Tumor antigen peptide AMZ2P1-neo and use thereof

Abstract

The application discloses a tumor antigen peptide AMZ2P1-neo and application thereof, and the amino acid sequence of the tumor antigen peptide AMZ2P1-neo is shown as SEQ ID NO. 1. The tumor antigen peptide AMZ2P1-neo provided by the application has excellent immunogenicity, can promote T cells to secrete IFN-gamma, and promote the killing effect of T cells on a uveal melanoma cell line; further, under the co-stimulation of CD3 / CD28, the killing effect of T cells on the uveal melanoma cell line is stronger. The tumor antigen peptide AMZ2P1-neo can be used as a new antigen of uveal melanoma, and can be used as a target for clinical treatment or diagnosis of uveal melanoma, and can be used for preparing a uveal melanoma treatment drug.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, specifically to a tumor antigen peptide AMZ2P1-neo and its applications. Background Technology

[0002] Tumor neoantigens are polypeptides encoded by tumor cells due to events such as gene mutations or viral integration, which are not present in normal tissues. They can be recognized by the immune system through the body's inherent antigen presentation system, triggering an immune response. Tumors resulting from mutations exist only in cancer cells, making tumor neoantigens ideal targets for cancer immunotherapy. Unlike other immunotherapies (such as checkpoint inhibitors), the immune response to tumor neoantigens is highly specific and does not attack normal tissues. Because tumor neoantigens are related to the clinical response to tumor immunotherapy, they can serve not only as potential biomarkers to predict patient responses to treatments such as immune checkpoint blockade, but also as therapeutic targets.

[0003] Uveal melanoma (UM) is the most common intraocular malignancy in adults. It has been reported that nearly 50% of UM patients eventually metastasize and survive for less than a year. Due to the current lack of effective treatments for UM, further understanding of its development and treatment response mechanisms is essential. Recent studies have shown that tumor neoantigen-targeted therapy is a promising direction for the treatment of UM. In fact, clinical therapies based on T-cell receptors (TCRs) utilizing neoantigens, such as GP100 [NCT03070392] and PRAME [NCT02743611], are currently under investigation. However, these tumor neoantigens are not specific to UM and are also present in cutaneous melanoma. Unlike cutaneous melanoma, ocular uveal melanoma has a relatively low tumor mutation burden (TMB) and a unique tumor microenvironment (TME) that significantly affects treatment efficacy. The subsequent unresponsiveness of UM to drug therapy can be attributed to these TME characteristics. Currently, despite ongoing research, the specific and druggable neoantigen targets for uveal melanoma remain unclear.

[0004] Therefore, there is an urgent need for a new tumor antigen peptide as a target to treat uveal melanoma. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiency in the prior art of lacking tumor antigen peptides for the effective treatment of uveal melanoma.

[0006] To achieve the above objectives, the present invention provides a tumor antigen peptide AMZ2P1-neo, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the complex formed by the binding of the tumor antigen peptide AMZ2P1-neo to MHC molecules can be recognized by CD3 and / or CD28 cytotoxic T cells.

[0008] Preferably, the MHC molecule includes at least MHC-I or MHC-II molecules.

[0009] The present invention also provides a nucleic acid that encodes the tumor antigen peptide AMZ2P1-neo as described above.

[0010] The present invention also provides an expression vector that expresses the nucleic acid as described above.

[0011] The present invention also provides a recombinant host cell, wherein the recombinant host cell comprises at least one of the tumor antigen peptide AMZ2P1-neo as described above, the nucleic acid as described above, and the expression vector as described above.

[0012] The present invention also provides an application of the tumor antigen peptide AMZ2P1-neo, wherein the application is to prepare a therapeutic antitumor drug using any of the tumor antigen peptides AMZ2P1-neo described above.

[0013] Preferably, the tumor includes at least one of uveal melanoma and cutaneous melanoma.

[0014] Preferably, the antitumor drug further comprises CD3 and / or CD28 monoclonal antibodies.

[0015] Preferably, the antitumor drug further comprises a pharmaceutically acceptable carrier or excipient.

