Tumor antigen screening method based on HLA typing

Abstract

The invention discloses a tumor antigen screening method based on HLA (human leukocyte antigen) typing. According to the method disclosed by the invention, on the basis of biosignal screening, antigen presentation and T cell stimulation immune screening are carried out through human body antigen presenting cells (APC). The immortalized B cells are used as antigen presenting cells and are long-acting and stable, APC cells of different HLA types are respectively set for antigen screening by referring to high-frequency HLA type arrangement characteristics, the coverage is wide, and the specificity is high; according to the method disclosed by the invention, rapid antigen screening can be carried out on a high-frequency antigen of a population queue or a personalized antigen of a single sample, and the high-immunogenicity antigen screened by the method disclosed by the invention can be used for tumor vaccine or TCR-T construction, so that tumor neoantigen screening can be carried out more rapidly and accurately by the method disclosed by the invention; the screened target spot can be used as a tumor treatment target spot applied to tumor vaccines, CAR-T or TCR-T cell therapy and the like.

Description

Technical Field

[0001] The present invention relates to a method for screening tumor antigens based on HLA typing, belonging to the field of biotechnology. Background Art

[0002] Tumor neoantigens are antigens unique to tumors and not present in normal human tissues, and can be used as potential targets for tumor treatment. Most of the previous methods for antigen screening did not consider HLA typing, were simple and crude, and lacked rigorous verification of truly simulating the in vivo tumor immune process. In addition, most current tumor antigens refer to antigens derived from mutations. For tumors with a low mutation burden, their clinical application prospects are often limited. With the continuous development of high-throughput sequencing technology, the sequencing depth has gradually increased, the understanding of tumor neoantigens has become more and more complete, and the sources of tumor antigens have also expanded to gene fusion, alternative splicing, and protein modification, etc., greatly enriching the acquisition ways of tumor neoantigens and providing the possibility for screening specific and broad-spectrum tumor neoantigens urgently needed clinically.

[0003] Most previous studies used dendritic cells (DCs) for antigen screening and immune verification experiments in tumor antigen screening. However, due to disadvantages such as a small number (less than 3% in peripheral blood PBMC), a short survival period (the longest survival period is 2 weeks), and the inability to be immortalized, dendritic cells limit their application in tumor antigen screening.

[0004] Most traditional rapid antigen detection methods did not consider the HLA (human leucocyte antigen) typing of the immune cells (donors) used in the detection, and their immune verification lacked pertinence, and most of the obtained results were false positives.

[0005] In addition, the previous screening of single antigens by the method of peptide pulsing was difficult to meet the requirement of high-throughput screening of a large number of current neoantigens. Therefore, on this basis, researchers explored the feasibility of using the method of electroporating a mixed antigen mRNA sequence. However, DC cells are sensitive to the state and have extremely poor transfection tolerance. Most cells die within a short time after electroporation and cannot efficiently present antigens, seriously hindering the current R & D process of cell therapy.

[0006] And the co-culture of previous antigen-presenting cells (APCs) and T cells took a long time, with a 2-week antigen-specific T cell acquisition cycle, seriously blocking the subsequent preparation process of cell therapy drugs and unable to meet the treatment needs of advanced cancer patients. Existing laboratories or companies need to complete the screening as soon as possible and then prepare when preparing tumor vaccines or CAR-T or TCR-T. The existing experimental methods seriously delay the industrial or scientific research process.

[0007] Specific HLA typing selectively presents high-affinity antigen peptides that match HLA, and the peptide-HLA complex is exposed on the cell membrane surface, enabling tumor vaccines or TCR-T cell therapies to exert their effects. According to current data, the high-frequency HLA types among Chinese cancer patients are mainly HLA-A typing, and the high-frequency rankings are HLA-A*11:01 (20.893%), HLA-A*24:02 (15.545%), HLA-A*02:01 (12.036%), HLA-A*02:07 (8.418%), HLA-A*33:03 (8.229%) in sequence. The rest are supplemented by HLA-B and HLA-C subtypes (such as Figure 1 shown). Given the diverse and widespread HLA typing types, existing in vitro antigen screening methods are difficult to verify the immunogenicity of tumor neoantigens with a comprehensive, accurate, long-term and stable antigen verification system.

