Method for screening new antigen polypeptide sequence of malignant solid tumor

Abstract

The invention relates to the technical field of new antigen screening, and particularly discloses a malignant solid tumor new antigen polypeptide sequence screening method, which comprises the following steps: obtaining a new antigen prediction result based on HLA class I molecule binding; screening to obtain candidate new antigen related mutations; extracting a corresponding polypeptide sequence from a neoantigen prediction result to obtain an MHC class I potential neoantigen polypeptide set; generating all 15 peptide sequences by taking the mutation site as the center, and predicting the binding capacity of the 15 peptide sequences with MHC II molecules to obtain an MHC II potential new antigen polypeptide sequence set; adopting a maximum boundary expansion algorithm to obtain a long peptide sequence; synthesizing a polypeptide sequence to obtain a candidate polypeptide sequence; and evaluating the immunogenicity of the candidate polypeptide sequence to obtain a new antigen polypeptide sequence. According to the invention, the binding characteristics of the MHC class I molecules and the MHC class II molecules are considered at the same time, and the efficiency and the accuracy of new antigen screening can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of new antigen screening, and in particular to a method for screening new antigen polypeptide sequences of malignant solid tumors. Background Art

[0002] In recent years, with the rapid development of gene sequencing technology, artificial intelligence and bioinformatics, the field of personalized cancer treatment has ushered in major breakthroughs. Among them, the development of cancer vaccines based on tumor neoantigens has become a research hotspot. Neoantigens are polypeptide sequences produced by tumor cell-specific mutations and recognized by the immune system. As tumor-specific targets, they have great potential in the development of personalized tumor vaccines and T cell adoptive therapy.

[0003] However, the top-ranked neoantigens currently selected based on the prediction results did not show the expected immunogenicity in subsequent experimental verification. The reason is that most current methods still focus on short peptides (8-12 amino acids) that bind to MHC class I, and the direct use of these short peptides will have the following problems: 1) Short peptides can bind to the MHC of non-professional APCs, which lack the secondary signaling mechanism for complete T cell activation, resulting in poor T cell response or immune tolerance. 2) Short peptides will lead to HLA (human leukocyte antigen) type restrictions, because HLA is highly diverse in the population, and HLA is variable in the population. Mutations occurring in HLA (especially LOH) may lead to the loss of its antigen presentation function. 3) Short peptides are more easily digested by enzymes and eliminated from the body more quickly.

[0004] In recent years, many studies have confirmed that long peptides with a length of 15 to 31 amino acids can effectively enhance the immune response of CD4+ and CD8+ T cells because they can cover a wider range of HLA types and contain multiple epitope peptides, thereby inducing a more comprehensive and lasting immune response. However, the method of generating such long peptides based on existing new antigen prediction technology lacks systematicity and effectiveness. Summary of the invention

[0005] The present invention aims to solve the above problems. To this end, the present invention provides a method for screening new antigen polypeptide sequences for malignant solid tumors, which adopts a dual molecular docking screening mechanism and considers the binding characteristics of MHC class I and class II molecules at the same time, which can effectively improve the efficiency and accuracy of new antigen screening and provide reliable technical support for the development of personalized tumor vaccines.

[0006] The present invention provides a method for screening new antigen polypeptide sequences of malignant solid tumors, and the technical scheme adopted is as follows: comprising the following steps: Step 1: Obtain new antigen prediction results based on HLA class I molecule binding; Step 2: Based on the neoantigen prediction results, candidate neoantigen-related mutations are screened; Step 3: According to the candidate neoantigen-related mutations, the corresponding peptide sequences are extracted from the neoantigen prediction results to obtain a set of MHC class I potential neoantigen peptides; Step 4: According to the candidate neoantigen-associated mutation, a protein sequence at the mutation site is obtained, wherein the protein sequence is a polypeptide sequence of no less than 15 amino acids; Step 5: Based on the protein sequence, a sliding window method is used to generate all 15-peptide sequences centered on the mutation site; the binding ability of each 15-peptide sequence to the MHC class II molecule is predicted, and the 15-peptide sequences with a percentile rank of eluted ligand prediction score less than or equal to 1 are screened to obtain a set of MHC class II potential new antigen polypeptide sequences; Step 6: Based on the MHC class I potential new antigen peptide set and the MHC class II potential new antigen peptide sequence set, a maximum boundary extension algorithm is used to obtain a long peptide sequence; Step 7: Based on the long peptide sequence, synthesize the peptide sequence, screen the peptide sequence that meets the quality control requirements, and obtain the candidate peptide sequence; Step 8: Evaluate the immunogenicity of the candidate polypeptide sequence, and obtain the new antigen polypeptide sequence based on the evaluation results.

