Tumor neoantigen polypeptide aiming at BRD4: NUTM1 gene fusion mutation and application of tumor neoantigen polypeptide in NUT cancer treatment

Abstract

The invention discloses a tumor neoantigen polypeptide aiming at BRD4: NUTM1 gene fusion mutation and an application of the tumor neoantigen polypeptide in NUT cancer treatment. The tumor neoantigen polypeptide comprises: (1) a tumor neoantigen polypeptide with an amino acid sequence represented by SEQ ID No.2; and (2) polypeptides with the same or similar functions obtained by substitution and / or deletion and / or addition of at least one amino acid in the amino acid sequence. The antigen peptide disclosed by the invention can stimulate and activate T cells specifically aiming at BRD4:: NUTM1 gene fusion mutation of a human body in vitro and perform mass amplification for adoptive reinfusion treatment of a patient; the polypeptide has obvious immunogenicity, and the killing ability of T cells on NUT cancer cells with BRD4: NUTM1 gene fusion mutation is improved; meanwhile, the BRD4: NUTM1 fusion mutant tumor antibody can be synthesized on a large scale and is used for subsequent standardized and individualized immunotherapy of BRD4:: NUTM1 fusion mutant tumor patients.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy technology, and more specifically, to a neoantigen polypeptide targeting BRD4::NUTM1 gene fusion mutations and its application in the treatment of NUT cancer. Background Technology

[0002] NUT cancer is a rare, poorly differentiated malignant tumor with a poor prognosis. First reported in 1991, subsequent reports have primarily focused on midline structures such as the head, neck, and mediastinum, hence the name "midline cancer." NUT cancer can occur at any age, but is most prevalent in young adults. It is highly aggressive, often diagnosed at an advanced stage with distant metastases in most cases. Currently, there are approximately 100-200 new cases globally each year, but due to its rarity and underdiagnosis, the actual incidence rate may be underestimated. It is reported to account for 7% of poorly differentiated / undifferentiated cancers in children and young adults, with a median survival of only 6-9 months and a 5-year survival rate of <20%. Current standard treatments for NUT cancer include surgery, chemotherapy (such as cisplatin + etoposide), radiation therapy, and targeted therapy (such as BET inhibitors). Surgical treatment is generally suitable for tumors with small invasion areas and early stages; chemotherapy has low response rates and is prone to rapid drug resistance; radiotherapy is only suitable for local control and cannot prevent metastasis; targeted drugs can temporarily inhibit BRD4::NUTM1 function, but their efficacy as monotherapy is limited and recurrence is common. Therefore, developing new treatment methods for NUT cancer has significant clinical value and social implications.

[0003] With the development of tumor immunology, numerous clinical trials have confirmed that combining immunotherapy with chemotherapy and targeted therapy in neoadjuvant cancer treatment can not only improve patients' treatment response but also effectively prolong their survival. Tumor neoantigens are peptides presented on the surface of tumor cells, which bind to major histocompatibility complex (MHC) molecules and exist as protein complexes on the tumor cell surface. They can be specifically recognized by cytotoxic T-cell receptors (TCRs), thereby activating the T-cell immune response. Compared to traditional treatments, immunotherapy focuses on activating and enhancing the patient's own immune system to kill tumor cells, thus offering advantages such as precise treatment, fewer side effects, and longer-lasting effects. Furthermore, the body's immune system possesses the characteristic of immune memory; therefore, immunotherapy can help patients develop memory immunity, preventing tumor recurrence and metastasis.

[0004] NUT cancer is closely related to NUTM1 gene rearrangement. The NUTM1 gene, located on the long arm of chromosome 15, encodes the NUT protein expressed in testicular and ovarian germ cells. Chromosomal translocations / rearrangements lead to the fusion of the NUTM1 gene on chromosome 15q14 with different fusion partner genes, with the BRD4 fusion partner showing the worst prognosis. Approximately two-thirds of NUT cancer cases carry the BRD4::NUTM1 fusion gene. BRD4, a member of the BET protein family, acts as a coactivator in the transcription of cell cycle-related genes, participating in the regulation of cell growth, proliferation, cell survival, and inflammatory responses. Studies have shown that NUTM1 protects cancer cell telomeres by activating reverse transcriptase, thus immortalizing cancer cells. The BRD4::NUTM1 fusion protein disrupts squamous cell differentiation and promotes tumorigenesis. Zheng D et al. demonstrated using a mouse model that the BRD4::NUTM1 fusion gene can induce invasive cancer in mice and can induce midline NUT cancer from a wide range of tissues with strong phenotypic variability.