[0008] As an APC, B cells have advantages that traditional antigen-presenting cells do not have: ① B cells are the most effective antigen-presenting cells in the case of very low antigen concentration; ② B cells can be amplified: in a suitable culture system, with the addition of stimulants, the number of B cells can be amplified in large quantities in a short time to exert their effects; ③ B cells can be immortalized to ensure the repeated verification of single results; ④ B cells have antigen presentation markers, which can be quickly achieved through flow cytometry detection; ⑤ In the secondary immune response, B cells are the most important antigen-presenting cells.

[0009] Therefore, if a tumor neoantigen screening strategy based on the antigen-presenting function of B cells is developed, it can screen tumor neoantigens more quickly and accurately. The screened targets can be used as tumor treatment targets for applications such as tumor vaccines, CAR-T, or TCR-T cell therapies. Summary of the Invention

[0010] The object of the present invention is: aiming at the deficiencies of the prior art, the present invention provides a tumor antigen screening method based on HLA typing. The method of the present invention pre-checks through B cell surface markers to accelerate the screening process and greatly makes up for the deficiencies of previous antigen screening.

[0011] To achieve the above object, the present invention provides a tumor antigen screening method based on HLA typing, which is characterized by including the following steps:

[0012] Step 1: Obtain the peripheral blood of healthy people with different HLA typings, reserve B lymphocytes and T lymphocytes and freeze them, select B lymphocytes with different HLA typings for immortalization, and preserve and freeze the cells according to different HLA typings;

[0013] Step 2. Obtain tumor candidate antigens and detect their immunogenic presentation potential: Obtain high-throughput sequencing data from tumor samples obtained by surgical resection or puncture of patients. Perform potential antigen prediction according to the algorithm to obtain tumor candidate antigens. After synthesizing the candidate antigens into polypeptides, perform peptide loading, or use the method of electrotransferring the antigen mRNA sequence to introduce it into B cells. Subsequently, use anti-human CD86 + fluorescent antibody to detect the proportion of B lymphocytes; compared with the control group, the proportion of CD86+ B cells is significantly increased, that is, the candidate antigen has immunogenicity and can be presented by APCs;

[0014] Step 3. Detect the immune activation ability of tumor candidate antigens: Use the co-culture method of antigen-loaded APC cells and T cells for immunogenicity verification; subsequently, use enzyme-linked immunosorbent assay (ELISA) or ELISpot to detect the IFN-γ secretion of T cells.

[0015] Preferably, the specific steps of step 1 include: Isolate peripheral blood mononuclear cells (PBMCs) of healthy people with different HLA types, and use CD19 + magnetic beads for sorting. After sorting, use the CD40L culture system for cell culture; use the method of immortalized recombinant vector or infection with EBV virus to prepare immortalized B cells; after detecting the HLA type, perform cryopreservation.

[0016] Preferably, the immortalized recombinant vector has the immortalized sequence shown in SEQ ID NO: 1.

[0017] Preferably, the high-throughput sequencing in step 2 is whole-genome sequencing, whole-exome sequencing, or transcriptome sequencing.

[0018] Preferably, the APC cells and T cells are co-cultured at a ratio of 4:1.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Most traditional antigen screening methods obtain results by scoring and ranking through algorithms, lacking the screening of the normal human physiological immune process; the method involved in the present invention is based on bioinformatics screening and performs immune screening of antigen presentation by human antigen-presenting cells (APCs) and T cell stimulation, which is more in line with the normal tumor immune process;

[0021] (2) Currently, most in vitro antigen screening uses DCs for antigen presentation. However, DC cells cannot proliferate, and their survival time is only 2 weeks. They cannot be stably passaged or genetically edited, resulting in unstable "single-time" results and inability to be repeatedly verified. The present invention uses immortalized B cells as antigen-presenting cells, which are long-lasting and stable, and powerfully break through the key bottleneck in the current cell therapy R & D process;

[0022] (3) The present invention refers to the distribution characteristics of high-frequency HLA typing, and sets APC cells with different HLA typings for antigen screening respectively, with a wide coverage and high specificity;

[0023] (4) Diversified antigen loading methods: B cells not only have phagocytic ability and can perform peptide loading, but can also introduce the constructed antigen mRNA sequence into antigen-presenting B cells by electroporation for antigen presentation;

