Compositions and methods for the treatment or prophylactic treatment of cancer, including triple-negative breast cancer and / or lung cancer.

Abstract

Compositions and methods for treating, and for prophylactically treating, subjects diagnosed with or at risk of having breast cancer, including triple-negative breast cancer, and / or subjects diagnosed with or at risk of having lung cancer.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority based on U.S. Patent Application No. 63 / 495,005, filed on April 7, 2023, the entire content of which is hereby incorporated by reference into this specification.

[0002] [Description of Research or Development by Federal Government Support] None.

[0003] [Reference to Electronic Sequence Listing] The content of the electronic sequence listing (650053.01062.xml; size: 29,855 bytes; and creation date: April 5, 2024) is hereby incorporated by reference into this specification in its entirety.

Background Art

[0004] Breast cancer is a major malignancy in women, with an estimated 281,550 new cases and an estimated 43,600 deaths reported in the United States in 2021. Approximately 20% of breast cancers are classified as triple - negative breast cancer (TNBC) because they do not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), which includes the basal - like and more recently defined claudin - low subtypes as molecular subtypes. TNBC is also associated with African - American race, younger age, higher grade and mitotic index, and advanced stage at diagnosis.

Summary of the Invention

Means for Solving the Problems

[0005] Compositions and methods for treating a subject and / or for prophylactic treatment of a subject are disclosed herein.

[0006] In various aspects, the method for the treatment and / or prophylactic treatment of a subject is for the treatment and / or prophylactic treatment of cancer including breast cancer and / or lung cancer.

[0007] In various aspects, the method for the treatment and / or prophylactic treatment of a subject is for the treatment and / or prophylactic treatment of breast cancer including TNBC.

[0008] In various aspects, the method for the treatment and / or prophylactic treatment of a subject is for the treatment and / or prophylactic treatment of lung cancer.

[0009] One or more polypeptides comprising or consisting of a fragment of topoisomerase 2 alpha (Top2A) are disclosed herein. In various aspects, the one or more polypeptides include or consist of one or more of a polypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1; a polypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2; or a polypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 3. In one or more aspects, the one or more polypeptides include or consist of a polypeptide having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2 and a polypeptide having the sequence of SEQ ID NO: 3.

[0010] Compositions comprising one or more polypeptides are disclosed herein. In various embodiments, the one or more polypeptides comprise one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In one or more embodiments, the one or more polypeptides comprise one or more polypeptides having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, and a polypeptide having the sequence of SEQ ID NO: 3. In one or more embodiments, the composition may or may optionally include an adjuvant.

[0011] Compositions comprising one or more polynucleotides are also disclosed herein. In various embodiments, one or more polynucleotides encode one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0012] Disclosed herein are methods for treating a subject or for prophylactic treatment of a subject, comprising the step of administering a composition comprising one or more polypeptides to the subject. In various embodiments, the one or more polypeptides comprise one or more polypeptides that are at least 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 90%, 95%, or 99% identical to SEQ ID NO: 3. In various embodiments, the one or more polypeptides comprise a polypeptide having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, and / or a polypeptide having the sequence of SEQ ID NO: 3. In various embodiments, the composition may or may optionally include an adjuvant. In some embodiments, the subject has not been previously diagnosed with or treated for cancer, breast cancer, or TNBC. In various embodiments, the subject has been previously diagnosed with cancer, breast cancer, and / or TNBC. In one or more embodiments, subjects are diagnosed with breast cancer, and the breast cancer does not express one or more of the following: estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor 2 (HER2). In various embodiments, subjects exhibit one or more risk factors associated with TNBC, selected from pregnancy, multiple child birth, and obesity. In certain embodiments, at least one or at least two doses of a therapeutically effective amount of the composition are administered to the subjects. [Brief explanation of the drawing]