[0016] The beneficial effects of this invention are:

[0017] (1) This invention first discovered a specific tumor antigen peptide AMZ2P1-neo (KNILNGSRA) in uveal melanoma cell lines and tumor samples from uveal melanoma patients. After purification, the tumor antigen peptide AMZ2P1-neo (KNILNGSRA) binds to MHC molecules to form a complex. The complex can be specifically recognized by CD3 and / or CD28 cytotoxic T cells. The cytotoxic T cells secrete cytokines, which promote the killing effect of cytotoxic T cells on uveal melanoma cells. Furthermore, under the co-stimulation of CD3 / CD28, the killing effect of cytotoxic T cells on uveal melanoma cells is stronger.

[0018] (2) The tumor antigen peptide AMZ2P1-neo (KNILNGSRA) of the present invention can be used to prepare anti-tumor drugs, providing a novel small molecule drug with killing effect to enhance the immunotherapy of uveal melanoma. On the other hand, the discovery of the new tumor antigen peptide further enriches the tumor-specific neoantigen library of uveal melanoma. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the method for promoting the killing effect of T cells on tumors using the tumor antigen peptide AMZ2P1-neo (KNILNGSRA) of the present invention.

[0020] Figure 2 This is a schematic diagram illustrating how the three tumor antigen peptides SEPTIN6-neo, MZTB2-neo, and AMZ2P1-neo of the present invention stimulate PBMCs to secrete IFN-γ.

[0021] Figure 3 The figure shows the flow cytometry results of the three tumor antigen peptides SEPTIN6-neo, MZTB2-neo, and AMZ2P1-neo of the present invention promoting the killing effect of T cells on uveal melanoma cells. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and instruments used in the present invention are conventional reagents, methods and instruments in the art.

[0023] definition

[0024] Generally, the terms used in the claims and specification are intended to be interpreted as having the ordinary meaning as understood by one of ordinary skill in the art. For clarity, certain terms are defined below, and if there is a conflict between the ordinary meaning and the provided definition, the provided definition shall prevail.

[0025] As used in this article, the term "antigen" refers to a substance that induces an immune response.

[0026] As used herein, the term "tumor antigen peptide" refers to the candidate tumor antigen peptides screened in this application.

[0027] As used herein, the term “tumor neoantigen” is a new antigen that is present in the tumor cells or tissues of a subject but not in the corresponding normal cells or tissues of the subject.

[0028] As used in this article, the term "alternative splicing" refers to the fact that most eukaryotic gene transcription produces mRNA precursors that are spliced ​​in one way to produce one type of mRNA, thus producing only one type of protein. However, some genes produce mRNA precursors that can be spliced ​​in different ways to produce two or more types of mRNA, which is called alternative splicing.

[0029] Any term not directly defined herein shall be understood to have the meaning generally associated with it as understood within the field of this invention.

[0030] Tumor antigen peptides are peptides present on the surface of tumor cells that bind to major histocompatibility complex (MHC) molecules. MHC molecules include at least MHC class I and MHC class II molecules. After binding, they exist as protein complexes on the tumor cell surface and can be specifically recognized by cytotoxic T cell receptors (TCRs), thereby activating a T cell immune response. Tumor antigen peptides are not expressed on normal cells and are unlikely to trigger autoimmune responses. Therefore, tumor antigen peptides are promising immunotherapeutic targets in tumor vaccines and T cell-based cancer immunotherapies.

[0031] The acquisition of tumor antigen peptides is based on novel amino acid or protein sequences generated by tumor gene mutations. Under normal circumstances, aberrantly spliced ​​isoforms are degraded through meaningless-mediated mRNA decay, which is an mRNA quality control mechanism. Given that mRNA regulatory mechanisms in cancer are often dysregulated, aberrant subtypes can accumulate and form tumor-specific tumor antigen peptides. Therefore, tumor antigen peptides can not only serve as potential biomarkers to predict patient responses to treatments such as immune checkpoint blockades (ICBs), but also as therapeutic targets.

