step sl, designing tumor neogenetic antigen using antigen prediction software netMHC4.?
In a preferred embodiment of the present invention, the tumor tissue in vitro of tumour patient can be taken to do genetic analysis, found out non-synonymous
Gene mutation site (including the point mutation due to nucleic acid, insertion or missing and the amino acid sequence that is formed), uses antigen
Forecasting software netMHC4 prediction includes whole candidate tumor neoantigens of gene mutation site.It is then soft using antigen prediction
Part netMHC4 is designed and synthesized based on the binding ability of itself and ajor histocompatibility molecule comprising 15-30 amino acid length
Long peptide is as tumor neogenetic antigen.After designing tumor neogenetic antigen, those skilled in the art can use the prior art
In any known technology and methods synthesis purity > 95% related neoplasms neoantigen.
In step s 2, it is anti-that the tumor neogenetic is added in the cell culture fluid for suspending the peripheral blood mononuclear cells
Original carries out pre-stimulation and is cultivated.In one embodiment of the invention, peripheral blood mononuclear cells is isolated from peripheral blood
(Peripheral blood mononuclear cells, PBMC) is suspended with serum-free cell culture medium, and tumor neogenetic is added
Antigenic Peptide (the 15-30 amino acid long peptide comprising mutational site) pre-stimulation, at 37 DEG C, 5%CO2Incubator co-cultures 2 days.At this
In embodiment, stimulation incubation time be can be adjusted according to the actual situation.
In step s3, the peripheral blood mononuclear cells after pre-stimulation is transferred to the appearance for being coated with IFN γ antibody
In device, the tumor neogenetic antigen is added and be incubated for 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 the actual situation.
In step s 4, the IFN γ of the tumor specific T cells secretion is led to by the IFN γ antibody capture of cell peripheral
It crosses integrated enzyme reaction and generates macroscopic immunodotting.
In step s 5, the quantity of the immunodotting is counted, with described swollen based on the detection of the quantity of the immunodotting
The quantity of tumor neoantigen specific T-cells.The quantity of the tumor neogenetic T cells with antigenic specificity reflects patient's peripheral blood pair
The immune response of nascent tumor is strong and weak.
Therefore firstly, the peptide fragment of the tumor neogenetic antigen in the present invention is the total incubation of peripheral blood mononuclear cells
In the process, by the antigen presenting cell in peripheral blood mononuclear cells such as dendritic cells and B cell be cut into naturally and itself
The antigen fragment that ajor histocompatibility molecule is consistent, submission give the tumor neogenetic T cells with antigenic specificity.Therefore, it is examined
The peptide fragment of the tumor neogenetic antigen of the tumor neogenetic T cells with antigenic specificity identification measured is nature presentation, and
It and include that the tumor neogenetic antigentic specificity can ensure that by the unpredictable epitope arrived of Bioinformatic methods in this way
The natural sex of epitope of T cell identification and comprehensive.
Secondly, can detecte 3x10 using IFN γ enzyme-linked immunospot assay5A specific T-cells in PBMC, because
This its sensibility is high, is conducive to the tumor neogenetic T cells with antigenic specificity for detecting low ratio.And IFN γ enzyme linked immunological
What spotting method detected is after identifying tumour antigen and the tumor neogenetic antigen specific T of energy secreting function cell factor IFN γ is thin
Born of the same parents are functional detection, directly react the immune response of tumor neogenetic T cells with antigenic specificity.Tumor neogenetic antigen-specific
The immune response of property T cell can indicate by macroscopic immunodotting, quickly and easily quantitative detection patient peripheral blood
The immune response of tumor neogenetic antigen is directed in the immunogenicity and tumour patient peripheral blood of middle tumor neogenetic antigen.
In addition, the good tumor neogenetic antigen of immunogenicity is that patient's cancer cell is different from the distinctive target spot of normal cell,
It can be used as tumor vaccine or for expanding tumor specific T cells applied in immunotherapy of tumors.