[0013] [Figure 1A-1C]This section depicts data related to the overexpression of the Top2A gene in mouse and human TNBC tissues. (Figure 1A) Overexpression of the Top2A gene in M6 mouse mammary cancer cells versus M28 normal mammary cells; both cell lines were derived from C3(1) / Tag mice. (Figure 1B) Overexpression of the Top2A gene in human TNBC. (Figure 1C) Overexpression of the Top2A gene in African American (AA) TNBC. [Figure 2A] This paper depicts data related to the overexpression of the Top2A protein, as identified in human breast cancer tissue microarrays and mouse TNBCs derived from C3(1) / Tag mice. (Figure 2A) IHC staining of Top2A in breast cancer specimens of two types of human breast TMA (BRC961 and 962). a. Comparison of IHC staining results between normal and malignant tissues. **P<0.01. b. Comparison of IHC staining results in whole tissue, normal, inflammatory, hyperplastic, benign, non-invasive, malignant, and sarcoma. c. Representative IHC staining score index of Top2A in human breast TMA. [Figure 2B] This paper depicts data related to the overexpression of the Top2A protein observed in human breast cancer tissue microarrays and mouse TNBCs derived from C3(1) / Tag mice. (Figure 2B) Representative images of IHC staining of Top2A in 20-week-old C3(1) / Tag mammary gland tissue. a. Mammary gland tissue from a wild-type C3(1) / Tag mouse littermate. b-c. Mammary gland tissue from C3(1) / Tag mouse #1 and mouse #3, respectively. All images were scanned and captured using the NanoZoomer system (Hamamatsu Photonics, Hamamatsu, Japan). [Figure 2C]This paper depicts data related to the overexpression of the Top2A protein observed in human breast cancer tissue microarrays and mouse TNBC derived from C3(1) / Tag mice. (Figure 2C) Western blot analysis of protein expression in mouse breast cancer cell lines. (a) Representative image of the Western blot. (b) Top2A protein expression levels were evaluated by Western blot analysis in the following cell lines: M28, normal control; M27, weakly tumorigenic / benign tumor; M6, malignant tumor; M6C, metastatic tumor. [Figure 3A] This diagram depicts data related to in vivo screening of the Top2A peptide by ELISPOT in C3(1) / Tag-REAR mice. (Figure 3A) Representative immunogenicity heatmap for Top2A in FVB mice. The complete mouse Top2A protein sequence shows the identified immunogenicity "hotspots" for FVB mice. The colors represent the percentage of the highest score from three different algorithms for each amino acid, in order of rank score, from dark red to light blue. The color strata are as follows: dark red = ≥75% of the highest score; red = 50-75% of the highest score; orange = 40-50% of the highest score; yellow = 30-40% of the highest score; green = 20-30% of the highest score; blue = ≤20% of the highest score. [Figure 3B] This diagram illustrates data related to in vivo screening of Top2A peptides by ELISPOT in C3(1) / Tag-REAR mice. (Figure 3B) Selected peptide sequences of human Top2A based on a combined scoring system. Sequence IDs 1, 2, and 3 are shown in descending order. [Figure 3C]This section depicts data related to in vivo screening of Top2A peptides by ELISpot in C3(1) / Tag-REAR mice. (Figure 3C) IFN-γ-based ELISpot assay results. Splenocytes were collected from vaccinated mice and pulsed with a negative control peptide (HIV peptide), a positive control peptide (concanavalin A), and three individual Top2A peptides or combinations thereof (combos). After 72 hours of incubation, the ELISpot assay was performed, plates were scanned, and the number of spots was statistically analyzed. Data are shown as mean ± SE of wells replicated three times per group, n=5, ***, p<0.001. [Figure 4A] Data related to the in vivo immunogenicity of the Top2A peptide are depicted (Figure 4A & Figure 4B). Demonstration of Top2A peptide specificity using peptide-loaded MHC class II tetramers. A. Top2A peptide-specific tetramer staining of spleen CD4 T cells from mice vaccinated with Top2A. *p<0.05. [Figure 4B] Figures 4A and 4B illustrate data related to the in vivo immunogenicity of the Top2A peptide. Demonstration of Top2A peptide specificity using peptide-loaded MHC class II tetramers. B. Representative flow cytometry plots of Top2A tetramer + CD4 T cells. [Figure 4C] Data related to the in vivo immunogenicity of the Top2A peptide are depicted (Figure 4C & Figure 4D). The Top2A peptide induced proliferation of spleen CD4+ T cells isolated from vaccinated mice. C. Isolated CD4+ T cells were labeled with CFSE and stimulated with Top2A peptide-treated mature DCs. *p<0.05, **p<0.01, ***p<0.001. [Figure 4D]Data related to the in vivo immunogenicity of the Top2A peptide are depicted. (Figures 4C & 4D) The Top2A peptide induced proliferation of spleen CD4+ T cells isolated from vaccinated mice. D. Representative flow cytometry histogram of CFSE-stained CD4+ T cells. Percentages indicate the frequency of dividing CD4+ T cells. [Figure 5A] Data illustrating Top2A vaccination inhibited tumor growth in C3(1) / Tag-REAR mice are shown. (Figure 5A) Experimental design and timeline of vaccine administration. [Figure 5B] Data illustrating Top2A vaccination inhibited tumor growth in C3(1) / Tag-REAR mice (Figure 5B). Tumor growth curve after M6 tumor cell inoculation. After implantation, tumor diameter was measured using a digital caliper, and tumor volume was calculated using the following formula: maximum diameter × (minimum diameter)² × 0.4. Data are shown as mean ± SE, *P<0.05, **P<0.01, ***P<0.001. [Figure 5C] Data illustrating Top2A vaccination inhibited tumor growth in C3(1) / Tag-REAR mice are depicted (Figure 5C). The weight of each tumor was measured at the experimental endpoint. Data are shown as mean ± SE, n=6-7 mice per group, *P<0.05. [Figure 5D] Data illustrating that Top2A vaccination inhibited tumor growth in C3(1) / Tag-REAR mice are depicted (Figure 5D). Top2A vaccination significantly increased the percentage of functionally activated CD4+ and CD8+ cells in the spleen. Cells isolated from the spleens of Top2A-vaccinated mice and adjuvant control mice were intracellularly stained for granzyme B, IFN-γ, and TNFα. *P<0.05, **P<0.01. [Figure 6A-6B]This paper depicts data demonstrating that Top2A vaccination of C3 / Tag genetically engineered mice prevented mammary tumor development. (Figure 6A) Experimental design for this experiment. (Figure 6B) Overview of tumor development in vaccinated C3 / Tag genetically engineered mice at 20 weeks of age. Estimated tumor volume is depicted based on palpable tumor development; diameter was measured using a digital caliper and tumor volume was calculated using the following formula: maximum diameter × (minimum diameter)² × 0.4. *P<0.05. [Figure 6C-6D] Data illustrating that Top2A vaccination of C3 / Tag genetically modified mice prevented mammary tumor development are depicted. (Figure 6C) Immunohistochemical (IHC) analysis was performed to determine invasive CD4 and CD8 T cells infiltrating the tumor tissue. The number of positive cells in the field of view is expressed as the number of CD4+ and CD8+ cells per 1 mm2 of tumor area. **p<0.01. (Figure 6D) Representative images of H&E and IHC staining for CD4 and CD8 in tumors vaccinated with Top2A. All images were scanned using the NanoZoomer system (Hamamatsu Photonics, Hamamatsu, Japan). [Figure 7] This study describes data related to cytokine production by spleen T cells derived from Top2A-vaccinated mice. Twelve cytokines were analyzed using the QIAGEN ELISArray kit. Splenocytes were collected from either Top2A-vaccinated mice or adjuvant-only mice and co-cultured with the Top2A vaccine for 72 hours. The supernatant was collected and assayed using the QIAGEN ELISArray kit according to the manufacturer's instructions. Data are shown as mean ± SE, for CpG only (n=3) and Top2A (n=3). *P<0.05, ***P<0.001 (compared to CpG only). [Figures 8A-8B]Data illustrating that C3(1) / Tag mice vaccinated with Top2A were protected from secondary exposure to M6 cells. (Figure 8A) Schematic diagram of the experimental design and timeline of vaccine administration. (Figure 8B) Tumor growth curve after M6 tumor cell dissemination in 22-week-old C3(1) / Tag mice. 5 × 10⁵ cells were injected into the #4 mammary fat body of female C3(1) / Tag mice. After implantation, tumor diameter was measured using a digital caliper, and tumor volume was calculated using the following formula: maximum diameter × (minimum diameter)² × 0.4. Control C3(1) / Tag female mice (n=3) were unvaccinated. Top2A C3(1) / Tag female mice (n=3) were vaccinated with Top2A. No tumors were present in the #4 mammary fat body at the time of M6 inoculation. [Figure 8C] Data illustrating that C3(1) / Tag mice vaccinated with Top2A were protected from secondary exposure to M6 cells are depicted (Figure 8C). The weight of each tumor was measured at the end of the experiment. [Figure 8D] Data illustrating that C3(1) / Tag mice vaccinated with Top2A were protected from secondary exposure to M6 cells (Figure 8D). Top2A vaccination increased the number of CD4+ and CD8+ tumor-infiltrating lymphocytes in the treated animals. (a) Representative images of CD4 and CD8 IHC staining in tumor tissue. (b) Quantification of CD8 IHC images. The number of positive cells in the field of view is expressed as the number of CD4+ and CD8+ cells per 1 mm2 of tumor area. All images were scanned using a NanoZoomer system (Hamamatsu Photonics, Hamamatsu, Japan). All data are shown as mean ± SE, *P<0.05, **P<0.01, ***p<0.001. [Figure 9A] This depicts data related to clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (Figure 9A) Expression of marker genes for CD8+T, CD4+T, DNT, DC, macrophages, and neutrophils in mouse TNBC tumor samples from mice vaccinated with control Top2A. [Figure 9B] This depicts data related to clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (Figure 9B) Landscape of the overall immune cell population from tumor samples of control and Top2A-vaccinated mice. [Figure 9C] This depicts data related to clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (Figure 9C) Expression of marker genes for CD8+T, CD4+T, DNT, DC, and macrophages in lymph node samples from control and Top2A-vaccinated mice. [Figure 9D] This depicts data related to clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (D) Landscape of immune cells in lymph nodes of control and Top2A-vaccinated mice. [Figure 10A-10B] This section depicts data demonstrating the efficacy of the Top2A vaccine in CD8+ T cell subsets in breast tumor (Figures 10A-10D) and lymph node (Figures 10E-10H) tissues. (Figure 10A) Canonical marker expression was selected from CD8+ T cell subsets based on scRNA-seq data. (Figure 10B) Heatmap of marker expression for each CD8+ T cell subset. [Figure 10C-10D] Data illustrating the efficacy of the Top2A vaccine on CD8+ T cell subsets in breast tumor (Figures 10A–10D) and lymph node (Figures 10E–10H) tissues are depicted. (Figure 10C) Distribution of each CD8+ T cell subset; (Figure 10D) Percentage change in CD8+ T cell subsets across control and TOP2A vaccine-treated groups. [Figures 10E-10F]This section depicts data demonstrating the efficacy of the Top2A vaccine in CD8+ T cell subsets in breast tumor (Figures 10A-10D) and lymph node (Figures 10E-10H) tissues. (Figure 10E) Canonical marker expression was selected from CD8+ T cell subsets based on scRNA-seq data. (Figure 10F) Heatmap of marker expression for each CD8+ T cell subset. [Figure 10G-10H] Data illustrating the efficacy of the Top2A vaccine on CD8+ T cell subsets in breast tumor (Figures 10A–10D) and lymph node (Figures 10E–10H) tissues are depicted. (Figure 10G) Distribution of each CD8+ T cell subset; (Figure 10H) Percentage change in CD8+ T cell subsets across control and TOP2A vaccine-treated groups. [Figures 11A-11B] Data illustrating the effect of Top2A vaccine treatment on changes in CD4+ T cell subsets in breast tumor (Figures 11A–11D) and lymph node (Figures 11E–11H) tissues are shown. (Figure 11A) Canonical marker expression was selected based on scRNA-seq data for CD4+ T cells. (Figure 11B) Heatmap of marker expression for each CD4+ T cell subset. [Figure 11C-11D] Data illustrating that Top2A vaccine treatment affected changes in CD4+ T cell subsets in breast tumor (Figures 11A–11D) and lymph node (Figures 11E–11H) tissues. (Figure 11C) Distribution of each CD4+ T cell subset; (Figure 11D) Percentage change in CD4+ T cell subsets across control and TOP2A vaccine-treated groups. [Figure 11E-11F] Data illustrating the effect of Top2A vaccine treatment on changes in CD4+ T cell subsets in breast tumor (Figures 11A–11D) and lymph node (Figures 11E–11H) tissues are shown. (Figure 11E) Canonical marker expression was selected based on scRNA-seq data for CD4+ T cells. (Figure 11F) Heatmap of marker expression for each CD4+ T cell subset. [Figure 11G-11H]Data illustrating that Top2A vaccine treatment affected changes in CD4+ T cell subsets in breast tumor (Figures 11A–11D) and lymph node (Figures 11E–11H) tissues. (Figure 11G) Distribution of each CD4+ T cell subset; (Figure 11H) Percentage change in CD4+ T cell subsets across control and TOP2A vaccine-treated groups. [Figure 12A] This depicts data related to the distribution of CD4+ T cell receptor (TCR) clone types in mouse mammary tumor samples from Top2A-vaccinated and unvaccinated control mice. (Figure 12A) Distribution of CD4+ TCR clone types in mammary tumor tissue from Top2A-vaccinated mice. [Figure 12B] This depicts data related to the distribution of CD4+ T cell receptor (TCR) clone types in mouse mammary tumor samples from Top2A-vaccinated and untreated control mice. (Figure 12B) Distribution of CD4+ TCR clone types in mammary tumor tissue from untreated CpG control mice. [Figures 13A-13B] This paper describes data related to TCR-peptide binding prediction for three Top2A peptide epitopes assayed using tetramers. Predicted binding of the following peptides to CD4+ TCR clone types detected in mammary tumor tissue from Top2A-vaccinated mice: (A) First Top2A peptide - KDIVALMVRRAYDIA (SEQ ID NO: 1); (B) Second Top2A peptide - ILNWVKFKAQVQLNKK (SEQ ID NO: 2). [Figure 13C] This paper describes data related to TCR-peptide binding prediction for three Top2A peptide epitopes assayed using tetramers. Predicted binding of the following peptides to CD4+ TCR clones detected in mammary tumor tissue from Top2A-vaccinated mice: (C) Third TOP2A peptide - KKWKVKYYKGLGTSTSK (SEQ ID NO: 3). [Figures 14A-14B]Data illustrating the efficacy of Top2A vaccine treatment in DNT cells (CD4-CD8-double-negative T cells) in breast tumor samples are depicted. (Figure 14A) UMAP plots were generated from scRNA-seq data using canonical T cell marker expression. (Figure 14B) Heatmaps of marker expression for each DNT cell subset. [Figure 14C-14D] This section depicts data demonstrating the efficacy of Top2A vaccine treatment on DNT cells (CD4-CD8-double-negative T cells) in breast tumor samples. (Figure 14C) Distribution of each DNT cell subset. (Figure 14D) Percentage change in DNT cell subsets across control and Top2A vaccine treatment groups. [Figures 15A-15B] Data illustrating the efficacy of Top2A vaccine treatment in DNT cell subsets in lymph node tissue are depicted. (Figure 15A) UMAP plots were generated from scRNA-seq data using canonical marker expression on the surface of DNT cells. (Figure 15B) Heatmaps of markers expressed by each DNT cell subset. [Figures 15C-15D] The data illustrates the effect of Top2A vaccine treatment on DNT cell subsets in lymph node tissue. (Figure 15C) UMAP plot distribution of each DNT cell subset. (Figure 15D) Percentage change in DNT cell subsets across control and TOP2A vaccine treatment groups. [Figures 16A-16B] Data illustrating the efficacy of the Top2A vaccine in macrophages in breast tumor samples are depicted. (Figure 16A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of macrophages. (Figure 16B) Heatmaps based on marker expression for each macrophage subset, M1 (antitumor) and M2 (tumor-promoting) macrophages. [Figures 16C-16D]Data illustrating the efficacy of the Top2A vaccine on macrophages in mammary tumor samples are depicted. (Figure 16C) UMAP plot distribution of M1 and M2 macrophage subsets. (Figure 16D) Percentage change in M1 and M2 macrophage subsets in control vs. TOP2A vaccine-treated mice. [Figures 17A-17B] Data illustrating the efficacy of Top2A vaccine treatment in macrophages in lymph nodes are depicted. (Figure 17A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of macrophages. (Figure 17B) Heatmaps based on marker expression for M1 and M2 macrophage subsets. [Figures 17C-17D] The data illustrates the effect of Top2A vaccine treatment on macrophages in lymph nodes. (Figure 17C) UMAP plot distribution of M1 and M2 macrophage subsets. (Figure 17D) Percentage change in M1 and M2 macrophage subsets in control vs. TOP2A vaccine-treated mice. [Figures 18A-18B] This section depicts data demonstrating the efficacy of the Top2A vaccine in dendritic cells (DCs) in breast tumor samples. (Figure 18A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of DCs. (Figure 18B) Heatmaps based on marker expression for each DC subset, conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). [Figures 18C-18D] Data illustrating the efficacy of the Top2A vaccine in dendritic cells (DCs) in mammary tumor samples are depicted. (Figure 18C) UMAP plot distribution of cDC and pDC subsets. (Figure 18D) Percentage change in cDC and pDC subsets in control vs. TOP2A vaccine-treated mice. [Figures 19A-19B]Data illustrating the efficacy of Top2A vaccine treatment in dendritic cells (DCs) in lymph node samples are depicted. (Figure 19A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the DC surface. (Figure 19B) Heatmaps based on marker expression for cDC and pDC subsets. [Figures 19C-19D] The data illustrate the effect of Top2A vaccine treatment on DCs in lymph node samples. (Figure 19C) UMAP plot distribution of cDC and pDC subsets. (Figure 19D) Percentage change in cDC and pDC subsets in control vs. TOP2A vaccine-treated mice. [Figures 20A-20B] Data illustrating the efficacy of Top2A vaccine treatment on neutrophils in breast tumor samples are depicted. (Figure 20A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of neutrophils. (Figure 20B) Heatmaps based on marker expression for neutrophil subsets, stage I and stage II. [Figures 20C-20D] Data illustrating the effect of Top2A vaccine treatment on neutrophils in mammary tumor samples are depicted. (Figure 20C) UMAP plot distribution of stage I and stage II neutrophil subsets. (Figure 20D) Percentage change in stage I and stage II subsets in control vs. TOP2A vaccine-treated mice. [Figure 21A] Figure 21A shows the assay plate and depicts data related to the peptide IFN-γ ELISPOT assay as detailed in Example 13. [Figure 21B] Figure 21B is a table listing the peptides that were tested. [Figure 22] Figure 21A shows a bar graph of the IFN-γ ELISPOT assay. [Figures 23A-23C]The experimental design and data showing that the Top2A vaccine inhibited lung tumor progression in a synergistic model of lung cancer are depicted. (Figure 23A) Experimental design and timeline of vaccine administration. (Figure 23B) Tumor growth curve after inoculation of LKR13 tumor cells. After implantation, tumor diameter was measured using a digital caliper and tumor volume was calculated using the following formula: maximum diameter × (minimum diameter)² × 0.4. Data are shown as mean ± SE, **P<0.01, p-value calculated using one-way ANOVA. (Figure 23C) Survival analysis of LKR13 tumor cell-inoculated mice (n=5). Statistical significance was calculated using the log-rank (Mantel-Cox) test, ****P<0.0001. [Figures 23D-23F] The experimental design and data showing that the Top2A vaccine inhibited lung tumor progression in a synergistic model of lung cancer are depicted. (Figure 23D) Experimental design and timeline of vaccine administration. (Figure 23E) LLC (Figure 23E) Tumor growth curve after tumor cell inoculation. After implantation, tumor diameter was measured using a digital caliper and tumor volume was calculated using the following formula: maximum diameter × (minimum diameter)² × 0.4. Data are shown as mean ± SE, **P<0.01, p-value calculated using one-way ANOVA. (Figure 23F) LLC (Figure 23F) Survival analysis of tumor cell-inoculated mice (n=5). Statistical significance was calculated using the log-rank (Mantel-Cox) test, ****P<0.0001. [Figure 24A] OVA & TOP2A mRNA vaccine in a lung metastasis model. (Figure 24A) mRNA vaccine design. [Figure 24B] OVA & TOP2A mRNA vaccine in a lung metastasis model. (Figure 24B) Using lung metastasis as the endpoint, C57BL / 6 mice were inoculated with B16 melanoma cells and immunized with OVA-mRNA LNP. [Figure 24C]OVA & TOP2A mRNA vaccine in a lung metastasis model. (Figure 24C) Using lung metastasis as the endpoint, SV129 mice were inoculated with LKR13 lung tumor cells and immunized with TOP2A-mRNA-LNP. ***P<0.001. [Figure 25] IEDB predictions of binding affinity to TOP2A. The binding affinity predicted by the IEDB algorithm is plotted based on the percentile ranking of each amino acid and mapped to the amino acid sequence of TOP2A, with red indicating more alleles binding to the region. [Figure 26] World population coverage calculated by the IEDB. [Figure 27A] Expression of cyclin E and KIF15 in human and C3(1) / Tag mouse-derived TNBCs, and immune responses to selected epitopes. (Figure 27A) Overexpression of cyclin E and KIF15 in human TNBCs. [Figures 27B-27C] Expression of cyclin E and KIF15 in human and C3(1) / Tag mouse-derived TNBCs, and immune responses to selected epitopes. (Figures 27B & 27C) Overexpression of cyclin E in C3(1) / Tag mouse-derived TNBCs. [Figures 27D-27E] Expression of cyclin E and KIF15 in human and C3(1) / Tag mouse-derived TNBCs, and immune responses to selected epitopes. (Figures 27D and 27E) Overexpression of KIF15 in C3(1) / Tag mouse-derived TNBCs. [Figure 28A] Cyclin E2 and KIF15 vaccination are effective in inhibiting TNBC growth in a C3(1)Tag-RARE syngeneic mouse model (Figure 28A), IFNγ ELISPOT in splenocytes after completion of four immunization cycles. HIV p52 was used as a negative control. [Figure 28B]Cyclin E2 and KIF15 vaccination is effective in inhibiting TNBC growth in a C3(1)Tag-RARE syngeneic mouse model. N=5 mice / group; B&C, mean tumor volume (mm3±SEM) from mice injected with adjuvant (CpG) and cyclin E2 peptide (Figure 28B) in C3(1)Tag mice, n=5 mice / group; *P<0.05. [Figure 28C] Cyclin E2 and KIF15 vaccination are effective in inhibiting TNBC growth in a C3(1)Tag-RARE syngeneic mouse model. N=5 mice / group; B&C, mean tumor volume (mm3±SEM) from mice injected with adjuvant (CpG) and KIF15 peptide (Figure 28C) in C3(1)Tag mice, n=5 mice / group; *P<0.05. [Figure 29] The preventive effect of the combination of cyclin E2, KIF15, and TOP2A vaccine on TNBC development in the C3(1) / Tag GEM model. N=9-10 mice / group; *P<0.05. [Figure 30A] TOP2A vaccination induced an immune response and prevented mammary tumor development in C3 / Tag mice. (Figure 30A) Representative immunogenicity heatmap for TOP2A in FVB mice. The complete mouse TOP2A protein sequence shows identified immunogenicity "hotspots" for FVB mice. The colors, from dark red to light blue, represent the percentage of the highest score from three different algorithms for each amino acid, in order of rank score. [Figures 30B-30C]TOP2A vaccination induced an immune response and prevented mammary tumor development in C3 / Tag mice. (Figure 30B) Representative IFN-γ-based ELISpot assay results showing the T cell response to specific TOP2A peptides derived from mouse splenocytes. (Figure 30C) IFN-γ-based ELISpot assay results. Splenocytes were collected from vaccinated mice and pulsed with a negative control peptide (HIV peptide), a positive control peptide (concanavalin A), three individual TOP2A peptides, or combinations thereof (combo). Data are shown as mean ± SE of wells replicated three times per group, n=5, ****p<0.0001. [Figures 30D-30E] TOP2A vaccination induced an immune response and prevented mammary tumor development in C3 / Tag mice. (Figure 30D) Experimental design for this experiment. (Figure 30E) Overview of tumor development in 20-week-old vaccinated C3 / Tag genetically engineered mice. Estimated tumor volume is depicted based on palpable tumor development. *p<0.05. [Figures 30F-30G] TOP2A vaccination induced an immune response and prevented mammary tumor development in C3 / Tag mice. (Figure 30F) Representative IHC staining and quantitative data for tumor-infiltrating (infiltration) CD4+ cells from mammary tumor tissue. (Figure 30G) Representative IHC staining and quantitative data for tumor-infiltrating CD8+ cells from mammary tumor tissue. The number of positive cells in the field of view is expressed as the number of CD4+ cells (Figure 30F) and CD8+ cells (Figure 30G) per 1 mm2 of tumor area. **p<0.01. [Figure 30H] TOP2A vaccination induced an immune response and prevented mammary tumor development in C3 / Tag mice (Figure 30H). Twelve cytokines were analyzed using the ELISArray kit, QIAGEN, to assess bulk cytokine production by T cells in the culture supernatant. Splenocytes were collected from either TOP2A-vaccinated mice or adjuvant-only mice and co-cultured with TOP2A vaccine for 72 hours: Adj (CpG only, n=3), TOP2A (n=3). [Figure 30I-30K]TOP2A vaccination induced an immune response and prevented mammary tumor development in C3 / Tag mice. (Figures 30I-30K) Body weight, ALT, and AST levels at 20 weeks of age. Data are shown as mean ± SE, *p<0.05, ***p<0.001 (two-sided t-test compared to CpG only). [Figure 31A] Figure 2. TOP2A vaccination inhibited tumor growth in the syngeneic C3(1) / Tag-REAR mouse model. (Figure 31A) Experimental design and timeline of vaccine administration. [Figure 31B] Figure 2. TOP2A vaccination inhibited tumor growth in the syngeneic C3(1) / Tag-REAR mouse model. (Figure 31B) Tumor growth curve after M6 tumor cell inoculation. After implantation, tumor diameter was measured using a digital caliper, and tumor volume was calculated using the following formula: maximum diameter × (minimum diameter)² × 0.4. Data are shown as mean ± SE, *p<0.05, **p<0.01, ***p<0.001 (two-sided t-test). [Figure 31C] Figure 2 shows that TOP2A vaccination inhibited tumor growth in the syngeneic C3(1) / Tag-REAR mouse model. (Figure 31C) The weight of each tumor was measured at the experimental endpoint. Data are shown as mean ± SE, n=6-7 mice per group, *P<0.05. [Figures 31D-31F] Figure 2. TOP2A vaccination inhibited tumor growth in the syngeneic C3(1) / Tag-REAR mouse model. (Figures 31D-31F) TOP2A vaccination significantly increased the percentage of functionally activated D4+ and CD8+ cells in the spleen. Cells isolated from the spleen were stained for intracellular markers (granzyme B, IFN-γ, and TNFα). *p [Figure 31G-31I] Figure 2. TOP2A vaccination inhibited tumor growth in the syngeneic C3(1) / Tag-REAR mouse model. (Figures 31G-31I) TOP2A vaccination significantly increased the percentage of functionally activated D4+ and CD8+ cells in the spleen. Cells isolated from the spleen were stained for intracellular markers (granzyme B, IFN-γ, and TNFα). *p [Figure 32A] Figure 3 shows that C3(1) / Tag mice vaccinated with TOP2A were protected from secondary exposure to M6 cells. (Figure 32A) Schematic diagram of the experimental design and timeline of vaccine administration. [Figures 32B-32C] Figure 3. C3(1) / Tag mice vaccinated with TOP2A were protected from secondary exposure to M6 cells. (Figure 32B) Tumor growth curve after M6 tumor cell inoculation in 22-week-old C3(1) / Tag mice. 5 × 10⁵ cells were injected into the #4 mammary fat body of female C3(1) / Tag mice. After implantation, tumor diameter was measured using a digital caliper, and tumor volume was calculated using the following formula: maximum diameter × (minimum diameter)² × 0.4. Control C3(1) / Tag female mice (n=4) were not vaccinated. C3(1) / Tag female mice (n=3) were vaccinated with TOP2A. No tumors were present in the #4 mammary fat body at the time of M6 inoculation. (Figure 32C) The weight of each tumor was measured at the end of the experiment. (Figures 32D-32E) TOP2A vaccination was effective in improving CD4+ in treated animals. [Figures 32D-32E] Figure 3 shows that C3(1) / Tag mice vaccinated with TOP2A were protected from secondary exposure to M6 cells (Figures 32D-32E). TOP2A vaccination increased the number of CD4+ (Figure 32D) and CD8+ (Figure 32E) tumor-infiltrating lymphocytes in the treated animals. The number of positive cells in the field of view is expressed as the number of CD4+ and CD8+ cells per 1 mm2 of tumor area. All data are shown as mean ± SE, *p<0.05, **p<0.01, ***p<0.001 (two-sided t-test). [Figures 33A-33B] Figure 4. Efficacy of TOP2A vaccine in CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33A) Canonical marker expression was selected from CD8+ T cell subsets based on scRNA-seq data. (Figure 33B) Heatmap of marker expression for each CD8+ T cell subset. [Figures 33C-33D]Figure 4. Effect of TOP2A vaccine on CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33C) Distribution of each CD8+ T cell subset; (Figure 33D) Percentage change in CD8+ T cell subsets across control and TOP2A vaccine-treated groups. [Figures 33E-33F] Figure 4. Efficacy of TOP2A vaccine in CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33E) Canonical marker expression was selected from CD8+ T cell subsets based on scRNA-seq data. (Figure 33F) Heatmap of marker expression for each CD8+ T cell subset. [Figure 33G-33H] Figure 4. Effect of TOP2A vaccine on CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33G) Distribution of each CD8+ T cell subset; (Figure 33H) Percentage change in CD8+ T cell subsets across control and TOP2A vaccine-treated groups. [Figure 33I-33J] Figure 4. Efficacy of TOP2A vaccine in CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33I) Canonical marker expression was selected from CD4+ T cells based on scRNA-seq data. (Figure 33J) Heatmap of marker expression for each CD4+ T cell subset. [Figures 33K-33L] Figure 4. Effect of TOP2A vaccine on CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33K) Distribution of each CD4+ T cell subset; (Figure 33L) Percentage change in CD4+ T cell subsets across control and TOP2A vaccine-treated groups. [Figure 33M-33N] Figure 4. Efficacy of TOP2A vaccine in CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33M) Canonical marker expression was selected from CD4+ T cells based on scRNA-seq data. (Figure 33N) Heatmap of marker expression for each CD4+ T cell subset. [Figures 33O-33P]Figure 4. Effect of TOP2A vaccine on CD8+ T and CD4+ T cell subsets in breast tumors and lymph node tissue. (Figure 33O) Distribution of each CD4+ T cell subset; (Figure 33P) Percentage change in CD4+ T cell subsets across control and TOP2A vaccine-treated groups. [Figure 34A] CD4+ TCR clone type distribution in mouse mammary tumor samples, and TCR-peptide binding prediction for three TOP2A peptides. (Figure 34A) Distribution of CD4+ TCR clone types in mammary tumor tissue from TOP2A-vaccinated mice. [Figure 34B] Distribution of CD4+TCR clone types in mouse mammary tumor samples, and prediction of TCR-peptide binding for three TOP2A peptides. (Figure 34B) Distribution of CD4+TCR clone types in mammary tumor tissue from untreated CpG control mice. Predicted binding of the following peptides to the CD4+TCR clone types detected in mammary tumor tissue from TOP2A-vaccinated mice. [Figures 34C-34D] CD4+ TCR clonal distribution in mouse mammary tumor samples, and TCR-peptide binding prediction for three TOP2A peptides. (Figure 34C) First TOP2A peptide - KDIVALMVRRAYDIA (SEQ ID NO: 1); (Figure 34D) Second TOP2A peptide - ILNWVKFKAQVQLNKK (SEQ ID NO: 2). [Figure 34E] CD4+ TCR clonal distribution in mouse mammary tumor samples, and TCR-peptide binding prediction for three TOP2A peptides. (Figure 34E) Third TOP2A peptide - KKWKVKYYKGLGTSTSK (SEQ ID NO: 3). [Figures 35A-35B] Overexpression of the TOP2A gene in mouse and human TNBC tissue. (Figure 35A) Overexpression of the TOP2A gene in M6 mouse mammary cancer cells versus M28 normal mammary cells; both cell lines were derived from C3(1) / Tag mice. (Figure 35B) Overexpression of the TOP2A gene in human TNBC. [Figure 35C]Overexpression of the TOP2A gene in mouse and human TNBC tissue. (Figure 35C) Overexpression of the TOP2A gene in African American (AA) TNBC. [Figures 35D-35F] Overexpression of the TOP2A gene in mouse and human TNBC tissue. (Figures 35D-35F) Immunohistochemistry of TOP2A expression in mammary tissue of C3(1) / Tag mice. (Figure 35D) Expression in mammary tissue from wild-type littermates of C3(1) / Tag mice. (Figure 35E) Expression in ductal carcinoma in situ (DCIS). (Figure 35F) Expression in invasive cancer. All images were scanned and captured using the NanoZoomer system (Hamamatsu Photonics, Hamamatsu, Japan). [Figures 36A-36B] IFN-γ-based ELISpot assay in C3(1) / Tag mice. (Figure 36A) Representative IFN-γ-based ELISpot assay results showing T cell response to specific TOP2A peptides derived from mouse splenocytes. (Figure 36B) IFN-γ-based ELISpot assay results. Splenocytes were collected from vaccinated mice and pulsed with a negative control peptide (HIV peptide), a positive control peptide (concanavalin A), three individual TOP2A peptides, or combinations thereof (combo). After 72 hours of incubation, the ELISpot assay was performed, plates were scanned, and the number of spots was statistically analyzed. Data are shown as mean ± SE of wells replicated three times per group, n=5, **, p<0.01****, p<0.0001 (two-sided t-test). [Figure 37A] Clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (Figure 37A) Expression of marker genes for CD8+T, CD4+T, DNT, DC, macrophages, and neutrophils in mouse TNBC tumor samples from mice vaccinated with control TOP2A. [Figure 37B]Clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (Figure 37B) Landscape of the overall immune cell population from tumor samples of control and TOP2A-vaccinated mice. [Figure 37C] Clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (Figure 37C) Expression of marker genes for CD8+T, CD4+T, DNT, DC, and macrophages in lymph node samples from control and TOP2A-vaccinated mice. [Figure 37D] Clustering analysis of scRNA-seq data from TNBC and lymph node tissue immune cells sorted by flow cytometry. (Figure 37D) Landscape of immune cells in lymph nodes of control and TOP2A-vaccinated mice. [Figure 38A] Efficacy of TOP2A vaccine treatment in DNT cells (CD4-CD8-double-negative T cells) in breast tumor samples. (Figure 38A) UMAP plots were generated from scRNA-seq data using canonical T cell marker expression. [Figure 38B] Efficacy of TOP2A vaccine treatment in DNT cells (CD4-CD8-double-negative T cells) in breast tumor samples. (Figure 38B) Heatmap of marker expression for each DNT cell subset. [Figure 38C] Efficacy of TOP2A vaccine treatment on DNT cells (CD4-CD8-double-negative T cells) in breast tumor samples. (Figure 38C) Distribution of each DNT cell subset. [Figure 38D] Effect of TOP2A vaccine treatment on DNT cells (CD4-CD8-double-negative T cells) in breast tumor samples. (Figure 38D) Percentage change in DNT cell subsets across control and TOP2A vaccine treatment groups. [Figure 39A]Effect of TOP2A vaccine treatment on a subset of DNT cells in lymph node tissue. (Figure 39A) UMAP plots were generated from scRNA-seq data using canonical marker expression on the surface of DNT cells. [Figure 39B] Efficacy of TOP2A vaccine treatment in DNT cell subsets in lymph node tissue. (Figure 39B) Heatmap of markers expressed by each DNT cell subset. [Figure 39C] Efficacy of TOP2A vaccine treatment in DNT cell subsets in lymph node tissue. (Figure 39C) UMAP plot distribution of each DNT cell subset. [Figure 39D] Effect of TOP2A vaccine treatment on DNT cell subsets in lymph node tissue. (Figure 39D) Percentage change in DNT cell subsets across control and TOP2A vaccine treatment groups. [Figures 40A-40B] Efficacy of TOP2A vaccine in macrophages in breast tumor samples. (Figure 40A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of macrophages. (Figure 40B) Heatmaps based on marker expression for each macrophage subset, M1 (antitumor) and M2 (tumor-promoting) macrophages. [Figures 40C-40D] Effect of TOP2A vaccine on macrophages in mammary tumor samples. (Figure 40C) UMAP plot distribution of M1 and M2 macrophage subsets. (Figure 40D) Percentage change in M1 and M2 macrophage subsets in control vs. TOP2A vaccine-treated mice. [Figure 41A-41B] Efficacy of TOP2A vaccine treatment in macrophages in lymph nodes. (Figure 41A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of macrophages. (Figure 41B) Heatmaps based on marker expression for M1 and M2 macrophage subsets. [Figures 41C-41D]Effect of TOP2A vaccine treatment on macrophages in lymph nodes. (Figure 41C) UMAP plot distribution of M1 and M2 macrophage subsets. (Figure 41D) Percentage change in M1 and M2 macrophage subsets in control vs. TOP2A vaccine-treated mice. [Figures 42A-42B] Efficacy of TOP2A vaccine in dendritic cells (DCs) in breast tumor samples. (Figure 42A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of DCs. (Figure 42B) Heatmaps based on marker expression for each DC subset, conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). [Figures 42C-42D] Efficacy of TOP2A vaccine on dendritic cells (DCs) in mammary tumor samples. (Figure 42C) UMAP plot distribution of cDC and pDC subsets. (Figure 42D) Percentage change in cDC and pDC subsets in control vs. TOP2A vaccine-treated mice. [Figures 43A-43B] Efficacy of TOP2A vaccine treatment in DCs in lymph node samples. (Figure 43A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of DCs. (Figure 43B) Heatmaps based on marker expression for cDC and pDC subsets. [Figures 43C-43D] Effect of TOP2A vaccine treatment on DCs in lymph node samples. (Figure 43C) UMAP plot distribution of cDC and pDC subsets. (Figure 43D) Percentage change in cDC and pDC subsets in control vs. TOP2A vaccine-treated mice. [Figure 44A-44B] Efficacy of TOP2A vaccine treatment in neutrophils in breast tumor samples. (Figure 44A) UMAP plots based on scRNA-seq data were generated using canonical markers expressed on the surface of neutrophils. (Figure 44B) Heatmaps based on marker expression for neutrophil subsets, stage I and stage II. [Figures 44C-44D]Effect of TOP2A vaccine treatment on neutrophils in mammary tumor samples. (Figure 44C) UMAP plot distribution of stage I and stage II neutrophil subsets. (Figure 44D) Percentage change in stage I and stage II subsets in control vs. TOP2A vaccine-treated mice. [Figure 45] Figure 45. Flow gating strategies used for flow cytometry in Figures 31D to 31I. [Modes for carrying out the invention]