[0032] Due to their immense potential in biomarker discovery and vaccine development, tumor antigen peptides are increasingly considered a promising direction for cancer immunotherapy. Tumor antigen peptides derived from alternative splicing isomers can serve as highly specific tumor antigens and are associated with clinical responses to tumor immunotherapy. However, research findings on tumor antigen peptides for cutaneous melanoma are not applicable to uveal melanoma; targeted therapies for cutaneous melanoma have poor therapeutic effects on uveal melanoma, and currently no effective tumor antigen peptides have been identified for uveal melanoma. Therefore, this invention investigates uveal melanoma to elucidate a novel library of cancer type-specific alternative splicing antigens, identify effective tumor antigen peptides, and explore their potential clinical applications.

[0033] Unless otherwise specified, the raw materials and reagents used in the following examples were obtained by researchers in the art through conventional commercial channels. Unless otherwise specified, the experimental steps involved in the examples were conventional technical operations performed by researchers in the art.

[0034] (I) Experimental cells, experimental materials and experimental instruments

[0035] 1. Experimental cells

[0036] Tumor samples from 5 patients with uveal melanoma (from the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine), 4 uveal melanoma cell lines MEL290, MEL92.1, OMM2.3, and MUM2B (provided by Dr. Martine J Jager of the Department of Ophthalmology at Leiden University Medical Center, Netherlands), and peripheral blood mononuclear cells (PBMCs).

[0037] 2. Experimental materials: CD3, CD28, interleukin-2, complete culture medium, carboxysuccinimide ester (CFSE) dye, PBS buffer, urea lysis buffer, 0.1% formic acid, acetonitrile.

[0038] 3. Experimental instruments: Novocyte FACS instrument, Easy-nLC1200 system, Q Exactive plus mass spectrometer (purchased from Thermo Scientific, Bremen, Germany).

[0039] (ii) Alternative splicing produces different alternative splice isomers.

[0040] Alternative splicing (AS) is a fundamental biological event in eukaryotes. After gene transcription, initial RNA, or RNA precursor, is produced. Then, through alternative splicing, different exons are selectively rejoined, resulting in different splice isoforms. In this way, a single gene can produce multiple different transcripts. These transcripts have their own specific expression and function at different stages of cell / individual differentiation and development, and in different tissues. This greatly enriches the types and quantities of coding and non-coding RNAs, thereby increasing the complexity of the transcriptome and proteome.

[0041] Alternative splicing is considered a major contributor to tumor-specific tumor antigen peptides. Studies have found that aberrant autosomal syndrome (AS) is associated with tumor-specific mesenchymal imbalance (TME) regulation and patient survival. However, due to technical limitations, previous AS studies in UM relied heavily on de novo reconstruction of short sequences from public databases (such as TCGA). The sequence lengths provided by these reads are insufficient to accurately identify AS events and novel splice isoforms. Novel splice isoforms discovered in previous studies have not been experimentally validated or functionally investigated.

[0042] Experimental results

[0043] This invention is the first to employ nanopore technology, specifically nanopore long-read sequencing, to detect UM (tumor antigen peptides), accurately identifying novel alternative splicing isomers. Based on these novel alternative splicing isomers, a new candidate library of tumor antigen peptides for UM was compiled. Twelve candidate tumor antigen peptides were obtained, including SEPTIN6 (amino acid sequence MADAGAMAATDIARQVGEGCRT), MZT2B (amino acid sequence RGAGLGRALHGAAPRMGQRL), and AMZ2P1 (amino acid sequence KNILNGSRA).

[0044] (III) Protein mass spectrometry detection showed that tumor antigen peptides were specifically expressed in tumor patient samples and tumor cell lines.

[0045] according to Figure 1 The following experiments were conducted using the procedure shown. High-resolution proteomic analysis was performed on tumor samples from 5 uveal melanoma patients and 4 uveal melanoma cell lines to screen for candidate tumor antigen peptides commonly expressed by all samples.

[0046] 1. Experimental Methods

[0047] Sample processing of uveal melanoma cell lines: Four cell types (MEL290, MEL92.1, OMM2.3, and MUM2B) were removed from the culture dishes. PBS was added to wash away any remaining culture medium, followed by lysis buffer (400-700 μL for a 25 mm culture dish). Cell scraping was then performed directly on the surface of the culture dish using a cell scraper. After scraping, the lysis buffer and cell samples were collected into EP tubes and lysed on ice (using 8M urea) to obtain peptide fragments from the uveal melanoma cell line samples.