[0007] Furthermore, the neoantigen prediction results include mutations, peptide sequences and their corresponding prediction scores as well as the patient's HLA typing results.

[0008] Furthermore, the neoantigen prediction result is a prediction result obtained by a neoantigen prediction method for malignant solid tumors.

[0009] Further, in step 2, the screening criteria for selecting new antigen-related mutations are: satisfying any of the following criteria: Standard 1: The core peptide segment is ranked within the top N1 in the neoantigen prediction results. In the neoantigen prediction results, the polypeptide sequence with the highest score corresponding to the mutation is its core peptide segment; and the expression level of the mutant gene TPM is greater than or equal to 0.5; and in the RNA sequencing data, the sequencing depth of the mutation site is less than 100 layers, or the sequencing depth of the mutation site is greater than or equal to 100 layers, and the mutation frequency is greater than 0; Criterion 2: The mutated gene has been confirmed as a known driver gene.

[0010] Furthermore, in step 3, For candidate neoantigen-associated mutations that meet criterion 1, extract the corresponding peptide sequences with scores higher than N2 in the neoantigen prediction results; For candidate neoantigen-associated mutations that meet criterion 2, all corresponding polypeptide sequences in the neoantigen prediction results are extracted.

[0011] Furthermore, in step 4, For a single amino acid substitution mutation, the polypeptide sequence including the mutation site and no more than 14 amino acids upstream and downstream thereof is extracted with the mutation site as the center to obtain the protein sequence; For insertion / deletion mutations, the extracted polypeptide sequence is no more than 14 amino acids upstream of the mutation site to the stop codon, and the polypeptide sequence containing the core peptide segment is selected to obtain the protein sequence. In the new antigen prediction results, the polypeptide sequence with the highest score corresponding to the mutation is its core peptide segment.

[0012] Furthermore, if there is no corresponding MHC class II molecule for the candidate neoantigen-associated mutation, the set of MHC class II potential neoantigen polypeptide sequences corresponding to the candidate neoantigen-associated mutation is empty.

[0013] Furthermore, in step 6, for each candidate neoantigen-associated mutation, a MHC class I long peptide sequence is constructed using a maximum boundary extension algorithm based on the MHC class I potential neoantigen peptide set; the 15 peptide sequences in the MHC class II potential neoantigen peptide sequence set are docked with the MHC class I long peptide sequence in sequence to obtain a long peptide sequence.

[0014] Furthermore, quality control requirements include: white or off-white powder; theoretical molecular weight of ±1.0Da; purity greater than or equal to 95.0%; acetic acid less than or equal to 20%, TFA less than or equal to 1.0%; acetonitrile less than or equal to 0.041%, N,N-dimethylformamide less than or equal to 0.088%, ether less than or equal to 0.5%, dichloromethane less than or equal to 0.06%, methanol less than or equal to 0.3%; heavy metals less than or equal to 10ppm; moisture less than or equal to 10%; bacterial endotoxin less than or equal to 5EU / mg; microbial TAMC less than or equal to 200cfu / g, TYMC less than or equal to 50cfu / g.

[0015] Furthermore, in step 8, Elispot technology is used to verify whether the candidate polypeptide sequence can activate T cells and induce them to secrete cytokines, and evaluate its immunogenicity.

[0016] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention adopts a dual molecular docking screening mechanism, taking into account the binding characteristics of MHC class I and class II molecules at the same time, significantly improving the accuracy of new antigen prediction. The present invention adopts a maximum boundary expansion algorithm, integrating the MHC class I potential new antigen polypeptide set and the MHC class II potential new antigen polypeptide sequence set to achieve intelligent optimization of candidate sequences.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is a flow chart of the method provided by the present invention.

[0020] Figure 2 It is a comparison chart of the results of immune response detection of the new antigen polypeptide provided by the present invention.

[0021] Figure 3 It is a statistical graph of the results of the immune response detection of the new antigen polypeptide provided by the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0023] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0024] Combine the following Figures 1 to 3 The present invention is further described in detail, and a method for screening a new antigen polypeptide sequence of a malignant solid tumor of the present invention is described: In this embodiment, Figure 1As shown, a method for screening new antigen polypeptide sequences for malignant solid tumors is provided, comprising the following steps: Step 1: Obtain new antigen prediction results based on HLA class I molecule binding.