[0014] [Detailed explanation] (Summary) Approximately 20% of breast cancers are classified as triple-negative breast cancer (TNBC) because they do not express the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This includes the basal cell-like and, more recently, the claudin-low molecular subtype. TNBC is also associated with African American race, young age, higher grade and mitotic index, as well as advanced stage at diagnosis. Specific risk factors associated with TNBC include germ factors (pregnancy and multiple births) and obesity. In TNBC patients, the 5-year survival rate is significantly lower than in other forms of breast cancer, including the ER+PR+HER- (luminal A) and HER+ subtypes. Luminal A tumors are treated with targeted therapies, such as hormonal agents (tamoxifen, aromatase inhibitors), while HER+ breast cancer is treated with antibodies or small molecule inhibitors. In contrast, early and intermediate stages of TNBC are routinely treated with standard cytotoxic chemotherapy, which results in strong early regression in most patients. However, resistance develops in the majority of patients. Current challenges in treating TNBC necessitate new approaches to improve clinical outcomes.

[0015] Cancer vaccines have so far been primarily used to treat active, late-stage cancers, which likely provides limited efficacy due to immunosuppression inherent in advanced malignancies. Immunosuppression can involve multiple mediators, including regulatory T cells (Tregs), inhibitory macrophages, and other suppressors. One potentially more effective way to use cancer vaccines lies in prophylactic or early progression prevention settings. Several reports have provided proof-of-concept support for the use of peptide vaccines targeting overexpressed autoantigens as immunoprevention for breast cancer (Lollini et al., 2006; Disis et al., 2013; Ebben et al., 2015; Pan J et al., 2017). Molecular analysis of tumors has identified many genes overexpressed in breast cancer that can be utilized as tumor antigens and potential vaccine candidates. Currently, the most common tumor antigens used in cancer immunotherapy are upregulated autoproteins such as HER2. Vaccination with peptides targeting overexpressed HER2 / neu in humans has been shown to be effective and well-tolerated (Schneble et al., 2014; Lowenfeld et al., 2016). Mutant epitopes are recognized by the immune system as exogenous "neoantigens" and induce a type 1 immune response, while epitopes derived from non-mutated autoantigens are more likely to activate T helper 2 (Th2) cytokines such as interleukin (IL)-10 and IL-6, which can inhibit cytotoxic T lymphocyte (CTL) proliferation and function. Recently, attempts have been made to specifically identify Th1-selective epitopes derived from non-mutated autoantigens that can induce neoantigen-like responses. When used in vaccines, Th1-selective epitopes can induce uncompetitive type 1 immunity and may be effective in preventing cancer growth in preclinical models. If a Th2-inducible epitope derived from the same protein is included in the vaccine, immunized Th2 cells can suppress the Th1-mediated antitumor effect (Cecil et al., 2014; Disis et al., 1996).If the antigen is expressed in the early stages of cancer development, the vaccine may be useful in prevention.

[0016] In this disclosure, we used the Cancer Genome Atlas (TCGA) database, in conjunction with transcriptomics and / or proteomics analyses of normal, versus, and malignant human breast tissue, to identify genes highly expressed in malignant tissue, and found that topoisomerase 2 alpha (Top2A) is highly expressed in human TNBC. Top2A is a key enzyme known to be involved in DNA replication and cancer cell proliferation, and is a target of several cytotoxic agents that directly or indirectly affect Top2A. Recent studies have suggested that Top2A has potential applications in breast cancer detection and management (Klintman et al., 2016). As described herein, we developed one or more polypeptides that can be used as Top2A peptide vaccines and evaluated their immunogenicity and prophylactic efficacy against TNBC in a mouse model. As described below, single-cell RNA sequencing (scRNA-sq) analysis showed that the Top2A vaccine induced antitumor CD4+ Th1 cells and cytotoxic CD8+ T cells in mouse mammary tumor and lymph node tissue samples. Furthermore, it was demonstrated that the Top2A vaccine induces a potent Top2A-specific memory immune response that prevents secondary exposure tumor development. Finally, testing of T cell receptor (TCR) sequences from CD4 TIL cells in tumors derived from vaccinated mice revealed the presence of TIL cells with TCR sequences for all immunization peptides. In addition to breast cancer, TOP2A is highly overexpressed / amplified in many tumor types, including lung cancer. As described herein, a significant prophylactic effect was found in a lung cancer mouse model vaccinated with the compositions disclosed herein. Given the extensive and unexpected data, the compositions and / or polypeptides disclosed herein, which can be used as Top2A vaccines, are highly immunogenic and effective in the treatment and / or prevention of cancers, including breast cancer and lung cancer, such as TNBC.

[0017] (Definitions and Terminology) The disclosed polypeptides, compositions, and methods for treating or providing a preventive treatment for one or more cancers, such as breast cancer and / or TNBC, may be further described using the following definitions and terminology. The definitions and terminology used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting.

[0018] As used herein and in the claims, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise.

[0019] Where used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. Where there is a use of these terms that is not obvious to those skilled in the art even considering the context in which they are used, “about” and “approximately” will mean up to plus or minus 10% of a particular term, and “substantially” and “significantly” will mean more than plus or minus 10% of a particular term.

[0020] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as “open” transitional clauses that allow for the inclusion of further additional components to the components enumerated in the claims. The terms “consist” and “consisting of” should be interpreted as “closed” transitional clauses that do not allow for the inclusion of additional components other than those enumerated in the claims. The term “essentially consisting of” should be interpreted as partially closed and allowing only the inclusion of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0021] The phrase "etc." should be interpreted as "for example, including." Furthermore, the use of any illustrative words, including but not limited to "etc.," is merely intended to shed more light on the invention and, unless otherwise claimed, does not impose any limitation on the scope of the invention.

[0022] Furthermore, in instances where conventions similar to "at least one of A, B, and C, etc." are used, such constructions are generally intended in a sense that a person skilled in the art would understand the convention (for example, "a system having at least one of A, B, and C" would include, but not be limited to, A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or a system having A, B, and C together). It will further be understood by a person skilled in the art that virtually any disjunct word and / or phrase presenting two or more alternative terms should be understood, whether in the specification or in the drawings, as construing the possibility of including one of the terms, either one of the terms, or both of the terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0023] All words such as "at most," "at least," "greater than," "less than," and so on, refer to a range that includes the listed numbers and can later be broken down into ranges and subranges. A range includes each of its individual members. For example, a group with 1 to 3 members refers to a group with 1, 2, or 3 members. Similarly, a group with 6 members refers to a group with 1, 2, 3, 4, or 6 members, and so on.

[0024] The modal verb "may" refers to a preferred use or selection of one or more options or choices among several described embodiments or features contained therein. If no options or choices are disclosed with respect to a particular embodiment or feature contained therein, the modal verb "may" refers to a positive action regarding the manner in which a described embodiment or feature contained therein is made or used, or a definitive decision to use a specific skill with respect to a described embodiment or feature contained therein. In this latter context, the modal verb "may" has the same meaning and implications as the auxiliary verb "can".

[0025] In the context of two or more nucleic acid or polypeptide sequences, the term “identical” or percentage “identical” means two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum match across a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection (i.e., identity of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more across a designated region of the entire nucleic acid or polypeptide sequence, or of individual parts or domains of the nucleic acid or polypeptide). Such sequences are then said to be “substantially identical.” In the context of nucleic acids, this definition also refers to the complement of the check sequence. For example, in embodiments, the identity exists over a region ranging in length from about 5, 10, 15, 20, 50, 100, or 1000 amino acids, or at least about 5, 10, 15, 20, 50, 100, or 1000 amino acids, to about 220, 100, or 1000, less than about 220, 100, or 1000 amino acids or nucleotides, or at least about 220, 100, or 1000 amino acids or nucleotides. If necessary, the identity exists over a region ranging in length from at least about 5, 10, 15, or 16 amino acids (see, for example, SEQ ID NO: 1), to about 100, about 20 to about 75, or about 30 to about 50 amino acids or nucleotides. Non-limiting examples of polypeptide sequences provided herein include sequences substantially identical to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. For example, polypeptides that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 are provided herein. Polypeptides having a difference of 1, 2, 3, 4, 5, 6, or 7, 8, 9, or 0 amino acids compared to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 are also intended herein.

[0026] For sequence comparison, typically one sequence functions as the reference sequence compared to the check sequence. When using a sequence comparison algorithm, the check and reference sequences are input into the computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the check sequence compared to the reference sequence based on the program parameters.

[0027] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST2.0 algorithms described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. As those skilled in the art will acknowledge, software for performing BLAST analysis is available through the National Center for Biotechnology Information (NCBI) website. In embodiments, BLAST and BLAST2.0 are used with the parameters described herein to determine the percent sequence identity of nucleic acids and proteins. In embodiments, the BLAST algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive threshold score T when aligned with words of the same length in the database sequence. In embodiments, T is referred to as the neighbor word score threshold (see Altschul et al., above). In embodiments, such initial neighbor word hits serve as seeds for initiating a search to find longer HSPs containing them. In embodiments, word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. In embodiments, the cumulative score is calculated for nucleotide sequences using parameters M (reward score for matched residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). In embodiments, for amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. In embodiments, the extension of word hits in each direction is stopped when the cumulative alignment score falls by a content X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is reached.In the embodiment, the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. In the embodiment, the NCBI BLASTN or BLASTP program is used for sequence alignment. In the embodiment, the BLASTN or BLASTP program uses the defaults used by NCBI. In the embodiment, the BLASTN program (for nucleotide sequences) uses, as default: word size (W) of 28; expected threshold (E) of 10; maximum match within the query range set to 0; match / mismatch score of 1, -2; linear gap cost; filter for low complexity regions used; and mask for lookup tables only used. In the embodiment, the BLASTP program (for amino acid sequences) uses, as default: word size (W) of 3; expected threshold (E) of 10; maximum match within the query range set to 0; BLOSUM62 matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)); gap cost of 11 present and 1 extended; and conditional composition score matrix adjustment.

[0028] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetic to the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0029] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as later modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0030] Amino acids may be referred to herein by their commonly known three-letter codes or by the single-letter codes recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred to by their commonly accepted single-letter codes.

[0031] The term "conservatively modified variant" applies to both amino acids and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially identical sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at all positions where alanine is specified by a codon, the codon can be changed to any of the corresponding codons listed without altering the encoded polypeptide. Such nucleic acid variants are "silent variants," which are a type of conservatively modified variant. All nucleic acid sequences in this specification that code for polypeptides also represent all possible silent variants of the nucleic acid. Those skilled in the art will recognize that by modifying each codon in a nucleic acid (with the exception of AUG, usually the sole codon for methionine, and TGG, usually the sole codon for tryptophan), a functionally identical molecule can be obtained. Therefore, each silent variant of the nucleic acid encoding the polypeptide is implicit in each of the described sequences with respect to the expression product, not to the actual probe sequence.

[0032] With regard to amino acid sequences, those skilled in the art will recognize that individual substitutions to peptide, polypeptide, or protein sequences that alter a single amino acid are “conservatively modified variants” in which the alteration results in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are, in addition to, polymorphic variants, interspecific homologs, and alleles, and do not exclude them.