[0024] (5) Rapid detection: Whether using peptide loading or electroporating antigen sequences, anti-human CD86 can be used quickly after antigen loading + to detect the proportion of B lymphocytes, and the increase in the CD86 + proportion indicates successful antigen loading and also proves that these antigens have immunogenicity; the above-mentioned CD86 + B cells are antigen-presenting B lymphocytes (BAPC); the whole process takes no more than 48 hours. In the past, screening for tumor neoantigens often used HLA tetramer replacement experiments combined with DC-T cell co-culture experiments, with unstable effects and a time required of more than 2 weeks;

[0025] (6) Direct and objective reaction: The most direct way to verify immunogenicity is to observe the effector function of T cells; co-culture with T cells can detect IFN-γ secretion (using ELISA or ELISpot), or detect the proportion of 41-BB + or CD137 + in CD8 + T cells. An increase in the proportion of 41-BB+ or CD137+ cells or an increase in the mean fluorescence intensity value indicates that the added antigen has immunogenicity and can activate the normal immunity of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows the HLA typing ratio of the Chinese population.

[0027] Figure 2 is a schematic diagram of the tumor neoantigen screening process.

[0028] Figure 3 shows the flow cytometry detection results of sorted B lymphocytes (CD19 + cells).

[0029] Figure 4 are the results of CD86 after peptide loading and mRNA electroporation of antigen sequences detected by flow cytometry + in B cells.

[0030] Figure 5 is the detection of the secretion of IFN-γ in T cells by ELISpot after co-culture with T cells;

[0031] Figure 6 is the plasmid map of the immortalized recombinant vector. Specific embodiments

[0032] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows.

[0033] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0034] Example

[0035] A method for screening tumor antigens based on HLA typing, the process is as Figure 2 shown, including the following steps:

[0036] ① B cell separation and culture: Separate peripheral blood mononuclear cells (PBMC) from healthy people with different HLA typings, and use CD19 + magnetic beads for sorting, and use the CD40L culture system for cell culture after sorting; Figure 3 Flow cytometry results of sorted B lymphocytes (CD19 + cells).

[0037] ② B cell immortalization: Select B lymphocytes with appropriate HLA typings, and use an immortalized recombinant vector (which has an immortalized sequence as shown in SEQ ID NO: 1, and its plasmid map is as Figure 6 shown) to become immortalized B cells after electroporation (it is also possible to immortalize by infecting with EBV virus), and preserve and freeze the cells according to different HLA typings.

[0038] ③ According to the tumor samples obtained by surgical resection or puncture of the patient, perform high-throughput sequencing (whole genome sequencing, whole exome sequencing or transcriptome sequencing) data, and perform potential antigen prediction according to the algorithm; the algorithm can refer to the algorithms disclosed in the existing literature. In this example, the potential antigen sources consider the sources of tumor neoantigens that may be generated such as tumor mutations, gene fusions, and alternative splicing. The mutation prediction algorithm uses Mutect [1] ; the gene fusion prediction algorithm uses fusionSTAR[3] ; The alternative splicing prediction algorithm uses rMAST [2] for peptide prediction.

[0039] ④ Based on the prediction results obtained from the above algorithm, the potential translated peptides are respectively subjected to HLA affinity calculation and antigen presentation potential calculation, and the peptides with high affinity and high presentation potential are retained [4-5] as potential antigenic peptides.

[0040] ⑤ The candidate antigen can be synthesized into a polypeptide (the polypeptide can be synthesized by conventional solid-phase synthesis method) and added to the APC cell culture supernatant or the synthesized mRNA sequence is electroporated into B cells for antigen loading;

[0041] ⑥ Flow cytometry is performed on APC cells: anti-human CD86 fluorescent antibody is used for flow cytometry to detect CD86 + B lymphocyte ratio. Figure 4 is the result of CD86 + B cells after peptide loading and mRNA electroporation of antigen sequence by flow cytometry detection.

[0042] ⑦ APC cells and T cells are co-cultured, and ELISpot or ELISA is used to detect the antigen presentation ability of APCs loaded with different antigens (the standard is to detect that the secretion of IFN-γ exceeds the control secretion level). Figure 5 is the detection of the secretion of IFN-γ by T cells using ELISpot after co-culture with T cells.

[0043] Whether it is a high-frequency antigen in a population cohort or an individualized antigen in a single sample, rapid antigen screening can be carried out according to the above process.

