METHOD AND PLATFORM FOR DETECTING IMMUNOGENICITY OF TUMOR NEOANTIGEN

Abstract

A method and a platform for detecting an immunogenicity of a tumor neoantigen are provided. Specifically, the detection method includes the following steps: culturing human peripheral blood monocytes ex vivo for 13 days, adding an antigenic peptide fragment of human influenza virus and stimulating and activating cytokines, antigenic peptides, and immunoadjuvants during the 13 days, and finally conducting enzyme-linked immunospot (ELISPOT) chromogenic reaction and instrument-based scanning, counting, and analysis to detect the immunogenicity of tumor neoantigen. An application of the detection method and platform in biomedicine is provided. Compared with the prior art, the detection method and platform have advantages and characteristics of a short detection period, high convenience, low consumption of experimental cells, and low detection cost. Therefore, the detection method and platform can be used for ex vivo high-throughput assay for the immunogenicity of the tumor neoantigen.

Claims

1. A method for detecting an immunogenicity of a tumor neoantigen, comprising the following steps: (1) day 1: adding human interleukin (IL)-4 into a Roswell Park Memorial Institute (RPMI) complete medium per well to allow a concentration of the IL-4 to be 20 ng/ml to obtain a first culture solution, adding granulocyte-macrophage colony-stimulating factor (GM-CSF) into the first culture solution to allow a concentration of the GM-CSF to be 100 ng/ml to obtain a second culture solution, culturing 510.sup.5 thawed human peripheral blood monocytes (PBMCs) in a 24-well plate containing 500 l of the second culture solution per well to obtain a second culture solution containing PBMCs, and incubating the second culture solution containing PBMCs in a cell incubator for 48 h at 37 C.; (2) day 3: adding an antigenic peptide fragment of a first human influenza virus into the second culture solution containing PBMCs per well with human IL-7 and polyinosinic-polycytidylic acid (Poly I:C) to obtain a third culture solution containing PBMCs per well, allowing a concentration of the human IL-7 to be 5 ng/ml and a concentration of the Poly I:C to be 20 g/ml in the third culture solution containing PBMCs per well, wherein the first antigenic peptide fragment in the third culture solution per well has a first plurality of concentrations; (3) day 5: adding 500 l of the RPMI complete medium supplemented with 10 ng/ml of the human IL-7, 10 ng/ml of IL-15, and 40 U/ml of human IL-2 into each well of the 24-well plate to obtain a fourth culture solution containing PBMCs per well, allowing a total volume of the fourth culture solution containing PBMCs per well to be 1 ml; (4) day 8: replacing 500 l of the fourth culture solution per well with 500 l of the RPMI complete medium supplemented with 10 ng/ml of the human IL-7, 10 ng/ml of the IL-15, and 40 U/ml of the human IL-2; (5) day 11: collecting non-adherent cells from the 24-well plate, washing the non-adherent cells with the RPMI complete medium twice, and culturing the non-adherent cells in 500 l of the RPMI complete medium without any cytokine and stimulating factor for 48 h, to obtain human PBMC-derived T lymphocytes; and (6) day 13: taking out a plurality of enzyme-linked immunospot (ELISPOT) strips from a kit, adding each of the ELISPOT wells with 200 l of the RPMI complete medium supplemented with 210.sup.5 cells of the thawed human PBMCs and 110.sup.5 cells of the human PBMC-derived T lymphocytes, and a second antigenic peptide fragment of the human influenza virus to obtain a fifth culture solution per ELISPOT well, wherein the second antigenic peptide fragment of the fifth culture solution per ELISPOT strip has a second plurality of concentrations; mixing the fifth culture solution per ELISPOT well uniformly, incubating the fifth culture solution per ELISPOT well in the cell incubator for 22 h at 37 C., and subsequently conducting an ELISPOT assay on the the fifth culture solution per ELISPOT well.

2. The method according to claim 1, wherein the RPMI complete medium in steps (1) and (6) is supplemented with 10% (v/v) heat inactivated human serum AB, 100 U/ml penicillin/streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM 2-[4-(2-hydroxyerhyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2.5 g/ml amphotericin B, and 0.05 mM 2-mercaptoethanol.

3. The method according to claim 1, wherein the first antigenic peptide fragment of the human influenza virus in step (2) is a sequence fragment at positions 58-66 of a sequence of Influenza Matrix M1, and the first plurality of concentrations include 0.25 ng/ml, 1 ng/ml, and 2 ng/ml.

4. The method according to claim 1, wherein the second antigenic peptide fragment of the human influenza virus in step (6) is a sequence fragment at positions 58-66 of a sequence of Influenza Matrix M1, and the second plurality of concentrations include 0.25 ng/ml, 1 ng/ml, and 2 ng/ml.