[0033] The following eight groups each contain amino acids that are conserved substitutions with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0034] As used herein, "breast cancer" refers to malignant cancer of the cells of the breast.

[0035] As used herein, "triple-negative breast cancer (TNBC)" refers to breast cancer that does not express the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC can encompass basal cell-like and claudin-low molecular subtypes.

[0036] As used herein, "lung cancer" refers to malignant cancer of the cells of the lung.

[0037] The term "subject" can be used interchangeably with the terms "individual" and "patient," and includes both human and non-human subjects. In some embodiments, the subject may be any mammal.

[0038] As used herein, the terms “to treat” or “treatment” encompass both “preventive” and “curative” treatments. “Preventive” treatment is intended to mean delaying the onset of a disease, symptoms of a disease or medical condition, suppressing any symptoms that may appear, or reducing the risk of the onset or recurrence of a disease or symptom. “Curative” treatment includes reducing the severity of an existing disease, symptom or condition or preventing its worsening. Thus, treatment includes remission or prevention of worsening of an existing disease symptom, prevention of the onset of additional symptoms, remission or prevention of the underlying systemic cause of the symptoms, inhibition of a disorder or disease, e.g., suppression of the onset of a disorder or disease, alleviation of a disorder or disease, regression of a disorder or disease, alleviation of a condition caused by a disease or disorder, or cessation of symptoms of a disease or disorder.

[0039] Non-limiting examples of treatment for a subject include, but are not limited to, the step of administering a composition comprising one or more polypeptides to a subject, wherein the one or more polypeptides comprises or consist of one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In one or more embodiments, the one or more polypeptides comprises or consist of one or more polypeptides having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, or a polypeptide having the sequence of SEQ ID NO: 3. In various embodiments, the composition may also include an adjuvant. Another non-limiting example of treatment for a subject includes, but is not limited to, administering to a subject a composition comprising a polynucleotide encoding one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In various embodiments, the polynucleotide may be mRNA.

[0040] Non-limiting examples of prophylactic treatment for a subject include, but are not limited to, the step of administering a composition comprising one or more polypeptides to the subject, wherein the one or more polypeptides comprises or consist of one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In one or more embodiments, the one or more polypeptides comprises or consist of one or more polypeptides having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, or a polypeptide having the sequence of SEQ ID NO: 3. In various embodiments, the composition may also include an adjuvant. Another non-limiting example of a prophylactic treatment for a subject includes, but is not limited to, administering to the subject a composition comprising a polynucleotide encoding one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In various embodiments, the polynucleotide may be mRNA.

[0041] As used herein, the term “adjuvant” refers to a component used in certain vaccines to produce a stronger immune response in the vaccinated subject. By example, but not as an limitation, an adjuvant may include one or more oligonucleotides containing CpG dinucleotides (CpG oligodeoxynucleotides), Montanide, aluminum, monophosphoryl lipid A (MPL), MPL+aluminum salts, and oil-in-water emulsions comprising, for example, squalene, MPL, and QS-21.

[0042] As used herein, “therapeutic dose” means an amount of one or more therapeutic agents, such as the compositions described herein, that is effective in treating a disease or disorder and / or in preventing a disease or disorder. For example, a therapeutic dose of one or more of the compositions described herein may be an amount effective in inducing an immune response from a subject, such as by stimulating a local and / or systemic antigen-specific CD4+ and CD8+ T cell response, which may also result in an increase of one or more of IFN-γ, TNF-α, IL-2, or IL-23. Moreover, or instead, for example, a therapeutic dose of one or more of the compositions described herein may be an amount effective in reducing the number of cancer cells, reducing tumor size, inhibiting or slowing tumor growth and / or metastasis, and / or preventing the development of specific cancer cells, such as breast cancer cells, including TNBC cells.

[0043] (composition) In certain embodiments, one or more polypeptides comprising or consisting of a fragment of topoisomerase 2 alpha (Top2A) are disclosed herein. In various embodiments, one or more polypeptides comprise one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In one or more embodiments, one or more polypeptides comprise one or more polypeptides having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, or a polypeptide having the sequence of SEQ ID NO: 3.

[0044] In various embodiments, the composition may instead or in addition contain one or more polypeptides disclosed in Table 1 below, and / or have sequences that are at least 80%, 85%, 90%, 95%, or 99% identical to one or more sequences in Table 1.

[0045] [Table 1]

[0046] In certain embodiments, the composition may instead contain or consist of one or more polypeptides that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more of Sequence IDs 1-3, 4, 6, 8, 9, 20-27, and 28-30.

[0047] In various embodiments, the composition may contain one or more fragments of topoisomerase 2 alpha (Top2A), and one or more other antigens, such as cyclin E2 and / or KIF15 fragments. In such embodiments, the composition may contain or consist of one or more polypeptides that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-3, 4, 6, 8, and 9; and one or more polypeptides that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more of SEQ ID NOs: 28-30. In various embodiments, the fragments of topoisomerase 2 alpha (Top2A), and one or more other antigens, such as cyclin E2 and / or KIF15 fragments, may be present in a single continuous polypeptide, and may or may not be separated by a linker.

[0048] In various embodiments, the composition may contain one or more MHC-II epitopes of Top2A and one or more MHC-I epitopes of Top2A. For example, in various embodiments, the composition may contain or consist of one or more polypeptides that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-3, 4, 6, 8, and 9; and one or more polypeptides that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more of SEQ ID NOs: 20-27. In various embodiments, the MHC-II epitopes and MHC-I epitopes may exist in a single continuous polypeptide, and may or may not be separated by a linker.

[0049] In various embodiments, the composition may include a polypeptide sequence disclosed herein or a polypeptide having two or more copies of a polypeptide sequence disclosed herein that represent at least 80%, 85%, 90%, 95%, or 99% of that sequence. In various embodiments, the copies may or may not be separated by a linker. For example, in one embodiment, the polypeptide may be at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 31.

[0050] In various embodiments, the composition may be a vaccine or a component of a vaccine.

[0051] In certain embodiments, a composition may be a vaccine or a component of a vaccine, comprising any form of vaccine that can provide one or more epitopes represented by one or more polypeptides described herein. For example, in one embodiment, a vaccine or component of a vaccine may include polynucleotides, mRNA and / or DNA that encode one or more polypeptides disclosed herein and are configured to be expressed in a subject. In the same or alternative embodiments, a vaccine or component of a vaccine may include polynucleotides, mRNA and / or DNA that encode one or more polypeptides that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. In various embodiments, the vaccine or vaccine components may include polynucleotides, mRNA and / or DNA encoding a polypeptide that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 31.

[0052] In various embodiments, if the composition contains a polynucleotide encoding one or more polypeptide sequences disclosed herein, or at least 80%, 85%, 90%, 95%, or 99% of one or more polypeptide sequences disclosed herein, the polynucleotide may be one or more mRNAs. In certain embodiments, one or more mRNAs or polynucleotides may be present in any suitable medium for administration to a subject. In one embodiment, one or more mRNAs or polynucleotides may be present in lipid nanoparticles (LNPs).

[0053] In one or more embodiments, the composition may contain an adjuvant or may contain one as needed. In various embodiments, the adjuvant may contain any adjuvant suitable for use in a vaccine. In certain embodiments, the adjuvant may contain one or more of the following: CpG dinucleotide (CpG oligodeoxynucleotide), Montanide, aluminum, monophosphoryl lipid A (MPL), MPL + aluminum salt, e.g., squalene, MPL and QS-21, in an oil-in-water emulsion.

[0054] (method) In various embodiments, methods are disclosed for treating subjects with cancer, including breast cancer and / or lung cancer, and / or for prophylactic treatment of subjects.

[0055] In various aspects, the methods for treating and / or preventing the treatment of the subject are for treating and / or preventing breast cancer, including TNBC.

[0056] In various aspects, the methods for treating and / or preventing the treatment of the subject are for treating and / or preventing lung cancer.

[0057] In various embodiments, the method may include the step of administering one or more of the compositions disclosed herein to a target.

[0058] In one or more embodiments, the methods disclosed herein include the step of administering a vaccine to a subject, the vaccine comprising one or more compositions disclosed herein.

[0059] As discussed above, in various embodiments, the compositions disclosed for use in the methods herein may include one or more polypeptides. In certain example embodiments, one or more polypeptides may include or consist of one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In one or more embodiments, one or more polypeptides may include or consist of a polypeptide having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, and / or a polypeptide having the sequence of SEQ ID NO: 3. In one or more embodiments, the composition may include or may optionally include an adjuvant. Additional example polypeptides and polynucleotides are described above and referenced herein.

[0060] In some embodiments, subjects have not been previously diagnosed with breast cancer and / or TNBC. In the same or alternative embodiments, subjects have not been treated for breast cancer and / or TNBC. In various embodiments, subjects have been previously diagnosed with breast cancer and / or TNBC. In one or more embodiments, breast cancer does not express one or more of the following: estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor 2 (HER2). In various embodiments, subjects exhibit one or more TNBC-related risk factors selected from pregnancy, multiple births, and obesity.

[0061] In certain embodiments, subjects have not been previously diagnosed with lung cancer. In the same or alternative embodiments, subjects have not been treated for lung cancer. In various embodiments, subjects have been previously diagnosed with lung cancer. In one or more embodiments, subjects exhibit one or more risk factors associated with lung cancer, including but not limited to being a smoker or former smoker.

[0062] In certain embodiments, at least one or at least two doses of a therapeutically effective amount of the composition are administered to a subject in accordance with the method described herein. In certain embodiments, one or more doses of a therapeutically effective amount of the composition may be administered over a desired time interval, for example, monthly, annually, or every two, three, four, five, or ten years.

[0063] In various embodiments, the step of administering the composition to a subject in the method described herein may result in increased levels of one or more of IFN-γ, TNF-α, IL-2, or IL-23 in the subject compared to an untreated control. In various embodiments, the untreated control may be another subject and / or a healthy person who has not received the composition, and / or a subject who has received a placebo. In certain embodiments, the step of administering the composition to a subject in the method described herein may result in stimulation of a local and / or systemic antigen-specific CD4+ and CD8+ T cell response compared to an untreated control. In one or more embodiments, the step of administering the composition to a subject in the method described herein may result in reduction, slowing, or inhibiting tumor growth, metastasis, and / or development, such as breast cancer tumor growth, metastasis, and / or development, including TNBC, compared to an untreated control. In the same or alternative embodiments, the step of administering the composition to a subject in the method described herein may result in reduction of one or more symptoms associated with breast cancer, including TNBC.

[0064] In certain embodiments, the methods disclosed herein are intended for combination therapy. For example, in one or more embodiments, the methods disclosed herein may include the step of administering one or more of the compositions described herein to a subject who is already receiving or concurrently receiving treatment for cancer, for example, breast cancer including TNBC. In various embodiments, a subject who is already receiving or concurrently receiving treatment may receive one or more cytotoxic chemotherapeutic agents. In certain embodiments, a subject receiving such combination therapy may experience increased reduction of cancer-related symptoms and / or further reduction, slowing or inhibition of cancer growth, metastasis or development compared to another subject who is not receiving combination therapy (for example, not receiving the compositions described herein but receiving other treatment for cancer). [Examples]

[0065] The following examples are for illustrative purposes only and are not intended to limit the scope of the claimed subject matter.

[0066] Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, accounting for approximately 20% of all breast cancers, and has a poor prognosis due to its aggressive behavior and lack of effective targeted therapies. In this study, using in silico analysis, we identified genes preferentially expressed in TNBC and found that topoisomerase 2 alpha (Top2A) is highly expressed at both the transcriptional and protein levels. Top2A is a key enzyme in DNA replication and a therapeutic target for breast cancer and other cancers. Overexpression of Top2A was confirmed in human breast cancer tissue microarrays and in mouse TNBC derived from C3(1) / Tag mice. Therefore, we identified Top2A-specific MHC II epitopes with optimal binding affinity using a combined scoring system that predicts its potential to induce a Th1 immune response. Furthermore, all peptides with identical amino acid sequences between humans and mice were selected for potential clinical translation. From the tested candidate Top2A peptides, three peptides were selected based on a strong IFN-γ ELISPOT response after immunization in tumor-naive mice and Top2A peptide-specific tetramer staining. The selected peptides were combined to form a multipeptide Top2A vaccine. Splenocytes collected from Top2A peptide-vaccinated animals showed a robust immune response to the immunization peptides, and in vitro stimulation of splenocytes with Top2A peptides increased Th1 cytokine secretion. The antitumor efficacy of the Top2A vaccine was demonstrated in syngeneic TNBC mouse models [C3(1) / Tag-REAR and M6 cells], where graft preprophylactic vaccination was associated with significantly reduced tumor growth compared to adjuvant controls. In genetically engineered mouse (GEM) models (C3(1) / Tag), the vaccine was >90% effective in preventing tumor development. No clear toxicity was observed with Top2A vaccination. Finally, when we examined the TCR sequences in CD4 TILs derived from vaccinated mice, we found that the TILs contained TCR sequences specific to all of the immunization peptides.These data indicate that the newly developed multipeptide Top2A vaccine is highly immunogenic, induces TILs with peptide-specific TCRs, and is highly effective in preventing and inhibiting TNBC development and progression in vivo.