[0005] Neoantigens encoded by tumor-specific mutations are key targets for effective T-cell-mediated immune and anti-tumor immune responses. Novel epitopes possess high immunogenicity and are considered excellent candidates for cancer vaccines. Previous studies have demonstrated that the antigenic potential of top-tier recurrent somatic mutations can be assessed by calculating the binding affinity between mutated epitopes and MHC class I, revealing that most recurrent mutations are predicted to be antigenic. A study by Riess JW reported on 31 patients with solid tumors carrying the BRD4::NUTM1 fusion mutation (16% diagnosed with NUT cancer). Through neoantigen prediction and MHC complex affinity modeling, the study predicted the binding of HLA-C*16:01 to the immunogenic neoantigen peptide: the fusion sequence VVSPP|ASAL. This showed a specific response to immune checkpoint inhibitors in head and neck cancer fusions with low TMB and low PD-L1 expression, demonstrating that fusion-related neoantigens are potentially tumor-specific targets that can trigger immune responses. However, studies on the practical application of this BRD4::NUTM1 gene fusion mutation as a potential target for tumor immunotherapy in NUT cancer have not yet been reported. Summary of the Invention

[0006] This invention provides a neoantigen polypeptide targeting the BRD4::NUTM1 gene fusion mutation and its application in the treatment of NUT cancer, which overcomes the above-mentioned defects of the prior art and provides a precise and efficient new target for the immunotherapy of NUT cancer.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0008] A tumor neoantigen polypeptide targeting the BRD4::NUTM1 gene fusion mutation, wherein the tumor neoantigen polypeptide is:

[0009] (1) The amino acid sequence is the tumor neoantigen polypeptide shown in MVQYKDSFL (SEQ ID No. 2);

[0010] (2) A polypeptide with the same or similar function obtained by substitution and / or deletion and / or addition of at least one amino acid in the amino acid sequence.

[0011] Among them, the aforementioned polypeptides with the same or similar functions refer to tumor antigen polypeptides that can activate patients with HLA type HLA-A*02:07 and accompanied by BRD4::NUTM1 gene fusion mutation to produce T cells specifically targeting tumors with the above mutation.

[0012] A nucleic acid molecule encoding a tumor neoantigen polypeptide.

[0013] A gene expression vector containing a nucleic acid molecule encoding a tumor neoantigen polypeptide.

[0014] A cell that contains tumor neoantigen peptides, nucleic acid molecules, or gene expression vectors.

[0015] The use of a tumor neoantigen polypeptide or nucleic acid molecule or gene expression vector or cell in any of the following:

[0016] (1) Application in the preparation of drugs for the prevention or treatment of NUT cancer with BRD4::NUTM1 gene fusion mutation;

[0017] (2) Application in the preparation of diagnostic reagents for NUT cancer with BRD4::NUTM1 gene fusion mutation;

[0018] (3) Application in the preparation of specific T cells for NUT cancer with BRD4::NUTM1 gene fusion mutation.

[0019] A specific type of T cell that is induced by a tumor neoantigen peptide and specifically targets the tumor neoantigen peptide.

[0020] A pharmaceutical composition for treating NUT cancer with a BRD4::NUTM1 gene fusion mutation, the pharmaceutical composition comprising a tumor neoantigen polypeptide or nucleic acid molecule or gene expression vector or cell or specific T cell, and a pharmaceutically acceptable carrier.

[0021] A vaccine that contains tumor neoantigen peptides or nucleic acid molecules.

[0022] A tumor neoantigen polypeptide targeting the BRD4::NUTM1 gene fusion mutation, wherein the tumor neoantigen polypeptide is obtained by the following method:

[0023] (1) The antigenic potential of top recurrent somatic mutations was assessed by calculating the binding affinity between the mutant epitope and major histocompatibility complex (MHC) class I.

[0024] (2) The immune epitope database (IEDB) algorithm was used to analyze the protein structure, surface accessibility and hydrophilicity of the antigen;

[0025] (3) Screening epitopes with potential immunogenicity from the predicted neoantigens, and designing peptides with specific amino acid sequences based on the identified antigenic recognition regions.