[0048] Tumor sample processing from uveal melanoma patients: Step 1: mince and remove blood. Place the sample in PBS buffer solution, mince, discard the discolored solution, replace with fresh PBS, and continue mincing. Repeat several times until the PBS solution no longer discolors. Step 2: weigh. For more accurate weighing, after washing, the sample can be blotted dry with filter paper before weighing. Step 3: fragmentation. Use ultrasound to fragment the tissue. Step 4: lysis. Add 5 mL of 8M urea lysis buffer to 1 g of tissue to obtain peptide fragments from the uveal melanoma patient tumor sample.

[0049] Peptides were analyzed using an Easy-nLC1200 system coupled with Q Exactive plus mass spectrometry (Thermo Scientific, Bremen, Germany). The peptide-containing fractions were injected and separated onto an analytical column (C18, 25 cm × 75 μm, 3 μm) using 0.1% formic acid solution at a flow rate of 300 nL / min and an acetonitrile gradient for 2 h. The mobile phase consisted of 0.1% formic acid (A) and 0.1% formic acid in 80% ACN (B). MS1 and MS2 scans were performed using Orbitrap. MS1 measurements were performed at a resolution of 70,000 m / s in the range of 350–1800 m / s with an AGC target of 3e6. MS2 measurements were performed at a resolution of 17,500 m / s with an AGC target of 1e5. The dynamic exclusion time was set to 30 s, and the normalized collision energy of the HCD was 28%.

[0050] 2. Experimental Results

[0051] After performing high-resolution proteomic analysis on tumor samples from four uveal melanoma cell lines and five uveal melanoma patients, three candidate tumor antigen peptides commonly expressed in all samples were screened out and designated as SEPTIN6-neo, MZTB2-neo, and AMZ2P1-neo, respectively. The corresponding 9-mer peptides were then synthesized in vitro.

[0052] (iv) The effect of different tumor antigen peptides on IFN-γ secretion levels

[0053] 1. Experimental Methods

[0054] The three tumor antigen peptides SEPTIN6-neo, MZTB2-neo, and AMZ2P1-neo were purified and used to stimulate PBMCs. The effects of the three tumor antigen peptides on IFN-γ secretion were detected by enzyme-linked immunospot assay (ELISPOT).

[0055] 2. Experimental Results

[0056] like Figure 2 As shown in the experiment, compared with the control group, the three tumor antigen peptides SEPTIN6-neo, MZTB2-neo and AMZ2P1-neo all increased the IFN-γ secretion of PBMCs.

[0057] (V) Detection of the ability of different tumor antigen peptides to promote T cell killing of tumors

[0058] 1. Experimental Methods

[0059] according to Figure 1The procedure involved stimulating peripheral blood mononuclear cells (PBMCs) with interleukin-2. One group received only the three tumor antigen peptides SEPTIN6-neo, MZTB2-neo, and AMZ2P1-neo, respectively, while the other group received co-stimulation with purified SEPTIN6-neo, MZTB2-neo, AMZ2P1-neo, and CD3 / CD28. Five days later, the stimulated PBMCs were collected as effector cells. Carboxysuccinimide ester (CFSE) is a fluorescent cell staining dye, 1×10⁻⁶. 7 MEL92.1 cells were washed in 1 μM CFSE containing PBS for 25 min, protected from light, to prepare CFSE-labeled target cells (i.e., CFSE-labeled MEL92.1 cells). After incubation, complete culture medium was added, and the cells were incubated at 37°C for 5 min. Then, the supernatant was removed, and the cells were resuspended in preheated fresh complete culture medium. In the T cell killing assay, effector cells (stimulated PBMCs) and target cells (CFSE-labeled MEL92.1 cells) were mixed at a ratio of 10:1. After 6 h, all cells were collected with 0.2% amylin-EDTA. CFSE-dead cells were analyzed by Novocyte FACS staining with propdidium iodidium (PI), and CFSE counts were performed. + PI + The proportion of cells and the formula for calculating cytotoxicity rate are: Cytotoxicity rate = (target cell death / % - spontaneous death / %) / (100 - spontaneous death / %) × 100%.