[0067] Breast cancer is the leading cause of cancer in women, with an estimated 281,550 new cases and 43,600 deaths reported in the United States in 2021 (Siege et al., 2021). Approximately 20% of breast cancers are classified as triple-negative breast cancer (TNBC) because they do not express the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This includes basal cell-like and, more recently, claudin-low subtypes (Prat et al., 2010). TNBC is also associated with racial predisposition to African American women, younger age, higher grade and mitotic index, and advanced stage at diagnosis (Jemal et al., 2011). Specific risk factors associated with TNBC include reproductive factors (pregnancy and multiple births) and obesity. In TNBC patients, the 5-year survival rate is significantly lower than in other forms of breast cancer, including ER+PR+HER- (luminal A) and HER+ subtypes (Onitilo et al., 2009; Harbeck et al., 2017). Luminal A tumors are treated with targeted therapies, such as hormones (tamoxifen, aromatase inhibitors), while HER+ breast cancer is treated with antibodies or small molecule inhibitors (Gao and Swain, 2018). In contrast, early and intermediate-stage TNBC are routinely treated with standard cytotoxic chemotherapy, which results in strong initial regression in most patients. However, resistance develops in most patients. The current challenges in treating TNBC necessitate new approaches to improve clinical outcomes.

[0068] Cancer vaccines have so far been primarily used to treat active, late-stage cancers, which likely provides limited efficacy due to immunosuppression inherent in advanced malignancies. Immunosuppression can involve multiple mediators, including regulatory T cells (Tregs), inhibitory macrophages, and other suppressors. One potentially more effective way to use cancer vaccines lies in prophylactic or early progression prevention settings. Several reports have provided proof of concept to support the use of peptide vaccines targeting overexpressed autoantigens as immunoprevention for breast cancer (Lollini et al., 2006; Disis et al., 2013; Ebben et al., 2015; Pan J et al., 2017). Molecular analysis of tumors has identified many genes overexpressed in breast cancer that can be utilized as tumor antigens and potential vaccine candidates. Currently, the most common tumor antigens used in cancer immunotherapy are upregulated autoproteins such as HER2. Vaccination with peptides targeting overexpressed HER2 / neu in humans has been shown to be effective and well-tolerated (Schneble et al., 2014; Lowenfeld et al., 2016). Mutant epitopes are recognized by the immune system as exogenous "neoantigens" and induce a type 1 immune response, while epitopes derived from non-mutated autoantigens are more likely to activate T helper 2 (Th2) cytokines such as interleukin (IL)-10 and IL-6, which can inhibit cytotoxic T lymphocyte (CTL) proliferation and function. Recently, attempts have been made to specifically identify Th1-selective epitopes derived from non-mutated autoantigens that can induce neoantigen-like responses. When used in vaccines, Th1-selective epitopes can induce uncompetitive type 1 immunity and may be effective in preventing cancer growth in preclinical models. If a Th2-inducing epitope derived from the same protein is included in the vaccine, immunized Th2 cells can suppress the Th1-mediated antitumor effect (Cecil et al., 2014; Disis et al., 1996).If the antigen is expressed in the early stages of cancer development, the vaccine may be useful in prevention.

[0069] In this study, the Cancer Genome Atlas (TCGA) database, along with transcriptomics and / or proteomics analyses of normal, versus malignant, human breast tissue, was used to identify genes highly expressed in malignant tissue. Topoisomerase 2 alpha (Top2A) was found to be highly expressed in human TNBC. Top2A is a key enzyme known to be involved in DNA replication and cancer cell proliferation, and is a target of several cytotoxic agents that directly or indirectly affect it. Recent studies have suggested that Top2A has potential applications in breast cancer detection and management (Klintman et al., 2016). A Top2A multipeptide vaccine was developed herein and evaluated for its immunogenicity and prophylactic efficacy against TNBC in a mouse model. Single-cell RNA sequencing (scRNA-sq) analysis showed that Top2A multipeptide vaccination induced antitumor CD4+ Th1 cells and cytotoxic CD8+ T cells in mouse breast tumor and lymph node tissue samples. Furthermore, the Top2A vaccine induces a potent Top2A-specific memory immune response that prevents secondary exposure tumor development. Finally, when TCR sequences derived from CD4 TIL cells in tumors from vaccinated mice were examined, TIL cells with TCR sequences for all of the immunizing peptides were found. In summary, the data demonstrate that the multipeptide Top2A vaccine is highly immunogenic and effective in preventing TNBC.

[0070] [material and method] Transgenic C3(1) / Tag mice and C3(1) / Tag-REAR (reorganization) mice were generously donated by Dr. Jeffery E. Green. FVB / N wild-type mice were purchased from Jackson Laboratory. Only F2 C3(1) / Tag or C3(1) / Tag-REAR generation mice were used for all experiments. Mice were maintained and bred at the Biomedical Resource Center of the Medical College of Wisconsin (MCW), Milwaukee, WI. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC).

[0071] (Cell lines) All M27 (weakly tumor-like / benign tumor), M6 (malignant tumor), M6C (metastatic tumor), and M28 (normal control) cells, derived from C3(1) / Tag mice, were provided by Dr. Jeffery E. Green (Holzer et al., 2003). The cell lines were maintained in high-glucose DMEM medium (Gibco) supplemented with 5% FBS, penicillin / streptomycin, and sodium pyruvate (Invitrogen).

[0072] Immunohistochemistry (IHC). IHC staining was performed by the Children's Research Institute Histology Core at MCW. Leica Bond Immunostainer Max (model #10897664) and Leica Bond Immunostainer RX systems (model #11784892) were used for human tissue microarrays (TMA) and mouse samples, respectively. Mouse mammary gland samples from C3(1) / Tag or C3(1) / Tag-REAR mice were inflated, formalin-fixed, and paraffin-embedded (Sakura Tissue Tek VIP5). 4 μm sections were used for IHC or hematoxylin-eosin (H&E) staining. The number of CD4+ and CD8+ tumor-infiltrating T lymphocytes (TILs) was determined as cells per 1 mm² of tumor area using CD4 antibody (Invitrogen 14-9766-82) and CD8 antibody (Invitrogen 14-0808-82). The entire slide was scanned using a NanoZoomer slide scanner (Hamamatsu). Subsequently, the tumor area was specifically highlighted and measured using NanoZoomer software.

[0073] (Human tissue microarray analysis) Two types of human TMA were purchased from US Biomax, Inc. (http: / / www.biomax.us / tissue-arrays / Breast / BRC961 and http: / / www.biomax.us / tissue-arrays / Breast / BRC962). Top2A antibody was purchased from Invitrogen, PAS-26255. Top2A expression in human TMA was assessed using a scoring system: score 0 = no staining; score 1 = faint staining; score 2 = moderate staining; and score 3 = strong staining. When assessing tissue staining intensity for each group, scores of 0 and 1 only were translated as weak; scores of 0, 1 and less than 10%; scores of 2 or 3 were translated as mild; and a score of 3 in more than 10% of samples in a given group was translated as high expression.

[0074] (ELISPOT Assay) Cell suspension derived from the entire spleen was filtered through a 70 μm cell filter (BD) and subjected to erythrocyte lysis using ACK lysis buffer. 1.5–3.0 × 10⁴ cells coated with anti-interferon-γ (IFN-γ) detection antibody were seeded into individual wells of a MAIPS4510 multiscreen 96-well plate containing medium with peptide, concanavalin A (positive control), HIV peptide (negative control), or antigen-free (negative control). After 72 hours of incubation, the plate was washed and incubated overnight at 4°C with secondary antibody (BD). The wells were then washed with PBS and HRP streptavidin was added. After 1 hour of incubation, the plate was developed using AEC substrate for 5–25 minutes. The plate was imaged and the number of spots quantified using an automated plate reader system (CTL Technologies).

[0075] (Scoring System for Predicting MHC Class II Binding Epitopes) A ​​combined scoring system was used to identify selected antigen-specific MHC epitopes with optimal binding affinity. This method was published by Dr. Disis and colleagues (Park et al., 2008). Briefly, the following algorithms were used for prediction to identify antigen-specific MHC class II epitopes with optimal binding affinity and randomness across multiple alleles: NetMHCIIpan (https: / / services.healthtech.dtu.dk / service.php?NetMHCIIpan-4.0, Technical University of Denmark, Lyngby, Denmark) and Rankpep (http: / / imed.med.ucm.es / Tools / rankpep.html, University Computense Madrid, Harvard, Madrid, Spain). For each available MHC class II allele, 20 peptide sequences were initially selected based solely on their predicted binding affinity rank from each algorithm. Each sequence was approximately 15 amino acids long. A score was assigned to each individual amino acid within the selected peptide, with 1 representing the amino acid with the highest predicted binding affinity. The individual amino acid scoring accounted for multi-peptide overlaps occurring within and between algorithms. The scores for each amino acid (S) were summed across multiple MHC class II alleles from two algorithms. Next, the number of MHC class II alleles (N) for which each amino acid was predicted to have high-affinity binding was counted. The final score for each amino acid was calculated by multiplying S and N. To facilitate the identification of the most potentially immunogenic segments of a protein, each amino acid was assigned a color (from dark red to light blue) based on its final score percentile, with dark red representing the highest (≥75%) and light blue representing the lowest (<10%).The color stratification is as follows: dark red = ≥75% of the highest score; red = 50-75% of the highest score; orange = 40-50% of the highest score; yellow = 30-40% of the highest score; green = 20-30% of the highest score; blue = ≤20% of the highest score. Therefore, dark red corresponds to sequences in which multiple peptides obtained high scores both within and across algorithms. Light blue represents sequences with the lowest potential immunogenicity for any predicted highly bound peptide.

[0076] (Vaccine Preparation and Immunization) Mice were vaccinated with 50 μg of each peptide. Three different Top2A peptides were purchased from Genemed Synthesis and diluted in phosphate-buffered saline (PBS) to 50 μl / mouse. The peptides and an equal amount of adjuvant CpG (class B CpG oligonucleotide; mouse TLR9 ligand, catalog no. tlrl-1826, InvivoGen) were added to bring the total vaccine volume to 100 μl / mouse. 50 μg of CpG was used per mouse. The Top2A vaccine was administered subcutaneously to the mice according to the timelines shown in Figures 5A and 8A.

[0077] (Dendritic Cell Generation) Mouse DCs were generated from bone marrow stem cells as previously described (Li et al. 2021). Briefly, bone marrow cells were cultured at a density of 2 × 10⁵ cells / mL in 6-well plates in RPMI-1640 complete medium supplemented with 20 ng / mL GM-CSF (R&D Systems, Minneapolis, MN). On day 4, the medium was replaced with fresh medium containing 10 ng / mL GM-CSF. On day 8, immature DCs were collected, pooled, and pulsed with Top2A peptide at a concentration of 50 μg / mL. TNF-α (10 ng / mL) and IL-1β (10 ng / mL) (R&D Systems) were added, and after 48 hours of culture, mature DCs were collected and used.

[0078] (Tetramer Assay) Top2A-specific T cells were generated from splenocytes of Top2A-vaccinated FVB / N wild-type mice by repeated stimulation of autologous T cells with Top2A peptide-loaded mature DCs. Briefly, isolated CD4+ T cells (5 × 10⁵ / 500 μL / well) were co-cultured with Top2A peptide-loaded mature DCs (1 × 10⁵ / 500 μL / well) in T-medium (RPMI1640, 10% FBS, 1% penicillin / streptomycin, 1 × β-ME) containing 10 ng / ml IL-2 in a 24-well plate at 37°C in 5% CO₂ for 7-10 days. After culturing, T cells were collected and stained with three types of Top2A-specific tetramers (synthesized by the NIH Tetramer Core Facility at Emory University, Atlanta, GA).

[0079] (CD4+ T cell proliferation assay) Isolated CD4+ T cells were labeled with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 μM; Invitrogen) for 10 minutes at 37°C, washed, and then seeded in 24-well plates in T-medium containing 10 ng / ml IL-2 (5 × 10⁵ / 500 μL / well). Mature DCs pulsed with Top2A peptide (1 × 10⁵ / 500 μL / well) were added to the plates and cultured for 4 days at 37°C in 5% CO₂. Cell proliferation was tracked by detecting CFSE dilution using flow cytometry analysis. Results are expressed as the average count per minute for three replicated cultures.

[0080] (In vivo oncogenicity assay) In a syngeneic model, C3(1) / Tag-REAR mice were generated from a C3(1) / Tag-established line by the loss of multiple copies of the original C3(1) / Tag antigen transgene (Aprelikova et al., 2016). M6 cells derived from C3(1) / Tag transgenic mammary tumors were implanted into the mammary fat pads of C3(1) / Tag-REAR mice. The M6 ​​cells were washed and 1 × 10⁶ cells were placed in 100 μl PBS. 6The cells were resuspended in PBS at a density of 100 cells and injected into the #4 mammary fat pad of female C3(1) / Tag-REAR mice. After implantation, the tumor diameter was measured using calipers, and the tumor volume was calculated using the following formula: maximum diameter × (minimum diameter). 2 ×0.4. In the spontaneous GEM model, C3(1) / Tag mice were treated with the Top2A peptide vaccine according to the experimental design in Figure 8A. C3(1) / Tag mice were sacrificed at 20 weeks of age to estimate tumor development. Tumor volume was measured using calipers and calculated using the following formula: maximum diameter × (minimum diameter) 2 ×0.4.

[0081] (Cytokine Analysis) The Mouse Th1 / Th2 / Th17 Cytokines Multi-AnalyteELISArray™ kit (Qiagen) was used for cytokine analysis. The cytokines represented by this array are IL2, IL4, IL5, IL6, IL10, IL12, IL13, IL17A, IL23, IFN-γ, TNFα, and TGFβ1. Splenocytes from various groups of mice were stimulated with various peptides for 72 hours, and the culture supernatant was collected and assayed according to the manufacturer's instructions.