[0026] A method for verifying the in vitro immunogenicity of a tumor neoantigen peptide, the method comprising the following steps:

[0027] (1) In vitro isolation of peripheral blood mononuclear cells (PBMCs) from tumor patients;

[0028] (2) Incubate PBMCs with tumor neoantigen peptides;

[0029] (3) T cell IFN-γ secretion was detected by enzyme-linked immunospot assay (ELISPOT) to assess the immunogenicity of tumor neoantigen peptides.

[0030] The above-described technical solution of the present invention has the following beneficial effects:

[0031] (1) The antigen peptide of the present invention can stimulate and activate human T cells specifically targeting the BRD4::NUTM1 gene fusion mutation in vitro and expand them in large quantities for adoptive infusion therapy in patients.

[0032] (2) The new antigenic peptide synthesized by the BRD4::NUTM1 gene fusion mutation can activate T lymphocytes that specifically target the mutant peptide in vitro and release the cytokine IFN-γ, indicating that the peptide has significant immunogenicity and enhances the killing ability of T cells against NUT cancer cells with BRD4::NUTM1 gene fusion mutation.

[0033] (3) The antigenic peptide of the present invention fills the gap in the treatment of NUT cancer patients with BRD4::NUTM1 gene somatic fusion mutations using personalized antigenic peptides.

[0034] (4) Meanwhile, the antigenic peptides of the present invention can be synthesized on a large scale and used in the standardized and individualized immunotherapy of patients with BRD4::NUTM1 fusion mutations. Attached Figure Description

[0035] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0036] Figure 1 Immunohistochemical staining of four molecular markers to assess the level of immune infiltration in a subset of NUT cancer patients according to this invention;

[0037] Figure 2 The figures show the results of the in vitro cell evaluation experiments for assessing the effectiveness of the synthetic polypeptide sequences No. 1 and No. 2 in this application. In the figures, lectin (PHA, phytohemagglutinin) was used as a positive control at a working concentration of 5 μg / mL; Medium was used as a negative control, consisting of an equal volume of sterile water.

[0038] Figure 3 This is a statistical chart of the ELISPOT results for the synthesized polypeptide sequences numbered 1 and 2 in Example 3 of this application. Where SFU / 10 6 Cells are defined as spots-forming units per million cells, with a plating cell count of 2 × 10⁻⁶. 5 Multiply by 5 to get (i.e., average number of spots multiplied by 5);

[0039] Figure 4 This is a schematic diagram illustrating the in vitro killing rate of NUT cancer cells by CTLs with specific synthetic polypeptide sequences (numbered 1 and 2) in Example 3 of this application. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0041] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use.

[0042] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the specification.

[0043] In all the examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Unless otherwise specified, the equipment used in the experiments is well known to those skilled in the art.

[0045] Example 1: Prediction and screening of tumor neoantigens using a tumor neoantigen prediction platform.

[0046] A total of 14 NUT cancer tumor tissue samples and corresponding adjacent normal tissues were collected clinically. All samples were verified to be positive for the BRD4::NUTM1 fusion gene by fluorescence in situ hybridization (FISH). Genomic DNA was extracted and whole exome sequencing and transcriptome sequencing were performed.

[0047] First, by pairwise analysis of whole exome and adjacent normal tissue, tumor-specific mutations (such as Indel) can be obtained. These mutated peptides constitute the first-stage tumor neoantigen database.

[0048] Secondly, gene expression levels (FPKM) are calculated based on transcriptome sequencing data of tumor samples. By comparing with a reference genome, neoantigens with FPKM < 10 in the first-stage tumor neoantigen database are filtered to ensure that the retained neoantigens have FPKM > 10 in tumor tissue, thus forming the second-stage tumor neoantigen database.

[0049] Finally, HLA genotyping was performed on each patient, and the Neocom algorithm was used to predict antigen affinity for each patient's HLA typing and the second-stage tumor neoantigen database. Peptides with IC50 < 50 nM were screened, and tumor neoantigens with IC50 values ​​< 500 nM were selected to form the final tumor neoantigen database.

[0050] In this invention, we screened a total of 5 neoantigen peptides as candidates (see Table 1).