[0025] In this embodiment, the neoantigen prediction result is a prediction result obtained by a neoantigen prediction method for malignant solid tumors. The neoantigen prediction result includes mutations, polypeptide sequences and their corresponding prediction scores, and the HLA typing results of the patient.

[0026] Specifically, the new antigen prediction results of this embodiment are the prediction results of a new antigen prediction method and system for malignant solid tumors disclosed in the Chinese invention patent with application publication number CN119446264A, and the subsequent screening process is also carried out based on the experimental results of the invention.

[0027] Step 2: Based on the neoantigen prediction results, candidate neoantigen-related mutations are screened.

[0028] The screening criteria for selecting new antigen-related mutations are: meeting any of the following criteria: Criteria 1: Conditions 1 to 3 are met simultaneously.

[0029] Condition 1: The core peptide is ranked within the top N1 in the neoantigen prediction results, and in this embodiment, N1 is 50. In the neoantigen prediction results, the polypeptide sequences are divided by mutation, and the polypeptide sequence with the highest score corresponding to each mutation is taken as its core peptide.

[0030] Condition 2: The expression level TPM of the mutant gene is greater than or equal to 0.5.

[0031] Condition 3: In RNA sequencing data, when the sequencing depth of the mutation site is less than 100 layers, or when the sequencing depth of the mutation site is greater than or equal to 100 layers, the mutation frequency is greater than 0.

[0032] Criterion 2: The mutated gene has been confirmed as a known driver gene.

[0033] If the mutated gene has been confirmed as a known driver gene, it does not need to meet all three conditions of criterion 1 and can be directly identified as a candidate neoantigen-associated mutation.

[0034] According to the above screening criteria, a total of 32 candidate neoantigen mutations were identified. These mutations include: 29 mutations with core peptide scores ranked 1, 2, 5, 7, 9, 10, 12, 13, 14, 16, 17, 18, 19, 22, 27, 28, 30, 31, 32, 33, 38, 39, 41, 43, 46, 47, 48, 49, 50; and 3 mutations occurring in known driver genes SOCS1, EGFR and TP53, as shown in Table 1.

[0035] Table 1 Candidate neoantigen-related mutations Step 3: Based on the candidate neoantigen-related mutations, the corresponding peptide sequences are extracted from the neoantigen prediction results to obtain a set of MHC class I potential neoantigen peptides.

[0036] The extracted peptide sequences need to meet the following conditions: For candidate neoantigen-associated mutations that meet criterion 1, polypeptide sequences whose corresponding scores in the neoantigen prediction results (prediction scores in Table 2) are higher than N2 are extracted. N2 is an artificially selected score threshold, and in this embodiment, N2 is 0.5.

[0037] For candidate neoantigen-associated mutations that meet criterion 2, the mutations in known driver genes, whose corresponding polypeptide sequences are not restricted by a score of 0.5, are extracted, and all corresponding polypeptide sequences in the neoantigen prediction results are extracted.

[0038] Step 4: Based on the candidate neoantigen-associated mutation, obtain the protein sequence where the mutation site is located, and the protein sequence is a polypeptide sequence of not less than 15 amino acids.

[0039] Specifically, in step 4, For a mutation of a single amino acid substitution, the polypeptide sequence including the mutation site and no more than 14 amino acids upstream and downstream thereof is extracted with the mutation site as the center to obtain the protein sequence. Preferably, 14 amino acids are taken from the upstream and downstream, in which case the length of the protein sequence is at most 29 amino acids.

[0040] For insertion / deletion mutations, the extracted polypeptide sequence is no more than 14 amino acids upstream of the mutation site to the stop codon, and the polypeptide sequence containing the core peptide segment is selected to obtain the protein sequence. Preferably, 14 amino acids are taken upstream.

[0041] Step 5: Based on the protein sequence, a sliding window method was used to generate all possible 15-peptide sequences centered on the mutation site. NetMHCIIpan 4.3 software was used to predict the binding ability of each 15-peptide sequence to MHC class II molecules. The strength of the binding ability was evaluated by the percentile rank of the eluted ligand prediction score. The 15-peptide sequences with a percentile rank of the eluted ligand prediction score less than or equal to 1 were screened to obtain a set of MHC class II potential neoantigen peptide sequences. See Table 2 for details.

