Method for testing immunogenicity of neoantigen

Abstract

The invention discloses a method for testing neoantigen immunogenicity, which can effectively screen neoantigens with higher immunogenicity through bioinformatics analysis and in-vitro function verification. The method comprises the following steps: firstly, screening an antigen which is remarkably different from a self-proteome through sequence alignment, so as to improve the specificity and effectiveness of immunotherapy, and secondly, culturing a mononuclear cell in a culture medium containing a granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4), so as to rapidly induce the mononuclear cell to differentiate into an immature dendritic cell. And under the action of subsequent TNF-alpha, IL-1beta, IL-6 and PGE2, the dendritic cells are further mature, so that the dendritic cells have efficient antigen presentation capability.

Description

Technical Field

[0001] The present invention belongs to the field of immunogenicity testing, and in particular, relates to a method for testing the immunogenicity of a neoantigen. Background Art

[0002] Neoantigens are specific mutated protein fragments produced by tumor cells that are different from the self-proteome in normal cells and can therefore serve as targets for immune system recognition. Since neoantigens only exist in tumor cells, the immune response against these antigens is highly tumor-specific, making neoantigens an important target for tumor immunotherapy (such as personalized tumor vaccines and T cell therapy).

[0003] However, assessing the immunogenicity of neoantigens faces certain challenges. Genetic mutations in tumor cells may produce a large number of mutant proteins, but not all of these mutations are able to trigger an effective T cell immune response. Most mutations generate peptides that are very similar to the self-proteome, which cannot be recognized by the immune system as "non-self" or produce sufficient stimulatory signals to activate T cells. In addition, although some mutations can generate an immune response, the immune response they induce is too weak or short-lived to effectively eliminate tumor cells.

[0004] The immunogenicity of different neoantigens varies depending on multiple factors, including the similarity to the self-proteome, the structural importance of the mutation location, and whether these antigens can be effectively processed and presented by antigen-presenting cells (such as dendritic cells). Some neoantigens may be located in areas of the tumor that are not easily exposed or very close to self-antigens, making them indistinguishable or ineffective for the immune system to recognize. In addition, the tumor microenvironment is often immunosuppressive, which means that even if neoantigens can stimulate an immune response, T cell function may be limited or ineffective due to inhibitory factors in the tumor microenvironment.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for testing the immunogenicity of neoantigens, thereby solving the problems raised in the above-mentioned background technology.

[0007] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0008] A method for testing the immunogenicity of a neoantigen comprises: step (1): separating and extracting peripheral blood mononuclear cells (PBMC);

[0009] Step (2): isolating CD8-positive T cells or CD4-positive T cells from the PBMCs;

[0010] Step (3): culturing the monocytes in a culture medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce the monocytes to differentiate into immature dendritic cells;

[0011] Step (4): adding tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) to the immature dendritic cells during the culture process to obtain mature dendritic cells;

[0012] Step (5): incubating the mature dendritic cells with the screened neoantigen polypeptides to load the dendritic cells with the neoantigen polypeptides;

[0013] Step (6): co-incubating the mature dendritic cells loaded with the neoantigen polypeptide with the separated CD8-positive T cells or CD4-positive T cells in proportion;

[0014] Step (7): After the incubation period is over, the incubated cells are collected, and the specific immune response of T cells to the neoantigen polypeptide is detected to evaluate the immunogenicity of the neoantigen polypeptide.

[0015] Optionally, the step of isolating CD8-positive T cells or CD4-positive T cells from the PBMC in step (2) is:

[0016] The suspension containing PBMCs was mixed with magnetic beads with CD8- or CD4-specific antibodies attached to their surfaces;

[0017] Fixing the magnetic beads in a magnetic rack, removing unbound cells, and obtaining magnetic beads bound to CD8-positive or CD4-positive T cells;

[0018] Elute the bound T cells from the beads using elution buffer, collect and resuspend in culture medium

[0019] Optionally, during the incubation process in step (6), culture medium containing cytokines IL-2, IL-7 and IL-15 is added to the co-cultured cells every 24 hours to maintain and promote the survival and proliferation of T cells.

[0020] Optionally, the neoantigen polypeptide is a neoantigen polypeptide selected by bioinformatics analysis and structure prediction, which is significantly different from the self-proteome and has high immunogenicity, wherein the bioinformatics analysis includes the following steps:

[0021] The amino acid sequence of the neoantigen peptide was compared with the protein sequence in the self-proteome database using the BLAST algorithm. Then, the comparison score was calculated to evaluate the similarity between the neoantigen peptide and the self-proteome. Finally, the peptides with similarity below the preset threshold were selected as candidate neoantigens with potential immunogenicity.