Also, tumor neogenetic antigen predicts and synthesizes that method is simple using Bioinformatic methods.Tumor neogenetic antigen is cut
It cuts and submission is passed in confluent monolayer cells in antigen and is naturally done, do not need the polymer for spending high cost synthesis MHC- Antigenic Peptide.
It below will be by way of examples in detail to the vitro detection tumour of third embodiment according to the present invention
The method of neoantigen specific T-cells is described further.
The prediction and synthesis of embodiment 1, tumor neogenetic antigen
It takes tumor tissue in vitro to do genome analysis, finds out gene mutation site non-synonymous (including the point due to nucleic acid
Mutation, the amino acid sequence be inserted into or lacked and formed), it is based on using antigen prediction software netMHC4 comprising described non-
The antigen in equivalent gene mutational site and the binding ability of corresponding ajor histocompatibility molecule selection binding ability are best
Antigenic peptide sequence is as candidate tumor neogenetic Antigenic Peptide.The binding ability of candidate tumor neogenetic Antigenic Peptide and corresponding MHC is logical
It crosses and reaches stable MHC- Antigenic Peptide in conjunction with the expression of required Antigenic Peptide minimum concentration.Concentration is lower, indicates Antigenic Peptide and MHC points
The combination of son is more stable, its antigen originality is stronger, can preferably stimulate the proliferation of specific T-cells.
Then itself and corresponding Main Tissues are selected 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 candidate tumor neogenetic Antigenic Peptide
The binding force of MHC molecule is answered to be apparently higher than the Antigenic Peptide sequence of the binding force of corresponding not mutated peptide (i.e. primary peptide) and MHC molecule
Column are used as the tumor neogenetic antigen.It determines that candidate tumor neogenetic Antigenic Peptide is the distinctive target spot of tumour cell, can stimulate
The specific immune response for generating tumour cell targeting, without the normal cell of the primary peptide of recognition expression.
Subordinate list 1 is the two tumor neogenetic Antigenic Peptides come out using netMHC4 software screening method and they correspond to primary peptide
Peptide sequence.As shown in subordinate list 2, the two tumor neogenetic Antigenic Peptides are all higher than corresponding primary with the binding force of corresponding MHC molecule
The binding force of peptide and MHC molecule indicates that the two mutation produce required tumor neogenetic antigen.
The amino acid sequence of tumor neogenetic antigen and its corresponding primary peptide that table 1 is predicted
Note: runic simultaneously adds the lower amino acid to draw lines to be the mutational site of tumor neogenetic antigen
The binding ability (Kd, nM*) of tumor neogenetic antigen and corresponding MHC that table 2 is predicted
*: potential tumor neoantigen peptide and the binding ability of corresponding MHC pass through in conjunction with the MHC- Antigenic Peptide that reaches stable
Required Antigenic Peptide minimum concentration indicates that concentration is lower, indicates that the combination of peptide and MHC molecule is more stable, potentially antigenic originality
It is stronger.
In the tumor neogenetic antigen that prediction includes mutational site, designing as shown in Table 1 includes tumor neogenetic Antigenic Peptide
The long peptide of 15-30 amino acid length, the tumor neogenetic Antigenic Peptide for delivering the biotech firm synthesis purity > 95% of peptide synthesis are spare.
2 tumor neogenetic Antigenic Peptide pre-stimulation of embodiment
With an example tumour patient when knowing informing, patient's peripheric venous blood 10mL is extracted with heparin sodium anticoagulant tube.