5. The method according to claim 1, wherein the method is applied in constructing a high-throughput verification system of the immunogenicity of the tumor neoantigen.

6. The method according to claim 1, wherein the method is applied in biomedicine.

7. A platform for detecting an immunogenicity of a tumor neoantigen, comprising the following components: (1) an RPMI complete medium; (2) cytokines and stimulating factors; (3) polyinosinic-polycytidylic acid (Poly I:C); (4) an antigenic peptide fragment of a human influenza virus; and (5) an ELISPOT Reader System.

8. The platform according to claim 7, wherein the cytokines and the stimulating factors comprise human interleukin (IL)-2, human IL-4, human IL-7, human IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the antigenic peptide fragment of the human influenza virus is a sequence fragment at positions 58-66 of a sequence of Influenza Matrix M1.

9. The platform according to claim 7, wherein the RPMI complete medium is supplemented with 10% (v/v) heat inactivated human serum AB, 100 U/ml penicillin/streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM 2-[4-(2-hydroxyerhyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2.5 g/ml amphotericin B, and 0.05 mM 2-mercaptoethanol.

10. The platform according to claim 7, wherein the platform is applied in constructing a high-throughput verification system of the immunogenicity of the tumor neoantigen.

11. The method according to claim 2, wherein the method is applied in constructing a high-throughput verification system of the immunogenicity of the tumor neoantigen.

12. The method according to claim 3, wherein the method is applied in constructing a high-throughput verification system of the immunogenicity of the tumor neoantigen.

13. The method according to claim 4, wherein the method is applied in constructing a high-throughput verification system of the immunogenicity of the tumor neoantigen.

14. The method according to claim 2, wherein the method is applied in biomedicine.

15. The method according to claim 3, wherein the method is applied in biomedicine.

16. The method according to claim 4, wherein the method is applied in biomedicine.

17. The platform according to claim 8, wherein the platform is applied in constructing a high-throughput verification system of the immunogenicity of the tumor neoantigen.

18. The platform according to claim 9, wherein the platform is applied in constructing a high-throughput verification system of the immunogenicity of the tumor neoantigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows a standard operating procedure of the enzyme-linked immunospot (ELISPOT) platform of the present invention. The standard operating procedure takes 14 days in total, including 13 days for ex vivo culture of human peripheral blood monocytes (PBMCs) (during which a stimulation process of cytokines, antigenic peptides, and immunoadjuvants is carried out) and the last day for ELISPOT chromogenic reaction, counting, and analysis.

[0031] FIG. 2 shows the results of an enzyme-linked immunospot (ELISPOT) platform building experiment. The results are obtained by an ELISPOT assay detection after a reaction of equivalent human peripheral blood monocytes (PBMCs) with different concentrations of a peptide fragment of an influenza virus according to the standard operating procedure. The number of spots finally formed in each well of a Petri dish increases with the increase of the additional amount of the antigenic peptide fragment of the human influenza virus (Influenza Matrix M1), showing a good positive correlation. Thus, a standard curve can be determined. Phytohemagglutinin (PHA) is used as a positive control.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The following describes the present invention in detail through specific embodiments, but it should be noted that the following embodiments are merely exemplary. The present invention can also be implemented or applied through other different embodiments. Based on different viewpoints and applications, various modifications or amendments can be made to the embodiments without departing from the spirit of the present invention.

[0033] To enable those skilled in the art to understand the features and effects of the present invention, the following generally describes and defines the terms and dictions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention pertains. In addition to the specific methods, devices, and materials used herein, any method, device, and material equivalent or similar to those described in the embodiments of the present invention can be used to implement the present invention by those skilled in the art according to conventional knowledge and the description of the present invention.

[0034] The materials, reagents, etc. used in the following embodiments are all commercially available, unless otherwise specified.

EMBODIMENTS

[0035]

TABLE-US-00001 Supplier Reagent used in the experiment Human peripheral blood monocytes (PBMCs) Cellular Technology Limited RPMI 1640 Sigma-Aldrich Human serum AB Gemini Bio-Products Penicillin/streptomycin with glutamine Thermo Fisher Scientific Sodium pyruvate Thermo Fisher Scientific Non-essential amino acids Thermo Fisher Scientific 2-[4-(2-Hydroxyerhyl)-1- Thermo Fisher Scientific piperazinyl]ethanesulfonic acid (HEPES) Amphotericin B Sigma-Aldrich 2-Mercaptoethanol Thermo Fisher Scientific Human interleukin-4 (IL-4) GenScript Granulocyte-macrophage colony-stimulating PeproTech factor (GM-CSF) Antigenic peptide fragment of human influenza Cellular Technology virus (Influenza Matrix M1) Limited Human interleukin-7 (IL-7) PeproTech Polyinosinic-polycytidylic acid (Poly I:C) Tocris Human interleukin-15 (IL-15) GenScript Human interleukin-2 (IL-2) GenScript Human IFN- Single-Color ELISPOT Kit Cellular Technology Limited Device Immunospot S6 ENTRY Analyzer Cellular Technology Limited