[0022] Optionally, in step (6), the concentration of the antigen polypeptide is 10 μg / ml, and the antigen polypeptide is incubated with the dendritic cells for 2-4 hours.

[0023] Optionally, in step (6), the ratio of mature dendritic cells to separated CD8-positive T cells or CD4-positive T cells is 1:10.

[0024] Optionally, in step (7), the step of detecting the specific immune response of T cells to the neoantigen polypeptide and evaluating the immunogenicity of the neoantigen polypeptide is:

[0025] The incubated T cell samples were transferred to the pre-treated Elispot plate and the neoantigen peptide stimulators co-incubated with T cells were added to each well;

[0026] The Elispot plate was placed in an incubator at 37°C and 5% CO2 for 24 hours to allow T cells to be activated and secrete cytokines under antigen stimulation;

[0027] After the incubation, the cells were removed and the Elispot plate was washed with PBS to remove unbound cells and antibodies. Next, capture antibodies were added to each well to capture FN-γ secreted by activated T cells. Then, detection antibodies and enzyme substrates were added to visualize the spots on the plate through a color reaction.

[0028] The number of spots in each well was counted, and the immunogenicity of the neoantigen peptides was evaluated by calculating the frequency of specific T cell responses stimulated by each neoantigen peptide.

[0029] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art. Of course, any product implementing the present invention does not necessarily need to achieve all the advantages described below at the same time:

[0030] 1. Through bioinformatics analysis combined with in vitro functional verification, this method can effectively screen new antigens with high immunogenicity. By screening antigens with significant differences from the self-proteome through sequence alignment, the specificity and effectiveness of immunotherapy are improved. Secondly, by culturing monocytes in a culture medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), monocytes are rapidly induced to differentiate into immature dendritic cells. Under the subsequent action of TNF-α, IL-1β, IL-6 and PGE2, dendritic cells further mature, giving them efficient antigen presentation capabilities.

[0031] 2. Measure the specific response of T cells to neoantigens through Elispot or other immunoassay methods. The Elispot method can accurately measure the immune factors such as interferon γ (IFN-γ) released by T cells, which represents the activation of T cells. This method can effectively distinguish the immunogenicity of different neoantigens.

[0032] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings described below are only some embodiments. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0034] In the figure:

[0035] Figure 1 This is the Elispot test result of the embodiment of the present invention.

[0036] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0037] The present invention will now be described in further detail with reference to the accompanying drawings.

[0038] See also Figure 1 As shown, in this embodiment, a method for testing the immunogenicity of a neoantigen is provided, comprising the steps of (1): isolating and extracting peripheral blood mononuclear cells (PBMC);

[0039] Step (2): isolating CD8-positive T cells or CD4-positive T cells from the PBMCs;

[0040] Step (3): culturing the monocytes in a culture medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce the monocytes to differentiate into immature dendritic cells;

[0041] Step (4): adding tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) to the immature dendritic cells during the culture process to obtain mature dendritic cells;

[0042] Step (5): incubating the mature dendritic cells with the screened neoantigen polypeptides to load the dendritic cells with the neoantigen polypeptides;

[0043] Step (6): co-incubating the mature dendritic cells loaded with the neoantigen polypeptide with the separated CD8-positive T cells or CD4-positive T cells in proportion;

[0044] Step (7): After the incubation period is over, the incubated cells are collected, and the specific immune response of T cells to the neoantigen polypeptide is detected to evaluate the immunogenicity of the neoantigen polypeptide.

[0045] In this embodiment, the step of isolating CD8-positive T cells or CD4-positive T cells from the PBMC in step (2) is:

[0046] The suspension containing PBMCs was mixed with magnetic beads with CD8- or CD4-specific antibodies attached to their surfaces;

[0047] Fixing the magnetic beads in a magnetic rack, removing unbound cells, and obtaining magnetic beads bound to CD8-positive or CD4-positive T cells;

[0048] Elute the bound T cells from the beads using elution buffer, collect and resuspend in culture medium

[0049] In this embodiment, during the incubation in step (6), a culture medium containing cytokines IL-2, IL-7 and IL-15 is added to the co-cultured cells every 24 hours to maintain and promote the survival and proliferation of T cells.