[0042] Table 2 Candidate neoantigen-related mutation results If there is no corresponding MHC class II molecule for the candidate neoantigen-associated mutation, the set of MHC class II potential neoantigen polypeptide sequences corresponding to the candidate neoantigen-associated mutation is empty. In this embodiment, multiple candidate neoantigen-associated mutations such as MAMDC4, ITPKB, SLC25A22, IRF4, and SBF1 do not have corresponding MHC class II molecules.

[0043] Step 6: Based on the MHC class I potential new antigen peptide set and the MHC class II potential new antigen peptide sequence set, a maximum boundary extension algorithm is used to obtain a long peptide sequence; The specific process is as follows: for each candidate neoantigen-related mutation, based on the MHC class I potential neoantigen peptide set, the maximum boundary extension algorithm is used to construct the MHC class I long peptide sequence; the 15 peptide sequences in the MHC class II potential neoantigen peptide sequence set are docked with the MHC class I long peptide sequence in turn to obtain the long peptide sequence. See Table 2 for details.

[0044] Step 7: Based on the long peptide sequence, synthesize the polypeptide sequence, screen the polypeptide sequences that meet the quality control requirements, and obtain the candidate polypeptide sequences.

[0045] In this embodiment, the quality control requirements that need to be met simultaneously include: white or off-white powder; theoretical molecular weight of ±1.0Da; purity greater than or equal to 95.0%; acetic acid less than or equal to 20%, TFA less than or equal to 1.0%; acetonitrile less than or equal to 0.041%, N,N-dimethylamide less than or equal to 0.088%, ether less than or equal to 0.5%, dichloromethane less than or equal to 0.06%, methanol less than or equal to 0.3%; heavy metals less than or equal to 10ppm, heavy metals include cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel; moisture less than or equal to 10%; bacterial endotoxin less than or equal to 5EU / mg; microbial TAMC less than or equal to 200cfu / g, TYMC less than or equal to 50cfu / g.

[0046] As shown in Table 2 , 12 long peptide sequences failed to be synthesized, and finally the number of candidate peptide sequences that met the above quality control requirements was 20, which were marked as P002-P009, P011-P017, P0019-P022 and P024.

[0047] Step 8: Evaluate the immunogenicity of the candidate peptide sequence, and obtain the new antigen peptide sequence based on the evaluation results. Use Elispot technology to verify whether the candidate peptide sequence can activate T cells and induce them to secrete cytokines, and evaluate its immunogenicity.

[0048] This example uses Elispot technology to verify whether the 20 candidate polypeptide sequences successfully synthesized in step 7 can activate T cells and induce them to secrete cytokines (IFN-γ), thereby evaluating their immune response. The verification results are as follows Figure 2 , Figure 3 The negative / positive control and positive judgment criteria of the ELISPOT experiment are as follows: 1. Negative control group: Only DMSO (dimethyl sulfoxide) and normal saline (NS) were used as stimuli, and PBMCs (peripheral blood mononuclear cells) without pretreatment with mixed peptides were used as negative control group.

[0049] 2. Positive control group: Only DMSO and saline were used as stimuli, and PBMCs pre-treated with the patient's personalized neoantigen peptide mixture were used as the positive control group.

[0050] 3. Positive judgment criteria: 1) Weakly positive: The stimulation index (SI value) is greater than 1.5, and the average count of the stimulant Spot is higher than the positive control. 2) Positive: The stimulation index (SI value) is greater than 2, and the average count of the stimulant Spot is higher than the positive control.

[0051] SI value setting: the average Spot count value of the experimental group (including the positive control group) divided by the average Spot count value of the negative control group.

[0052] Figure 2 In Chinese, Mix-peptide means mixed polypeptide, and PHA means phytohemagglutinin in Chinese.