[0051] Table 1 Information on candidate tumor neoantigen peptides

[0052]

[0053]

[0054] Example 2: Assessment results of immune infiltration based on immunohistochemical staining

[0055] The tumor microenvironment immune infiltration was assessed in the above 14 NUT cancer patients. Specifically, immunohistochemical staining for four molecular markers (CD3 / CD8 / CD45RO / FOXP3) was performed. Figure 1 (Only some patients' staining results are listed in the text), and the analysis of 10 types of cell infiltration levels in tumor tissue was completed.

[0056] Immunostaining assessment revealed that all NUT cancer patients had some degree of immune infiltration, but the level of infiltration varied.

[0057] Example 3: In vitro cell experiments to evaluate the effectiveness of tumor neoantigen peptide sequences

[0058] 1. Collection of peripheral blood mononuclear cells (PBMCs)

[0059] Take 5 mL of peripheral blood from the patient and add an equal volume of PBS buffer. In a separate 50 mL centrifuge tube, add 6 mL of Ficoll (lymphocyte separation medium), add the diluted blood sample, centrifuge at 1500 rpm for 30 min at room temperature, remove the upper plasma layer, collect the middle white membrane layer (PBMC), and store in liquid nitrogen.

[0060] 2. T cell incubation:

[0061] PBMC cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. After centrifugation at 1600 rpm for 5 min at room temperature to remove the supernatant, the cells were resuspended in culture medium containing 5% fetal bovine serum, mixed, and then stained with trypan blue to count the number of viable cells. After counting, the cell concentration was adjusted to 2 × 10⁵ / mL and cultured in 24-well plates. The five synthetic peptides from Example 1 and cell growth factors IL-2 / IL-7 (working concentration 50 ng / mL) were added, and the cells were cultured at 37°C. After ten days, the cells were collected and washed with 3 times the volume of PBS buffer (centrifuged at 1600 rpm for 5 min).

[0062] 3. Enzyme-linked immunospot assay (ELISPOT) for detection and counting:

[0063] First, add 100 μL of coated antibody to each well of the ELISPOT plate and incubate overnight at 4-8°C. Second, add 200 μL of 1640 culture medium and incubate at room temperature for at least 30 min. Then, add the peptide from Example 1 (working concentration of 5 μg / mL), and set up positive and negative control groups (water) for each well. Incubate at 37°C (5% CO2) for 12-48 h. Finally, perform the colorimetric reaction by adding detection antibody and streptavidin to the wells, washing away excess antibody, adding the chromogenic substrate, washing three times with sterile water, drying the plate, and detecting and counting the number of positive spots under a dissecting microscope.

[0064] 4. Results Analysis:

[0065] The activation effects of five selected tumor neoantigen peptides on patient T cells were systematically analyzed using ELISPOT plasm count. Figure 2 The ELISPOT results for antigenic peptides numbered 1 and 2 are shown; spot counts are shown below. Figure 3Calculations showed that peptide #2 had an average of 138 positive spots and a spot-forming unit (SFU) / million cells ratio of 690, exceeding the positive threshold of 50 (Porter M et al., J Invest Dermatol. 2022). This indicates that the peptide can significantly activate T lymphocytes and release the cytokine IFN-γ. Specifically, the peptide carrying a fusion mutation between exon 9 of the BRD4 gene and exon 3 of the NUTM1 gene can stimulate an immune response, increasing the killing ability of T cells against cancer cells with the BRD4::NUTM1 gene fusion mutation. In contrast, peptide #1 had a positive SFU / million cells ratio of 32.5 (meaning that MVQYKDSFL induced IFN-γ secretion 19-fold higher than the control peptide (p<0.001)), below the positive threshold, indicating that it could not stimulate T lymphocyte activation and release the cytokine IFN-γ, and therefore lacked immunogenicity. Peptide #2 is derived from a fusion mutation of the NUTM1 and BRD4 genes.

[0066] Experimental results show that the peptide MVQYKDSFL (number 2) targeting the BRD4::NUTM1 somatic fusion mutation can significantly activate T lymphocytes in NUT cancer patients and release the cytokine IFN-γ. This peptide has significant immunogenicity.

[0067] This application demonstrates that a neoantigen peptide targeting the BRD4::NUTM1 gene fusion mutation site could be used in the future for standardized and individualized tumor immunotherapy of NUT cancer.