[0050] In this embodiment, the neoantigen polypeptide is a neoantigen polypeptide selected by bioinformatics analysis and structure prediction, which is significantly different from the self-proteome and has high immunogenicity, wherein the bioinformatics analysis includes the following steps:

[0051] The amino acid sequence of the neoantigen peptide is compared with the protein sequence in the self-proteome database using the BLAST algorithm. Then, the comparison score is calculated to evaluate the similarity between the neoantigen peptide and the self-proteome. Finally, the peptides with similarity lower than the preset threshold are selected as candidate neoantigens with potential immunogenicity. The expression is: Among them, S total is the total score used to assess the similarity between the neoantigen and the self-proteome. Lower values ​​indicate lower similarity between the two and thus higher immunogenic potential. N is the number of amino acids in the neoantigen peptide. This is the length of the neoantigen sequence and represents each amino acid position in the alignment. i is the position index, representing the iiith amino acid position in the neoantigen sequence. Each amino acid position is independently compared for the alignment score. H(i) is the match score in the BLOSUM or PAM score matrix, which quantifies the match score between the neoantigen peptide and the corresponding amino acid in the self-proteome. For example, the score in the BLOSUM62 matrix represents the conservation of amino acid substitutions. P(i) is the position-specific match frequency, which represents the frequency of a certain amino acid substitution in the self-proteome. It is used to adjust the weight of matches that occur at highly conserved positions. M(i) is the conservative substitution score, which is used to calculate the conservation of amino acid substitutions in the neoantigen peptide and the self-proteome. Highly conservative substitutions generally represent lower immunogenicity. L(i) is the score of the local sequence context, which is corrected based on the context of the amino acid site, taking into account the influence of neighboring amino acids on the substitution. For example, if adjacent amino acid substitutions occur at key structural sites, the score will be reduced. G(i) is the mismatch score, which is used to quantify the situation where the amino acids in the neoantigen and self proteome are completely different. A higher mismatch score increases the immunogenicity score. D(i) is the mismatch penalty, indicating that amino acid mismatches in important structural regions will be heavily penalized (for example, substitutions in key functional domains). σ gap (i) is the missing penalty factor, which is used to penalize the missing (gap) in the neoantigen peptide and the self-proteome sequence. Missing usually has a serious impact on the sequence structure, so a penalty needs to be added. α, β, γ are weight coefficients used to adjust the impact of different components. They can be adjusted according to specific comparison requirements. α is the weight of the matching component. β is the weight of the conservative replacement component. γ is the weight of the unmatched component. δ is the weight of the missing penalty term. is an exponential function of the missing penalty, indicating that as the number of missing values ​​increases, the impact of missing values ​​on the total score increases exponentially.

[0052] In this embodiment, the concentration of the antigen polypeptide in step (6) is 10 μg / ml, and the antigen polypeptide is incubated with the dendritic cells for 2-4 hours.

[0053] In this embodiment, the ratio of mature dendritic cells to separated CD8-positive T cells or CD4-positive T cells in step (6) is 1:10.

[0054] In this embodiment, the step (7) of detecting the specific immune response of T cells to the neoantigen polypeptide and evaluating the immunogenicity of the neoantigen polypeptide is as follows:

[0055] The incubated T cell samples were transferred to the pre-treated Elispot plate and the neoantigen peptide stimulators co-incubated with T cells were added to each well;

[0056] The Elispot plate was placed in an incubator at 37°C and 5% CO2 for 24 hours to allow T cells to be activated and secrete cytokines under antigen stimulation;

[0057] After the incubation, the cells were removed and the Elispot plate was washed with PBS to remove unbound cells and antibodies. Next, capture antibodies were added to each well to capture FN-γ secreted by activated T cells. Then, detection antibodies and enzyme substrates were added to visualize the spots on the plate through a color reaction.

[0058] The number of spots in each well was counted, and the immunogenicity of the neoantigen peptides was evaluated by calculating the frequency of specific T cell responses stimulated by each neoantigen peptide.

[0059] Embodiment 1:

[0060] Step 1: Isolation and extraction of peripheral blood mononuclear cells (PBMC);

[0061] Step 2: CD8-positive T cells or CD4-positive T cells are separated from the PBMCs, and then the suspension containing the PBMCs is mixed with magnetic beads having CD8- or CD4-specific antibodies attached to the surface; then, the magnetic beads are fixed in a magnetic rack, unbound cells are removed, and magnetic beads bound to CD8-positive or CD4-positive T cells are obtained; finally, the bound T cells are eluted from the magnetic beads using an elution buffer, and the cells are collected and resuspended in a culture medium.