With lymphocyte separation medium (Fresenius Kabi Norge AS, LymphoprepTM) isolated by density-gradient centrifugation method
After peripheral blood mononuclear cells PBMC, suspended with fresh serum-free medium (AIM-V of Life Technology company),
Cell density is in 1-3x106/mL.In serum-free medium, above-mentioned tumor neogenetic Antigenic Peptide or corresponding primary peptide is added
(1-10 μ g/mL), at 37 DEG C, 5%CO236-48h is cultivated in incubator.Detect patient P BMC is to the immune response of primary peptide
In order to check whether that the immune response to tumor neogenetic antigen is targeting mutant nucleotide sequence, to determine tumor neogenetic antigen specific T
Cell only selectively targeted identification mutant cell, that is, cancer cell without kill normal cell.One is additionally incorporated without related peptide (ratio
Such as a peptide fragment of the envelope protein of inhibition of HIV) it is used as negative control, peptide is not added as blank control, for assessing in PBMC
The background level of immune response.
Embodiment 3 identifies the tumor neogenetic antigen of the tumor neogenetic antigen using the detection of IFN γ enzyme-linked immunospot assay
Specific T-cells (U-CyTech BV, CT230-PR5)
Operating process refers to kit service manual.Specific experiment process is as follows:
It is soaked Elispot plate 1 hour with 70% ethanol water in room temperature, after then cleaning 2 times using PBS, adds IFN γ
Coated antibody be coated with Elispot plate, 4 DEG C of splice lid overnight incubation, clean 3 times with PBS, seal up close fluid-tight close the IFN γ resist
The unbonded position of body, 37 DEG C of splice lid are incubated for 1 hour；
The PBMC concentration that adjustment pre-stimulation is crossed is 1-3x106It is anti-to be separately added into corresponding 1-10ug/mL tumor neogenetic by/ml
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
Such as a peptide fragment of the envelope protein of inhibition of HIV) serum-free cell culture medium and blank serum-free cell culture medium；Often
Hole adds on 100ul serum-free cell culture medium to the Elispot plate being coated with (i.e. every hole 1-3x105Cell, each Antigenic Peptide
Do 2-3 parallel hole), 37 DEG C of splice lid, 5%CO2Cultivate 16-24h；
Serum-free cell culture medium described in reject is first cleaned 2 times with PBS, then is washed 5 times with cleaning solution, and it is even that biotin is added
The IFN γ of connection identifies antibody, is incubated for 1 hour at 37 DEG C；
It is washed 5 times with cleaning solution, the horseradish peroxidase of coupling streptavidin is added, covered 37 DEG C and be incubated for 1 hour；
It is washed 5 times with cleaning solution, the substrate of horseradish peroxidase is added, room temperature is protected from light 25-50 minutes, sufficiently clear with clear water
The front and back sides of board-washing, dry.
Immunodotting number (as shown in Fig. 3) in hole is counted with Elispot plate analysis device, in conjunction with 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 identifies 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.
Two not mutated parent tumor peptides can be identified as shown in attached drawing 3-4, in the tumour patient peripheral blood in present case
And the immunocyte number for secreting IFN γ does not have notable difference with negative control, so primary peptide does not have effective stimulus tumour special
The ability of specific T cell, i.e., no immunogenicity.And it can identify two tumor neogenetic Antigenic Peptides and secreting function cell factor
The immunocyte number of IFN γ is apparently higher than for their corresponding not mutated primary peptides or negative control, especially for
The immunodotting of nascent peptide 1 clearly (attached drawing 3), shows that the two potential tumor neoantigens have immunogenicity, can pierce
Swash T cell specific in patient's peripheral blood, can be used for preparing tumor specific T cells or tumor vaccine is exempted from applied to tumour
Epidemic disease treatment.
In conclusion the invention discloses use in IFN γ elispot assay detection tumour patient peripheral blood to swell
The reaction of tumor neoantigen specific T-cells, determines the immunogenicity of tumor neogenetic antigen, looks for for the immunotherapy of tumors of patient
Effective target spot, the production applied to clinical immunotherapy drug.