[0082] (Flow Cytometry) Cell pellets were incubated with the target surface marker at the recommended or dose-set concentration, incubated at 4°C for 30 minutes, and protected from light. After incubation, cells were washed and resuspended in FACS fixation buffer for either analysis or intracellular staining. To begin intracellular staining, cells were fixed with the Foxp3 / transcription factor staining buffer set (eBioscience) and stained with the target intracellular marker at the recommended or dose-set concentration, while protected from light at 4°C for at least 30 minutes. Samples were washed with permeabilization buffer and resuspended in FACS fixation buffer. Stained cells were fixed in 1% paraformaldehyde and permeabilized according to the manufacturer's instructions to assess the expression of intracellular targets granzyme B, IFN-γ, and TNF-α. Flow cytometry was performed using an LSR-II flow cytometer (BD). Data were analyzed using FlowJo software (Tree Star).

[0083] (Western blotting) Cells were lysed in 200 μl of 1×NP40 lysis buffer containing a proteinase inhibitor cocktail (Thermo-Fisher), incubated on ice for 20 minutes, centrifuged at 16,000 g for 30 minutes, and then normalized to protein concentration as determined by the BCA method (Fisher Scientific, Pittsburgh, PA), followed by boiling for 5 minutes. The normalized lysates were separated by 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Thermo-Fisher) and immunoblotted with Top2A antibody (PA5-26255, Thermo-Fisher) and GAPDH (sc-25778, Santa Cruz).

[0084] (Single-cell RNA sequencing (scRNAseq) and TCR sequencing (scTCRseq)) For single-cell sequencing, lymph node samples (one from the CpG control group and two from the TOP2A vaccine-treated group) and mammary tumor samples (one from the CpG control group and one from the TOP2A vaccine-treated group) from C3(1) / Tag mice were collected at the end of the study, chopped, and digested in mouse tumor dissociation buffer (Miltenyi Biotec, CA) at 37°C for 30 minutes to produce single-cell suspensions according to the manufacturer's instructions. The processed samples were directly stained with violet viability dye and APC anti-CD45, and CD45+ leukocytes were sorted using FACS. Next, the FACS-sorted CD45+ leukocytes were spun down at 300g for 5 minutes and manually counted using a Neubauer Chamber. Approximately 2.0 × 10⁶ 4 The cells were loaded onto a 10X Chromium controller according to the manufacturer's instructions, resulting in the recovery of approximately 1 × 10⁴ cells. For lymph node samples, a single-cell transcriptome library was generated using the Chromium Single Cell 3' v3 reagent kit (10x Genomics). For tumor samples, single-cell transcriptomes and single-cell TCR libraries were prepared using the 10x Chromium Single Cell 5' and VDJ library construction kits. All libraries were sequenced using the NextSeq 500 / 550 High Output kit v2 (150 cycles) (Illumina) according to the manufacturer's protocol.

[0085] (scRNA-seq data analysis) Raw sequencing data was demultiplexed and converted to a gene-barcode matrix using Cell Ranger (version 2.2.0), mkfastq, and the counting function (10x Genomics). The mouse reference genome mm10 was used for alignment. The data was further analyzed in R (version 3.4.0) using Seurat (version 3). The number of genes detected per cell, the number of unique molecular identifiers (UMIs), and the percentage of mitochondrial genes were plotted, outliers were removed, and doublets and dead cells were filtered out. Raw UMI counts were normalized and logarithmically transformed. Next, a combined analysis was performed to identify shared cell clusters present across different datasets. Principal component analysis was performed using variable genes, and the top 20 most statistically significant principal components were used for UMAP analysis.

[0086] (Statistical Analysis) All in vitro assays were replicated at least three times. Six to twelve mice were used per group for in vivo studies. A two-sided Student's t-test was used to assess the differences between the control group and each treatment group. A P-value < 0.05 was considered statistically significant.

[0087] [Example 1 - Gene Expression Analysis of Top2A] The Top2A gene was found to be overexpressed in various mouse mammary tumor cell lines as well as in human TNBC and lung tumors (Figure 1). In mice, the Top2A gene was overexpressed in mouse mammary tumor cell lines M27 (benign mammary tumor cell line), M6 (malignant mammary tumor cell line), and M6C (metastatic mammary tumor cell line), with the M28 mammary cell line used as a normal control (Figure 1A). The Top2A gene was also highly overexpressed in TCGA TNBC samples (12 normal mammary tissues and 137 tumor samples from TNBC patients) and only limitedly expressed in normal mammary tissue (Figure 1B). The applicant also performed expression analysis of TNBC in African American (AA) populations at high risk of TNBC. Similar to the general TNBC population, Top2A was significantly overexpressed in TNBC derived from the AA subpopulation (Figure 1C). These data indicate that the Top2A gene is overexpressed in TNBC isolated from both mice and humans.

[0088] [Example 2 - Increased Top2A protein expression in human and mouse TNBC tissue] Using human breast cancer tissue microarrays (TMA, BRC961 and 962, US Biomax, Inc.), the high expression of Top2A in human TNBC was verified. The TMA included 36 common cancer types (including TNBC) and 12 normal and non-malignant breast tissues. Using the scoring system shown in Figure 2A, Top2A protein expression levels were assessed, and it was found that Top2A was overexpressed in human malignant tissue (Figure 2A). In mice, mammary tissue samples, including tumors and corresponding normal mammary glands, were collected from 19-week-old C3(1) / Tag female mice and FVB / N wild-type mice and analyzed by IHC. Representative images of Top2A IHC staining are shown in Figure 2B, where Top2A was highly expressed in ductal carcinoma in situ (DCIS) and malignant tumor tissue from C3(1) / Tag mice. In contrast, Top2A expression was not detected in normal mammary gland tissue from wild-type mice. Overexpression of Top2A was confirmed in mouse mammary cancer cell lines M28, M27, M6, and M6C, as shown in Figure 2C.

[0089] [Example 3 - Identification of Th1 Top2A epitopes for vaccine design] Using a multi-scoring system combining multiple MHC class II peptide binding algorithms, three candidate peptides, #232 (p232-p246), #410 (p410-p425), and #604 (p605-p621) (Figures 3A and 3B), were identified and selected. The immunogenicity of the Top2A peptide was assessed in C3(1) / Tag-REAR mice by the IFN-γ ELISPOT assay (Figure 3C). Splenocytes from Top2A-vaccinated mice demonstrated a stronger immune response on average for IFN-γ secreting cells with approximately 400 spot numbers (SPW) per well, compared to less than 10 SPW for the negative control HIV peptide. Furthermore, the immunogenicity of all three combined peptides (combo) was tested. Unexpectedly, mice vaccinated with the combo showed a significantly stronger immune response than mice vaccinated with a single peptide (Figure 3C). The sequences of the three peptides were 100% homologous between human and mouse Top2A. Peptides #232, #410, and #604 were selected to formulate a multipeptide Top2A vaccine for prophylactic efficacy studies.

[0090] [Example 4 - In vivo immunogenicity of Top2A peptide] To test whether the Top2A peptide can induce peptide-specific CD4+ T cells, 50 μg of Top2A peptide in CpG adjuvant was subcutaneously injected into FVB / N wild-type mice (3 mice per peptide dose). One week after the fourth immunization, splenocytes were collected, CD4+ T cells were isolated, and the cells were restimulated in peptide-pulsed DCs for 4–7 days, followed by analysis of peptide reactivity. In vivo immunization successfully generated peptide-specific CD4+ T cells, as detected by flow cytometry using the Top2A peptide tetramer (Figures 4A and 4B). Furthermore, CD4 T cells from peptide-immunized mice showed significantly higher cell proliferation against Top2A peptide-pulsed mouse DCs than CD4 T cells from the CpG-only group (Figures 4C and 4D). These results indicate that the Top2A peptide was able to induce a peptide-specific T cell response in FVB / N wild-type mice.

[0091] [Example 5 - Top2A multipeptide vaccination inhibited tumor development in a syngeneic TNBC mouse model] We designed experiments using a syngeneic TNBC mouse model, C3(1) / Tag-REAR mice inoculated with M6 tumor cells. As described in Figure 5A, 7-week-old C3(1) / Tag-REAR mice received their first vaccination with a peptide and CpG oligodeoxynucleotide (CpG ODN) adjuvant, followed by three more vaccinations at one-week intervals. One week after the last vaccination, M6 cells were implanted into the #4 mammary fat body of the C3(1) / Tag-REAR mice. Additional vaccinations were administered at four-week intervals until the end of the experiment. Notably, Top2A vaccination significantly reduced tumor volume compared to CpG-only treated control mice (Figure 5A; p<0.05). The mean tumor size at the experimental endpoint was 757.2 mm3 in controls versus 413 mm3 in vaccinated animals. Top2A vaccination also reduced tumor weight by almost 40% (Figure 5C, p<0.05). Mice vaccinated with Top2A were also tested for the presence of a systemic immune response. Splenocytes collected from Top2A-vaccinated mice were stained for intracellular markers (granzyme B, IFN-γ, and TNFα) and analyzed by flow cytometry. A significant increase in CD4+ and CD8+ T cells expressing granzyme B, IFN-γ, and TNFα was observed in the spleens of Top2A-vaccinated animals compared to controls treated with CpG alone (Figure 5D). In summary, these results indicate that the Top2A peptide vaccine induces both systemic and local immune responses.

[0092] [Example 6 - Top2A vaccination induced a Th1 cytokine response] To assess Th1 and Th2 cytokine production in response to the Top2A vaccine, the production of 12 cytokines was measured from in vitro peptide-stimulated splenocytes isolated from vaccinated mice (Figure 7). The most abundant cytokines detected in response to the Top2A peptide pool were the Th1 cytokines IL-2 and IFN-γ, which increased approximately 7-fold and 3-fold, respectively, compared to the adjuvant control. In contrast, Th2 cytokine production (IL-4, IL-5, and IL-13) did not increase compared to the adjuvant control. Interestingly, the Top2A vaccine stimulated not only Th1 cytokine production (IL-2 and IFN-γ) but also IL-23, which is known to be produced by antigen-presenting cells. These data suggest that the Th1 immune response is predominantly induced by the Top2A-specific peptide vaccine.

[0093] [Example 7 - C3(1) / Tag mice vaccinated with Top2A were protected from secondary exposure to M6 cells.] We designed an experiment to examine the long-term immunological memory-protective effect of the Top2A vaccine against secondary tumor cell development. The first "exposure" was spontaneous tumor development in C3 / Tag mice (see Figure 8A for experimental design). Secondary tumor exposure involved transplantation of syngeneic M6 tumor cells into 22-week-old (4 weeks after the final vaccination) C3 / Tag mice. Notably, Top2A-vaccinated mice did not develop spontaneous tumors, and vaccinated mice had significantly smaller M6 TNBC tumors at the experimental endpoint (Figures 8B, 8C). The mean tumor size in vaccinated mice was 42 mm³ vs. 229.9 mm³ compared to adjuvant controls, respectively (Figure 8B; p<0.001). Tumor weight in Top2A-vaccinated mice was also significantly reduced by almost 85% (Figure 8C). The number of CD4+ and CD8+ TILs in mice vaccinated with Top2A was also significantly increased, as shown in Figure 8D. These results suggest that the Top2A vaccine helps induce long-term T-cell memory, thereby resisting tumor recurrence.

[0094] [Example 8 - Single-cell gene expression landscape in breast tumors and lymph node tissue] Using the Seurat package (Butler et al., 2018; Stuart et al., 2019), we performed detailed clustering of sequenced single cells derived from mouse mammary tumors and lymph node tissue. Single-cell gene expression data were aligned and projected in two dimensions using uniform manifold approximation and projection (UMAP) (Bech et al., 2018). Gene expression patterns of canonical markers were analyzed to characterize different types of immune cell populations in the tumor samples. Six immune cell populations were detected in the mammary tumor samples, including CD8+ T cells, CD4+ T cells, CD4 / CD8 double-negative T cells (DNTs), dendritic cells (DCs), macrophages, and neutrophils (Figure 9A, Figure 9B). Single-cell expression data derived from lymph node tissue were also projected by UMAP, and the identified immune cell populations included CD8+ T cells, CD4+ T cells, DNTs, DCs, and macrophages, as expected (Figure 9C, Figure 9D).

[0095] [Example 9 - Top2A vaccine treatment increased the percentage of tumor-specific cytotoxic CD8+ T cells in mouse mammary tumors and lymph node tissue.] To determine the effects of Top2A vaccine treatment on various T cell subsets, deep clustering of CD8+ T cells, CD4+ T cells, and DNT cells derived from mouse mammary tumors was performed. The location of CD8+ T cells was identified by canonical markers (Figure 10A). Unsupervised clustering of CD8+ T cells using the TILPRED program (https: / / github.com / carmonalab / TILPRED) (Carmona et al., 2020) identified four CD8+ T cell subsets with distinct transcriptomics profiles (Figures 10B, 10C). The CD8 subsets included effector memory (EM)-like, exhausted, memory-like, and naive CD8+ T cells. In cancer, EM-like and exhausted CD8+ T cells are involved in antitumor and tumor-promoting functions, respectively. Top2A vaccination significantly increased the abundance of antitumor EM-like CD8+ T cells in the mammary tumor microenvironment (TME) (Figure 10D). In contrast, exhausted CD8+ T cells were reduced by Top2A vaccine treatment. These data suggest that Top2A vaccine treatment improves the overall composition of beneficial antitumor CD8+ T cells in mammary tumors. In lymph node samples from mice with tumors, the applicants observed a similar effect of Top2A vaccine treatment on CD8+ T cell subsets. CD8+ T cells from lymph node samples were plotted (Figure 10E), and CD8+ T cell subsets were identified using corresponding markers (Figures 10F, 10G). Top2A vaccine treatment increased the abundance of EM-like CD8+ T cells and significantly reduced the proportion of exhausted CD8+ T cells in the lymph nodes (Figure 10H).