[0062] Step 3: culturing the monocytes in a culture medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce the monocytes to differentiate into immature dendritic cells;

[0063] Step 4: adding tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) to the immature dendritic cells during the culture process to obtain mature dendritic cells;

[0064] Step 5: incubating the mature dendritic cells with the screened neoantigen polypeptides to load the dendritic cells with the neoantigen polypeptides, wherein the concentration of the neoantigen polypeptides is 10 μg / ml, and incubating the dendritic cells with the neoantigen polypeptides for 4 hours;

[0065] Step 6: The mature dendritic cells loaded with neoantigen peptides were co-incubated with the isolated CD8-positive T cells or CD4-positive T cells at a ratio of 1:10. During the incubation process, the culture medium containing cytokines IL-2, IL-7 and IL-15 was added to the co-cultured cells every 24 hours to maintain and promote the survival and proliferation of T cells;

[0066] Step 7: After the incubation period, the incubated cells are collected, and the specific immune response of T cells to the neoantigen polypeptide is detected to evaluate the immunogenicity of the neoantigen polypeptide.

[0067] Laying method:

[0068] In the Elispot detection step, the incubated T cell sample is transferred to the pre-treated Elispot plate. The specific plating method is as follows:

[0069] Orifice plate layout:

[0070] 100,000 T cells / well and 10,000 DC cells / well loaded with neoantigen peptides were added to each well.

[0071] Positive control: PHA (phytohemagglutinin) was used as a stimulator to ensure that T cells could be activated.

[0072] Negative control: DC cells not loaded with neoantigen peptides were added to ensure that T cells were not activated.

[0073] Experimental group: DC cells loaded with different neoantigen peptides were added.

[0074] Specific layout:

[0075] T1+DC+#1(neoantigen 1)

[0076] T2+DC+#2(neoantigen 2)

[0077] T3+DC+PHA (positive control)

[0078] T4+DC+neg (negative control)

[0079] T5+Blank well (no cells)

[0080] Sequence Listing: The present invention has 5 different neoantigen polypeptides, and the sequence information is as follows:

[0081] No. Neoantigen Peptide Sequence Source Tumor Type Expected Immunogenicity

[0082]

[0083] Experimental results:

[0084] In the Elispot assay, the capture antibody used is an antibody against interferon gamma (IFN-γ) to detect the secretion of cytokines after T cells are activated. Through the color reaction, spots are formed at the bottom of the Elispot plate, representing each T cell that secretes IFN-γ.

[0085] Positive control (PHA) well: number of spots = 200 (positive)

[0086] Negative control: number of spots = 5 (negative)

[0087] Neoantigen #1: Number of spots = 120 (positive)

[0088] Neoantigen #2: Number of spots = 80 (positive)

[0089] Neoantigen #3: Number of spots = 130 (positive)

[0090] Neoantigen #4: Number of spots = 10 (negative)

[0091] Neoantigen #5: Number of spots = 150 (positive)

[0092] Result analysis:

[0093] Through the results of the Elispot experiment, it can be clearly observed that the neoantigen peptides #1, #2, #3, and #5 showed strong immunogenicity, showing significant T cell response frequency and higher cytokine (such as IFN-γ) secretion levels, which indicates that these peptides can effectively activate the body's immune system and induce specific immune responses. In contrast, the response of peptide #4 was relatively weak, as shown by a lower frequency of T cell response and a significantly weakened cytokine secretion level, indicating that its immunogenicity was low and it may not be able to effectively activate the immune system or induce a sufficient immune response.

[0094] Based on these data, it is speculated that neoantigens #1, #2, #3, and #5 have strong immunostimulatory potential and can effectively induce immune responses, so they have good potential vaccine development value. The immunogenicity of these neoantigens may come from their unique antigenic epitope structure, which can be efficiently recognized by T cell receptors and activate specific immune responses. This makes them particularly important in vaccine development, especially in the development of personalized vaccines for diseases such as tumors or viral infections, where they are expected to become ideal target antigens.