The present invention further discloses candidate tumor neogenetic antigen and is predicted and synthesized using Bioinformatic methods, method letter
It is single.The cutting of neoantigen and submission are passed in confluent monolayer cells in antigen to be naturally done, and does not need to spend high cost synthesis MHC-
The polymer of Antigenic Peptide.The immune response of neoantigen specific T-cells can indicate by macroscopic immunodotting, very
Intuitively reflect in the immunogenicity and tumour patient peripheral blood of tumor neogenetic antigen for the immune anti-of tumor neogenetic antigen
It answers.The good tumor neogenetic antigen of immunogenicity is that patient's cancer cell is different from the distinctive target spot of normal cell, be can be used as swollen
Tumor vaccine is applied in the production of immunotherapy of tumors drug for expanding tumor specific T cells.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. a kind of method of the vitro detection tumor neogenetic T cells with antigenic specificity of non-diagnostic purpose characterized by comprising
S1, tumor neogenetic antigenic stimulus is added in the cell culture fluid of peripheral blood mononuclear cells and carries out culture incubation；
S2, identify that the tumor neogenetic antigen specific T of the tumor neogenetic antigen is thin using the detection of IFN γ enzyme-linked immunospot assay
Born of the same parents；
Wherein the tumor neogenetic antigen includes Antigenic Peptide that sequence is LSKENSLIIQFTSFVAV and sequence is
The Antigenic Peptide of KLVVVGAAGVGKSAL.
2. the method for the vitro detection tumor neogenetic T cells with antigenic specificity of non-diagnostic purpose according to claim 1,
It is characterized in that, the step S1 includes:
S11, the tumor neogenetic antigen is designed using antigen prediction software netMHC4；
S12, it the tumor neogenetic antigen is added in the cell culture fluid for suspending the peripheral blood mononuclear cells is pierced in advance
Swash and is cultivated.
3. the method for the vitro detection tumor neogenetic T cells with antigenic specificity of non-diagnostic purpose according to claim 2,
It is characterized in that, the step S11 includes:
S111, tumor tissue in vitro is taken to do genetic analysis, to find out gene mutation site non-synonymous；
S112, using antigen prediction software netMHC4 based on the antigen comprising the gene mutation site non-synonymous with it is corresponding
The best antigenic peptide sequence of the binding ability selection binding ability of ajor histocompatibility molecule；
S113, itself and corresponding Main Tissues phase are selected 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 as the tumor neogenetic antigen.
4. the method for the vitro detection tumor neogenetic T cells with antigenic specificity of non-diagnostic purpose according to claim 2,
It is characterized in that, the step S12 includes:
S121, the peripheral blood mononuclear cells is suspended in serum-free cell culture medium, and it is anti-that the tumor neogenetic is added
Original carries out pre-stimulation；
S122, contain CO in room temperature2Incubator in cultivated.
5. the method for the vitro detection tumor neogenetic T cells with antigenic specificity of non-diagnostic purpose according to claim 4,
It is characterized in that, the step S2 includes
S21, the peripheral blood mononuclear cells after pre-stimulation is transferred in the container for being coated with IFN γ antibody, institute is added
It states tumor neogenetic antigen to be incubated for, so that by the tumor neogenetic antigentic specificity after the tumor neogenetic antigenic stimulus
T cell secretes IFN γ；
IFN γ described in S22, the IFN γ antibody capture, and newborn macroscopic immunodotting is generated by integrated enzyme reaction；
The quantity of S23, the statistics immunodotting, it is special to detect the tumor neogenetic antigen based on the quantity of the immunodotting
The quantity of specific T cell.
6. the method for the vitro detection tumor neogenetic T cells with antigenic specificity of non-diagnostic purpose according to claim 5,
It is characterized in that, the step S21 further comprises:
S211, it soaks Elispot plate at normal temperature using ethanol water, the Elispot plate is then cleaned using PBS；
S212 plus IFN γ antibody are to be coated with the Elispot plate, and then splice lid low temperature is incubated overnight, and are then cleaned with PBS
It seals up afterwards and closes fluid-tight and close the unbonded position of the IFN γ antibody, splice lid room temperature is incubated for；
After the concentration of the peripheral blood mononuclear cells after pre-stimulation in S213, the adjustment serum-free cell culture medium,
The corresponding tumor neogenetic antigen is then added and forms mixing serum-free cell culture medium；
S214, the mixing serum-free cell culture medium is added in the plate hole for the Elispot plate being coated with, covers plate
Contain CO in room temperature2Incubator in cultivated.