[0068] Example 4: Neoantigen-specific CTLs kill tumor cells in vitro

[0069] Synthetic peptides 1 and 2 from Example 1 of this application were presented on dendritic cells and co-cultured with cytotoxic T lymphocytes (CTLs). The resulting antigen peptide 1 and antigen peptide 2-specific CTLs were used as effector cells, with tumor cell line C666-1 as the target cell, and effector-target ratios set at 1:1 and 4:1, respectively. The LDH method was used to detect the killing level of CTLs on tumor cells, and the CTL killing efficiency was calculated. Figure 4 The results showed that neoantigen peptide 2 (MVQYKDSFL) significantly promoted the effective killing of tumor cells by CTLs at different effector-to-target ratios (MVQYKDSFL-CTL (effector-to-target ratio 4:1): killing rate 68% (control peptide 12%, p<0.01)). In other words, compared with the control peptide, MVQYKDSFL induced a 6-fold increase in CTL killing rate (p<0.01), confirming that this peptide can significantly enhance the specific killing ability of T cells against NUT cancer cells. These results indicate that DC-CTL cell immunotherapy targeting MVQYKDSFL has potential therapeutic efficacy for NUT cancer.

[0070] While specific embodiments of the invention have been described in detail by way of examples, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of the invention. Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims

1. A neoantigen polypeptide targeting BRD4::NUTM1 gene fusion mutations, characterized in that, The tumor neoantigen polypeptide is: (1) The amino acid sequence is the tumor neoantigen polypeptide shown in SEQ ID No. 2; (2) A polypeptide with the same or similar function obtained by substitution and / or deletion and / or addition of at least one amino acid in the amino acid sequence.

2. A nucleic acid molecule encoding a tumor neoantigen polypeptide as described in claim 1.

3. A gene expression vector, characterized in that, The gene expression vector comprises the nucleic acid molecule as described in claim 2.

4. A cell characterized in that, The cell contains the tumor neoantigen polypeptide of claim 1, or the nucleic acid molecule of claim 2, or the gene expression vector of claim 3.

5. The use of the tumor neoantigen polypeptide according to claim 1, the nucleic acid molecule according to claim 2, the gene expression vector according to claim 3, or the cell according to claim 4 in any of the following: (1) Application in the preparation of drugs for the prevention or treatment of NUT cancer with BRD4::NUTM1 gene fusion mutation; (2) Application in the preparation of diagnostic reagents for NUT cancer with BRD4::NUTM1 gene fusion mutation; (3) Application in the preparation of specific T cells for NUT cancer with BRD4::NUTM1 gene fusion mutation.

6. A specific T cell, characterized in that, The specific T cells are induced by the tumor neoantigen polypeptide of claim 1 and specifically target the tumor neoantigen polypeptide of claim 1.

7. A pharmaceutical composition for treating NUT cancer with BRD4::NUTM1 gene fusion mutation, characterized in that, The pharmaceutical composition comprises the tumor neoantigen polypeptide of claim 1, the nucleic acid molecule of claim 2, the gene expression vector of claim 3, the cell of claim 4, or the specific T cell of claim 6, and a pharmaceutically acceptable carrier.

8. A vaccine, characterized in that, The vaccine comprises the tumor neoantigen polypeptide of claim 1 or the nucleic acid molecule of claim 2.

9. The tumor neoantigen polypeptide targeting the BRD4::NUTM1 gene fusion mutation according to claim 1, characterized in that, The tumor neoantigen polypeptide was obtained through the following method: (1) The antigenic potential of top recurrent somatic mutations was assessed by calculating the binding affinity between the mutant epitope and major histocompatibility complex (MHC) class I. (2) The immune epitope database (IEDB) algorithm was used to analyze the protein structure, surface accessibility and hydrophilicity of the antigen; (3) Screening epitopes with potential immunogenicity from the predicted neoantigens, and designing peptides with specific amino acid sequences based on the identified antigenic recognition regions.

10. A method for verifying the in vitro immunogenicity of the tumor neoantigen polypeptide of claim 1, characterized in that, The method includes the following steps: (1) In vitro isolation of peripheral blood mononuclear cells (PBMCs) from tumor patients; (2) Incubate PBMCs with tumor neoantigen peptides; (3) T cell IFN-γ secretion was detected by enzyme-linked immunospot assay (ELISPOT) to assess the immunogenicity of tumor neoantigen peptides.

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