[0044] In the present invention, the candidate antigen sequence can be a recombinant peptide or can synthesize mRNA; if it is a peptide, B cells matching the HLA typing are selected, added to the B cell culture supernatant, and incubated for 24 h; if it is antigen mRNA, the mRNA sequence is introduced into B cells using an electroporator, and then continued to be cultured.

[0045] In the present invention, the B cells loaded with the candidate antigen are detected using anti-human CD86 + fluorescent flow antibody, and compared with the blank control group, the CD86 + cell ratio increases, which proves that the candidate antigen peptide has immunogenicity. The above cells can be sorted and then co-cultured with T cells.

[0046] In the present invention, after co-culture of the antigen-loaded B lymphocytes and T cells, ELISA or ELISpot is used to detect 41-BB + or CD137 +Cell ratio. Compared with the blank control group, if the proportion of 41-BB + or CD137 + cells increases, it proves that the candidate antigen peptide has strong immunogenicity.

[0047] The highly immunogenic antigen screened by the method of the present invention can be used for the construction of tumor vaccines or TCR-T.

[0048] References:

[0049] [1] Cibulskis K, Lawrence MS, Carter SL, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol.

[0050] 2013; 31(3): 213-219. doi:10.1038 / nbt.2514

[0051] [2] Shen S, Park JW, Lu ZX, et al. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc Natl Acad Sci U S A. 2014; 111(51): E5593-E5601. doi:10.1073 / pnas.1419161111

[0052] [3] Haas BJ, Dobin A, Li B, Stransky N, Pochet N, Regev A. Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods. Genome Biol. 2019; 20(1): 213.

[0053] [4]Reynisson B,Alvarez B,Paul S,Peters B,Nielsen M.NetMHCpan-4.1andNetMHCIIpan-4.0:improved predictions of MHC antigen presentation byconcurrent motif deconvolution and integration of MS MHC eluted liganddata.Nucleic Acids Res.2020;48(W1):W449-W454.

[0054] [5]Marty R,Kaabinejadian S,Rossell D,et al.MHC-I Genotype Restrictsthe Oncogenic Mutational Landscape.Cell.2017;171(6):1272-1283.e15.

[0055] As described above, it is only the preferred embodiment of the present invention and does not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for screening tumor antigens based on HLA typing, characterized in that: The following steps are involved: Step 1: Obtain peripheral blood from healthy individuals with different HLA typing, reserve B lymphocytes and T lymphocytes and freeze them, select B lymphocytes with different HLA typing for immortalization, and preserve and freeze cells according to different HLA typing; Step 2. Obtain candidate tumor antigens and test their immune presentation potential: Obtain tumor samples from patients through surgical resection or puncture for high-throughput sequencing data, predict potential antigens, obtain candidate tumor antigens, synthesize candidate antigens into peptides, or use electroporation of antigen mRNA sequences to introduce them into B cells, and then use anti-human CD86 + Fluorescent antibodies were used to detect the proportion of B lymphocytes. Compared with the control group, the proportion of CD86+B cells increased significantly, indicating that the candidate antigen was immunogenic and could be presented by APCs. Step 3. Detect the immune activation ability of candidate tumor antigens: Use antigen-loaded APC cells and T cells to co-culture to verify immunogenicity; then use enzyme linked immunosorbent assay (ELISA) or ELISpot to detect IFN-γ secretion of T cells.

2. The tumor antigen screening method according to claim 1, characterized in that: The step 1 specifically comprises: isolating healthy human peripheral blood mononuclear cells (PBMC) of different HLA typing, using CD19 + Magnetic beads are used for sorting, and after sorting, cells are cultured using a CD40L culture system; immortalized B cells are prepared using an immortalized recombinant vector or by infecting the EBV virus; after HLA typing, they are frozen.

3. The tumor antigen screening method according to claim 2, characterized in that: The immortalization recombinant vector has an immortalization sequence as shown in SEQ ID NO:

1.

4. The tumor antigen screening method according to claim 1, characterized in that: The high-throughput sequencing in step 2 is whole genome sequencing, whole exome sequencing or transcriptome sequencing.

5. The tumor antigen screening method according to claim 1, characterized in that: The APC cells and T cells were co-cultured at a ratio of 4:1.

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