Embodiment 1: Ex Vivo Culture of Human Peripheral Blood Monocytes (PBMCs)

[0036] (1) Day 1: 510.sup.5 thawed human PBMCs were cultured in a 24-well plate containing 500 l of an RPMI complete medium (supplemented with 10% (v/v) heat inactivated human serum AB, 100 U/ml penicillin/streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM 2-[4-(2-hydroxyerhyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2.5 g/mL amphotericin B, and 0.05 mM 2-mercaptoethanol) in each well. The medium had been added 20 ng/ml of human interleukin (IL)-4 and 100 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) were further added and incubated in a cell incubator for 48 h at 37 C.

[0037] (2) Day 3: different concentrations of antigenic peptide fragment of human influenza virus (sequence fragments 58-66 of Influenza Matrix M1; concentrations: 0.25 ng/ml, 1 ng/ml, and 2 ng/ml), 5 ng/ml human IL-7, and 20 g/ml polyinosinic-polycytidylic acid (Poly I:C) were added into the cell culture medium.

[0038] (3) Day 5: 500 l of an RPMI complete medium (supplemented with 10 ng/ml of human IL-7, 10 ng/ml of IL-15, and 40 U/ml of human IL-2) was added into each well of the 24-well plate to reach a total volume of 1 ml (a final concentration of each of human IL-7 and IL-15 was 5 ng/ml; a final concentration of human IL-2 was 20 U/ml).

[0039] (4) Day 8: the cell culture medium was changed every three days. 500 l of the stale culture medium was removed, and 500 l of a fresh RPMI complete medium (supplemented with 10 ng/ml of human IL-7, 10 ng/ml of IL-15, and 40 U/ml of human IL-2) was added.

[0040] (5) Day 11: all non-adherent cells were collected from the 24-well plate, washed with the RPMI complete medium twice, and cultured in 500 l of an RPMI complete medium without any cytokine and stimulating factor for 48 h, to obtain human PBMC-derived T lymphocytes.

[0041] (6) Day 13: an enzyme-linked immunospot (ELISPOT) strip was taken out from a kit and mounted on the strip plate of the kit. After placing for warming, 200 l of an RPMI complete medium (supplemented with 210.sup.5 thawed human PBMCs and 110.sup.5 human PBMC-derived T lymphocytes) and different concentrations of an antigenic peptide fragment of a human influenza virus (a sequence fragment at positions 58-66 of a sequence of Influenza Matrix M1; the different concentrations includes 0.25 ng/ml, 1 ng/ml, and 2 ng/ml) were added, mixed uniformly, incubated in the cell incubator for 22 h at 37 C., and subsequently subjected to an ELISPOT assay.

Embodiment 2: ELISPOT Chromogenic Reaction

[0042] Day 14: The ELISPOT strip plate placed in the cell incubator for 22 h was taken out. The cells and cell culture medium therein were discarded. The remaining steps were implemented with reference to the operating instructions of the Human IFN- Single-Color ELISPOT Kit:

[0043] (1) washing the ELISPOT strip plate twice with phosphate-buffered saline (PBS) and 0.05% Tween-PBS (the volume was 200 l per well), respectively;

[0044] (2) adding anti-human IFN- detection solution (the volume was 80 l per well), mixing uniformly, and keeping for 2 h at room temperature;

[0045] (3) washing the ELISPOT strip plate thrice with 0.05% Tween-PBS (the volume was 200 l per well)

[0046] (4) adding tertiary detection solution (the volume was 80 l per well), mixing uniformly, and keeping for 30 min at room temperature;

[0047] (5) washing the ELISPOT strip plate twice with 0.05% Tween-PBS and distilled water (the volume was 200 l per well), respectively;

[0048] (6) adding chromogen solution (the volume was 80 l per well), mixing uniformly, and placing in the dark for 15 min at room temperature;

[0049] (7) adding sufficient tap water to stop the chromogenic reaction, taking out the ELISPOT strip from the strip plate, and drying at room temperature; and

[0050] (8) scanning and analyzing the number of immunospots in each well of the Petri dish by Immunospot S6 ENTRY Analyzer.

[0051] The specific implementations and embodiments of the present invention are described in detail above, but the present invention is not limited to the above implementations and embodiments. Within the knowledge of a person of ordinary skill in the art, various modifications can further be made without departing from the spirit of the present invention.