[0060] 2. Experimental Results

[0061] like Figure 3 As shown, the amino acid sequences of all three tumor antigen peptides can promote the killing effect of T cells on uveal melanoma cells. Among them, the tumor antigen peptide AMZ2P1-neo (KNILNGSRA) has the strongest effect in promoting the killing of uveal melanoma cells by T cells. Furthermore, the killing effect of the tumor antigen peptide AMZ2P1-neo (KNILNGSRA) is further enhanced after co-stimulation with CD3 / CD28. The amino acid sequence of the tumor antigen peptide AMZ2P1-neo (KNILNGSRA) is shown in SEQ ID NO.1, specifically Lysine-Asparagine-Isoleucine-Leucine-Asparagine-Glycine-Serine-Arginine-Alanine. Therefore, under the co-stimulation of CD3 / CD28, the tumor antigen peptide AMZ2P1-neo (KNILNGSRA) promotes the killing effect of T cells more strongly.

[0062] In some embodiments, a nucleic acid is provided that encodes the tumor antigen peptide.

[0063] In some embodiments, an expression vector is provided that expresses the nucleic acid. The expression vector used in this invention can be of various types, depending on the host and parent used, such as plasmids, phage vectors, and viral vectors. The vector may appropriately include elements such as an inducible promoter, a gene encoding a signal sequence, a selection marker gene, and a terminator.

[0064] In some embodiments, a recombinant host cell is provided, the recombinant host cell comprising any one of the tumor antigen peptide, the nucleic acid, and the expression vector. The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Any cell can be used as long as the function of the peptide of the present invention is not impaired; representative examples include: *Escherichia coli*; *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; plant cells, etc.

[0065] In some embodiments, an antitumor drug is provided, which comprises at least the aforementioned tumor antigen peptide and a pharmaceutically acceptable carrier. The tumor antigen peptide of the present invention is used as the active ingredient of the antitumor drug. Each antigen peptide, generated through intracellular degradation, binds to MHC molecules to form a complex. This complex is presented at high density to the surface of antigen-presenting cells. Cytotoxic T cells recognize this complex and secrete cytokines, such as IFN-γ, thereby killing cancer cells. Pharmaceutically acceptable carriers include, but are not limited to, liposomes and hydrophilic polymers.

[0066] In summary, this invention screens an alternative splice isomer generated by alternative splicing to obtain a tumor antigen peptide AMZ2P1-neo (KNILNGSRA), which can promote the killing effect of cytotoxic T cells on uveal melanoma cells and has a certain degree of specificity. After the addition of CD3 / CD28 co-stimulation, the killing effect on uveal melanoma cell lines is stronger. It can be used to prepare anti-tumor drugs to guide the clinical treatment of uveal melanoma.

[0067] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A tumor antigen peptide AMZ2P1-neo, characterized by, The amino acid sequence of the tumor antigen peptide AMZ2P1-neo is shown as SEQ ID NO.

1.

2. A nucleic acid, characterized in that, The nucleic acid encodes the tumor antigen peptide AMZ2P1-neo as described in claim 1.

3. An expression vector, characterized by, The expression vector expresses the nucleic acid as described in claim 2.

4. A recombinant host cell, characterized in that, The recombinant host cell comprises at least any one of the tumor antigen peptide AMZ2P1-neo as described in claim 1, the nucleic acid as described in claim 2, and the expression vector as described in claim 3.

5. Use of a tumor antigen peptide AMZ2P1-neo, characterized in that, The use is for preparing a drug for treating uveal melanoma using the tumor antigen peptide AMZ2P1-neo as described in claim 1.

6. Use according to claim 5, wherein The drug for treating uveal melanoma further comprises CD3 and / or CD28 monoclonal antibodies.

7. The use according to claim 5, wherein the compound is ###0002### The drug for treating uveal melanoma further comprises a pharmaceutically acceptable carrier or excipient.

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