7. the method for the vitro detection tumor neogenetic T cells with antigenic specificity of non-diagnostic purpose according to claim 6,
It is characterized in that, the step S22 further comprises:
Then serum-free cell culture medium described in S221, reject is cleaned using PBS, then cleaned using cleaning solution；
S222, the IFN γ that biotin coupling is then added identify that antibody, room temperature are incubated for；
S223, it is cleaned using cleaning solution, the horseradish peroxidase of coupling streptavidin is then added, splice lid carries out
Room temperature is incubated for；
S224, it is cleaned using cleaning solution, the substrate of horseradish peroxidase is then added, room temperature is protected from light, then using clear
Washing lotion is sufficiently cleaned, and is dried.
8. a kind of kit of vitro detection tumor neogenetic T cells with antigenic specificity characterized by comprising tumor neogenetic is anti-
Elispot plate and integrated enzyme reaction reagent former, that be coated with IFN γ antibody；The tumor neogenetic antigen includes that sequence is
The Antigenic Peptide and sequence of LSKENSLIIQFTSFVAV is the Antigenic Peptide of KLVVVGAAGVGKSAL, and the integrated enzyme reaction reagent includes
The IFN γ of biotin coupling identifies the bottom of antibody, the horseradish peroxidase for being coupled streptavidin, horseradish peroxidase
Object and auxiliary agent.
9. a kind of tumor vaccine, which is characterized in that anti-including the tumor neogenetic for expanding tumor neogenetic T cells with antigenic specificity
Original, the tumor neogenetic antigen includes the Antigenic Peptide that sequence is LSKENSLIIQFTSFVAV and sequence is KLVVVGAAGVGKSAL
Antigenic Peptide.
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| WO2016145578A1 (en) | 2015-03-13 | 2016-09-22 | Syz Cell Therapy Co. | Methods of cancer treatment using activated t cells |
| WO2019183924A1 (en) | 2018-03-30 | 2019-10-03 | Syz Cell Therapy Co. | Improved multiple antigen specific cell therapy methods |
| WO2019196088A1 (en) * | 2018-04-13 | 2019-10-17 | Syz Cell Therapy Co. | Methods of obtaining tumor-specific t cell receptors |
| CN110408657B (en) * | 2018-09-30 | 2021-03-16 | 北京鼎成肽源生物技术有限公司 | Construction method of AFFT1 cells |
| CN109337930B (en) * | 2018-09-30 | 2020-10-30 | 北京鼎成肽源生物技术有限公司 | AFFT1 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 |
| CN109136282A (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 HAFFT1 cell |
| CN110514845B (en) * | 2019-08-22 | 2022-09-27 | 深圳新合睿恩生物医疗科技有限公司 | A kind of tumor neoantigen immunogenicity detection method and detection platform |
| CN110567861B (en) * | 2019-09-09 | 2021-12-21 | 浙江普罗亭健康科技有限公司 | Kit for screening antigenic peptide with immunogenicity based on mass flow detection technology and detection method |
| CN111690609B (en) * | 2020-06-30 | 2022-04-05 | 深圳裕泰抗原科技有限公司 | Method for testing immunogenicity of neoantigen |
| CN111909992B (en) * | 2020-08-13 | 2023-10-31 | 深圳裕泰抗原科技有限公司 | Method for simultaneously detecting immunogenicity of neoantigen and neoantigen-specific TCR (TCR) |
| CN112666358A (en) * | 2021-01-14 | 2021-04-16 | 深圳裕康医学检验实验室 | Method for detecting antigen immunogenicity by using peripheral blood |
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