[0053] This method targets all HLA subtypes of patients, contains multiple epitope peptides, and ensures that the generated long peptide sequence covers the minimum epitope of CD8+T cells. Peptide sequences that can be recognized by CD4+ T cells and bind to MHC class II molecules will be evaluated, and sequences with high immunogenicity will be integrated into the long peptide sequence to generate the final new antigen peptide sequence. Such sequences can activate both CD4+ and CD8+ T cell subsets. This dual activation mechanism not only enhances the intensity of the immune response, but also prolongs the duration of the immune response through the support of CD4+ T cells. This method combines bioinformatics prediction, peptide synthesis feasibility assessment, experimental verification, and immunological principles to systematically screen out peptide sequences containing multiple epitopes and with the best immunogenicity.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening new antigen polypeptide sequences for malignant solid tumors, characterized in that: The following steps are involved: Step 1: Obtain new antigen prediction results based on HLA class I molecule binding; Step 2: Based on the neoantigen prediction results, candidate neoantigen-related mutations are screened; Step 3: According to the candidate neoantigen-related mutations, the corresponding peptide sequences are extracted from the neoantigen prediction results to obtain the MHC class I potential neoantigen peptide set; Step 4: According to the candidate neoantigen-associated mutation, a protein sequence at the mutation site is obtained, wherein the protein sequence is a polypeptide sequence of no less than 15 amino acids; Step 5: Based on the protein sequence, a sliding window method is used to generate all 15-peptide sequences centered on the mutation site; the binding ability of each 15-peptide sequence to the MHC class II molecule is predicted, and the 15-peptide sequences with a percentile rank of eluted ligand prediction score less than or equal to 1 are screened to obtain a set of MHC class II potential new antigen polypeptide sequences; Step 6: Based on the MHC class I potential new antigen peptide set and the MHC class II potential new antigen peptide sequence set, a maximum boundary extension algorithm is used to obtain a long peptide sequence; Step 7: Based on the long peptide sequence, synthesize the peptide sequence, screen the peptide sequence that meets the quality control requirements, and obtain the candidate peptide sequence; Step 8: Evaluate the immunogenicity of the candidate polypeptide sequence, and obtain the new antigen polypeptide sequence based on the evaluation results.

2. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: The neoantigen prediction results include mutations, peptide sequences and their corresponding prediction scores, as well as the patient's HLA typing results.

3. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: The neoantigen prediction result is a prediction result obtained by a neoantigen prediction method for malignant solid tumors.

4. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: In step 2, the screening criteria for selecting new antigen-related mutations are: meeting any of the following criteria: Standard 1: The core peptide segment is ranked within the top N1 in the neoantigen prediction results. In the neoantigen prediction results, the polypeptide sequence with the highest score corresponding to the mutation is its core peptide segment; and the expression level of the mutant gene TPM is greater than or equal to 0.5; and in the RNA sequencing data, the sequencing depth of the mutation site is less than 100 layers, or the sequencing depth of the mutation site is greater than or equal to 100 layers, and the mutation frequency is greater than 0; Criterion 2: The mutated gene has been confirmed as a known driver gene.

5. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 4, characterized in that: In step 3, For candidate neoantigen-associated mutations that meet criterion 1, extract the corresponding peptide sequences with scores higher than N2 in the neoantigen prediction results; For candidate neoantigen-associated mutations that meet criterion 2, all corresponding polypeptide sequences in the neoantigen prediction results are extracted.

6. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: In step 4, For a single amino acid substitution mutation, the polypeptide sequence including the mutation site and no more than 14 amino acids upstream and downstream thereof is extracted with the mutation site as the center to obtain the protein sequence; For insertion / deletion mutations, the extracted polypeptide sequence is no more than 14 amino acids upstream of the mutation site to the stop codon, and the polypeptide sequence containing the core peptide segment is selected to obtain the protein sequence. In the new antigen prediction results, the polypeptide sequence with the highest score corresponding to the mutation is its core peptide segment.

7. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: If there is no corresponding MHC class II molecule for the candidate neoantigen-associated mutation, the set of MHC class II potential neoantigen polypeptide sequences corresponding to the candidate neoantigen-associated mutation is empty.

8. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: In step 6, for each candidate neoantigen-associated mutation, the MHC class I long peptide sequence is constructed using the maximum boundary extension algorithm based on the MHC class I potential neoantigen peptide set; the 15 peptide sequences in the MHC class II potential neoantigen peptide sequence set are docked with the MHC class I long peptide sequence in turn to obtain the long peptide sequence.

9. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: Quality control requirements include: white or off-white powder; theoretical molecular weight of ±1.0Da; purity greater than or equal to 95.0%; acetic acid less than or equal to 20%, TFA less than or equal to 1.0%; acetonitrile less than or equal to 0.041%, N,N-dimethylformamide less than or equal to 0.088%, ether less than or equal to 0.5%, dichloromethane less than or equal to 0.06%, methanol less than or equal to 0.3%; heavy metals less than or equal to 10ppm; moisture less than or equal to 10%; bacterial endotoxin less than or equal to 5EU / mg; microbial TAMC less than or equal to 200cfu / g, TYMC less than or equal to 50cfu / g.

10. The method for screening new antigen polypeptide sequences for malignant solid tumors according to claim 1, characterized in that: In step 8, Elispot technology is used to verify whether the candidate peptide sequence can activate T cells and induce them to secrete cytokines, and its immunogenicity is evaluated.

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