[0095] In addition, the strength of the immune response to these neoantigens may also provide a reference for further optimization. By screening and optimizing immune adjuvants, delivery systems, or combining other immune enhancement strategies, the vaccine efficacy of these neoantigens can be further improved, thereby enhancing the preventive or therapeutic effects of the vaccine. This also provides new ideas and directions for future vaccine design, especially in improving the specificity and effectiveness of vaccines and reducing side effects, and has broad application prospects.

[0096] The present invention is not limited to the above-mentioned embodiments. Anyone should be aware that any structural changes made under the enlightenment of the present invention, and any technical solutions that are the same or similar to the present invention, fall within the protection scope of the present invention. The technology, shape, and structural parts not described in detail in the present invention are all well-known technologies.

Claims

1. A method for testing the immunogenicity of a neoantigen, characterized in that: The following steps are involved: Step (1): separation and extraction of peripheral blood mononuclear cells (PBMC); Step (2): isolating CD8-positive T cells or CD4-positive T cells from the PBMCs; Step (3): culturing the monocytes in a culture medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce the monocytes to differentiate into immature dendritic cells; Step (4): adding tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) to the immature dendritic cells during the culture process to obtain mature dendritic cells; Step (5): incubating the mature dendritic cells with the screened neoantigen polypeptides to load the dendritic cells with the neoantigen polypeptides; Step (6): co-incubating the mature dendritic cells loaded with the neoantigen polypeptide with the separated CD8-positive T cells or CD4-positive T cells in proportion; Step (7): After the incubation period is over, the incubated cells are collected, and the specific immune response of T cells to the neoantigen polypeptide is detected to evaluate the immunogenicity of the neoantigen polypeptide.

2. The method for testing the immunogenicity of a neoantigen according to claim 1, characterized in that: The step of isolating CD8 positive T cells or CD4 positive T cells from the PBMC in step (2) is: The suspension containing PBMCs was mixed with magnetic beads with CD8- or CD4-specific antibodies attached to their surfaces; Fixing the magnetic beads in a magnetic rack, removing unbound cells, and obtaining magnetic beads bound to CD8-positive or CD4-positive T cells; Bound T cells were eluted from the beads using elution buffer, collected and resuspended in culture medium.

3. The method for testing the immunogenicity of a neoantigen according to claim 1, characterized in that: During the incubation process in step (6), culture medium containing cytokines IL-2, IL-7 and IL-15 is added to the co-cultured cells every 24 hours to maintain and promote the survival and proliferation of T cells.

4. The method for testing the immunogenicity of a neoantigen according to claim 1, characterized in that: The neoantigen polypeptide is selected through bioinformatics analysis and structural prediction, and has significant differences from the self-proteome and high immunogenicity, wherein the bioinformatics analysis includes the following steps: The amino acid sequence of the neoantigen peptide was compared with the protein sequence in the self-proteome database using the BLAST algorithm. Then, the comparison score was calculated to evaluate the similarity between the neoantigen peptide and the self-proteome. Finally, the peptides with similarity below the preset threshold were selected as candidate neoantigens with potential immunogenicity.

5. The method for testing the immunogenicity of a neoantigen according to claim 1, characterized in that: In step (6), the concentration of the antigen polypeptide is 10 μg / ml, and the antigen polypeptide is incubated with the dendritic cells for 2-4 hours.

6. The method for testing the immunogenicity of a neoantigen according to claim 1, characterized in that: In step (6), the ratio of mature dendritic cells to separated CD8-positive T cells or CD4-positive T cells is 1:

10.

7. The method for testing the immunogenicity of a neoantigen according to claim 1, characterized in that: In step (7), the step of detecting the specific immune response of T cells to the neoantigen polypeptide and evaluating the immunogenicity of the neoantigen polypeptide is as follows: The incubated T cell samples were transferred to the pre-treated Elispot plate and the neoantigen peptide stimulators co-incubated with T cells were added to each well; The Elispot plate was placed in an incubator at 37°C and 5% CO2 for 24 hours to allow T cells to be activated and secrete cytokines under antigen stimulation; After the incubation, the cells were removed and the Elispot plate was washed with PBS to remove unbound cells and antibodies. Next, capture antibodies were added to each well to capture FN-γ secreted by activated T cells. Then, detection antibodies and enzyme substrates were added to visualize the spots on the plate through a color reaction. The number of spots in each well was counted, and the immunogenicity of the neoantigen peptides was evaluated by calculating the frequency of specific T cell responses stimulated by each neoantigen peptide.

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