[0096] [Example 10 - Top2A vaccine treatment increased the abundance of CD4+ Th1 cells in mouse mammary tumors and lymph node tissue.] Functional CD4+ cells can play a major role in the antitumor response induced by Top2A vaccine treatment. We analyzed CD4+ T cells derived from breast tumors and identified three types of CD4+ T cell subsets, including CD4 Th1, CD4 Th17, and Treg cells (Figures 11A–11C). Top2A vaccine treatment significantly increased the proportion of CD4+ Th1 and Th17 cells in breast tumors while decreasing the abundance of Treg cells (Figure 11D). CD4+ Th1 cells overexpressed the marker genes perforin, IFNg, and TNF; CD4 Th17 T cells overexpressed IL17a and Rorc; and Treg cells overexpressed Foxp3 (Figure 11B). These results suggest that the antitumor function of CD4+ T cells was significantly enhanced by Top2A vaccine treatment. In lymph node samples, four types of CD4+ T cell subsets were identified: CD4 CM (central memory T cells), CD4 Th1, Treg, and CD4 HSP (CD4+ T cells overexpressing Hspa1a) (Figures 11E-11G). Similar to breast tumor samples, the applicants observed an increased frequency of CD4+ Th1 cells after Top2A vaccination (Figure 11H). Top2A vaccination also increased CD4 CM cells, but decreased immunosuppressive CD4 HSP cells (Figure 11H). The applicants' results suggest that CD4+ Th1 cells are involved in the antitumor response generated by the Top2A vaccine.

[0097] [Example 11 - Top2A vaccine treatment also affects other immune cells in mouse tumors and lymph node tissue.] Tumor and lymph node tissues were examined for changes in other immune cell populations after Top2A vaccination. For DNT cells (CD4 / CD8 double-negative T cells) in mouse mammary tumors and lymph node tissues, the cells were divided into three subsets: helper, cytotoxic, and native DNT cells (Figures 14 and 15) (Yang et al., 2021). Top2A vaccination significantly increased the abundance of cytotoxic DNT cells, while the treatment decreased the proportion of native DNT cells in mouse mammary tumor tissue (Figure 14D). The percentage of helper DNT cells was not altered by Top2A vaccination (Supplemental Figure 14D). In lymph node samples, Top2A vaccination increased the percentage of helper DNT cells but did not alter the percentage of cytotoxic DNT cells (Supplemental Figure 15D). These results suggest that Top2A vaccination specifically increases the abundance of cytotoxic DNT cells in mouse mammary tumors. Macrophages were subdivided into M1 and M2 macrophages according to their marker gene expression in both mammary tumor and lymph node tissue (Figures 16 and 17). In both mammary tumor and lymph node tissue, the abundance of antitumor M1 macrophages increased and protumor-promoting M2 macrophages decreased after Top2A vaccination (Figures 16D and 17D). These data suggest that macrophages may also be involved in the antitumor response induced by Top2A vaccination. Two subsets of DCs were detected in the tissues of treated mice based on analysis of corresponding marker genes: conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) (Figures 18 and 19) (Villar et al., 2020). Top2A vaccination significantly increased the proportion of cDCs in mammary tumor tissue but not in lymph nodes. cDCs are known to play a crucial role in antitumor immunity (Murphy et al. 2022). Finally, neutrophils were detected in breast tumor samples.Neutrophils can be divided into stage I and stage II subsets, representing progenitor and mature neutrophils, respectively (Giladi et al., 2022). Both neutrophil subsets were identified in breast tumor tissue, and Top2A vaccine treatment was found not to alter the proportions of these two subsets (Figures 20A-20C).

[0098] [Example 12 - TCR clonal type analysis revealed the emergence of new T cell clones as a result of Top2A vaccination.] To explore the underlying mechanisms of the antitumor response induced by Top2A vaccine treatment, we analyzed scTCRseq data from control and Top2A-vaccinated T cells. 201 CD4+ T cells from tumor tissue derived from Top2A-vaccinated mice and 57 CD4+ T cells from tumor tissue derived from CpG-control mice were sequenced. The top five most frequent TCR clone types (frequency >= 4) constituted 15.5% of the total CD4+ TCR clone types in the Top2A-vaccinated group; these clone types were not found in the CpG-control group, suggesting their involvement in the Top2A vaccine-generating CD4+ T cell immune response (Figure 12A). Almost all TCR clone types in vaccinated mice were unique compared to CpG controls (Figure 12). To predict the likelihood of TCR-peptide binding to Top2A vaccine peptides, we used ERGO (pEptide tcR matchinG predictiOn) software, a highly specific and comprehensive TCR-peptide binding predictor (Springer et al., 2020) (https: / / github.com / louzounlab / ERGO). For the first Top2A peptide - KDIVALMVRRAYDIA (SEQ ID NO: 1), the top four TCR clone types with the best binding scores were CAAKPINYGNEKITF_CASSIWVGPSQNTLYF (2 cells, clonal frequency: 1%), CAVYQGGRALIF_CASSQRGIWENTGQLYF (6 cells, clonal frequency: 3%), NA_CASSGLGGDTQYF (2 cells, clonal frequency: 1%), and CALGDPGNTRKLIF_CASSLGGTGQLYF (9 cells, clonal frequency: 4.5%) (Figure 13A).For the second Top2A peptide, ILNWVKFKAQVQLNKK (SEQ ID NO: 2), the top four clonal types with the highest binding scores were NA_CTCSVSYNSPLYF (2 cells, clonal frequency: 1%), NA_CASSHTNSDYTF (4 cells, clonal frequency: 2%), CAAKPINYGNEKITF_CASSIWVGPSQNTLYF (2 cells, clonal frequency: 1%), and CALGDPGNTRKLIF_CASSLGGTGQLYF (9 cells, clonal frequency: 4.5%) (Figure 13B). For the third Top2A peptide, KKWKVKYYKGLGTSTSK (SEQ ID NO: 3), the top four clonal types with the highest binding scores are NA_CASSHTNSDYTF (4 cells, clonal frequency: 2%), CAVRDSNYQLIW_CASSMGDNYAEQFF (2 cells, clonal frequency: 1%), NA_CASSGLGGDTQYF (2 cells, clonal frequency: 1%), and CAVYQGGRALIF_CASSQRGIWENTGQLYF (6 cells, clonal frequency: 3%) (Figure 13C). Notably, the top two most frequent TCR clone types, CALGDPGNTRKLIF_CASSLGGTGQLYF and CAVYQGGRALIF_CASSQRGIWENTGQLYF, both showed high binding affinity to the Top2A peptide KDIVALMVRRAYDIA (SEQ ID NO: 1) (Figure 13A), suggesting that this Top2A peptide plays a crucial role in the Top2a vaccine-induced antitumor T cell response.

[0099] [Consideration] Top2A is an enzyme that regulates the topological state of DNA. It catalyzes double-strand DNA breaks and promotes gene transcription during mitosis (Pei, et al., 2018), and has been linked to several malignant lesions, including breast, ovarian, and prostate cancer. Top2A is directly associated with tumor cell proliferation and invasiveness in breast cancer (Klintman et al., 2016), and its expression has been reported to be amplified in TNBC patients with high-risk factors such as large tumor size, high-grade tumors, and lymph node invasion (Zheng et al., 2016; Nakagawa et al., 2011).

[0100] In this specification, overexpression of the Top2A gene was identified in both mouse mammary tumor cell lines and human TNBC cancer samples (Figure 1). Top2A expression was confirmed in TNBC derived from AA women. Recently, the incidence of TNBC has been reported to be higher in AA women (Carey et al., 2006), possibly due to various sociological factors in addition to an increased likelihood of developing TNBC (Siddharth et al., 2018), and is associated with a worse prognosis compared to European American women (Carey et al., 2006). Top2a protein expression levels were assessed in human TNBC tissue and mouse TNBC tumors, and overexpression at the protein level was confirmed (Figure 2).

[0101] CD4 T cells can differentiate into various Th subsets that can induce, modulate, and maintain an immune response against tumor antigens. Such CD4 T cell subsets include Th1, Th2, and regulatory T cells (Treg). The Th1 subset produces IFN-γ, TNF-α, and IL-2, which regulate cellular immunity and play a crucial role in the anti-tumor immune response (Pan et al, 2016). The Top2a vaccine used in this study predominantly induced Th1 cell and APC responses without inducing a strong Th2 response (Figure 7). Type I cytokines secreted by Th1 cells, such as IFN-γ, can upregulate MHC class I expression on the APC membrane, along with tumor cells, potentially facilitating tumor recognition by CD8+ T cells (Zhou, 2009). Furthermore, Th1 cells can facilitate the cross-presentation of MHC class I-binding peptide antigens to CD8+ T cells (Matsuo et al., 2004; Disis et al., 2013). These mechanisms may be involved in generating a Th1 response associated with the Top2A vaccine, and ultimately, facilitating the generation of a CD8+ T cell response. Cytotoxic CD8+ T cells play a crucial role in the antitumor immune response by directly killing tumor cells (Martinez-Lostao et al., 2015). Our research demonstrates that the Top2A vaccine can stimulate both localized and systemic antigen-specific CD4+ and CD8+ T cell responses. A significant increase in IL-23 secretion was found from splenocytes isolated from Top2A-vaccinated mice. This is noteworthy because IL-23 is known to play a role in antitumor activity in both syngeneic and GEM models (Lo et al., 2003; Ma et al., 2020). On the other hand, no toxicity was observed in vaccinated mice. We designed 15-17 amino acid peptides with 100% sequence identity between human and mouse Top2a, enabling the transition of these peptides to clinical research.Overall, the data presented herein demonstrate that the multipeptide Top2A vaccine is highly immunogenic and has the potential to be effective in the immunoprevention of TNBC.

[0102] [Example 13 - Additional screening for Top2A peptide] In this embodiment, additional peptide candidates were identified using a multi-scoring system that combines multiple MHC class II peptide binding algorithms, as performed in Example 3 above. The additional 18 peptide candidates are listed in Table 2 below.

[0103] [Table 2]

[0104] The immunogenicity of the Top2A peptides in Table 2 was assessed in C3(1) / Tag-REAR mice by IFN-γ ELISPOT assay using a negative control of HIV peptides, as described above with reference to Example 3. The IFN-γ ELISPOT plate is depicted in Figure 21, and the quantified results are provided in Figure 22. As can be seen in Figure 22, some of the peptides did not show significant immunogenic potential. Some peptides in this example showed immunogenic potential and also demonstrated sufficient solubility to be selected as potential Top2A peptide candidates for the compositions disclosed herein - these include the peptides related to the data circled in the bar graph in Figure 22 (SEQ ID NOs: 4, 5, 6, 7, 8, and 9).

[0105] [Example 14 - Top2A peptide vaccination inhibited lung tumor progression in an isogenic model of lung cancer.] In addition to breast cancer, TOP2A is highly overexpressed / amplified in many tumor types, including lung cancer. Therefore, we hypothesized that TOP2A vaccination would exhibit similar immunoprophylactic efficacy against lung cancer. Using a similar approach, we examined the efficacy of the TOP2A vaccine in two syngeneic lung cancer mouse models (using LKR13 and LLC lung adenocarcinoma cell lines). Significant prophylactic effects were found in mice vaccinated with the TOP2A vaccine (Figures 23A-23F). In the LKR13 cell model, the mean tumor volume at day 31 was 446.3 mm3 in the CpG control versus 69.6 mm3 in the Top2A vaccine (Figure 23B, p<0.01). In the LLC cell model, the mean tumor volume at day 20 was 1330.2 mm3 in the CpG control versus 348.4 mm3 in the Top2A vaccine (Figure 23E, p<0.01). Furthermore, the survival of animals treated with the TOP2A vaccine was significantly prolonged compared to adjuvant-treated mice in both mouse LUAD models (Figure 23C & Figure 23F, p<0.001, log-rank test). Based on the unexpected results in this embodiment, the Top2A compositions disclosed herein are promising candidates for immunoprevention of lung cancer.

[0106] [Example 15 - Design and testing of an MHC II Top2A mRNA vaccine] Both OVA-mRNA and MHC II TOP2A-mRNA LNPs were generated using Moderna's LNPs (M-LNPs) (Figures 24A-24C) and evaluated in B16 (OVA) or LKR13 (TOP2A) lung metastasis models. mRNA vaccines containing TOP2A-specific MHC-II epitopes were similarly tested in syngeneic animal models compared to peptide vaccines. Both OVA (positive control) (Figure 24B) and TOP2A (Figure 24C) mRNA vaccines (10 μg / mouse sc per week for three doses) generated strong antitumor immunity and significantly reduced metastatic burden in lung metastasis syngeneic models. Significantly stronger antitumor efficacy (>90%) was demonstrated after vaccination with TOP2A-mRNA-LNP compared to peptide / adjuvant TOP2A vaccines (Figure 24C). In the case of TOP2A-mRNA-LNP, it contained MHC II epitopes derived from three epitopes isolated by the linker. The mRNA encodes the amino acid sequence (SEQ ID NO: 31) presented below. The lipid nanoparticles (LNPs) used were FDA-approved LNPs from Moderna called SM-102, which are synthetic aminolipids used alongside other lipids to constitute lipid nanoparticles. Previously, SM-102 was essential for the drug delivery system of the Moderna COVID-19 vaccine. SM-102 is an ionizable lipid that maintains a nearly neutral charge at physiological pH but becomes positively charged within the nanoparticle structure (where the amine group is protonated to form an ammonium cation). This allows it to effectively bind to the negatively charged mRNA backbone. The remainder of the nanoparticle consists of pegylated lipids that enhance particle stability, along with phospholipids and cholesterol molecules that contribute to the particle structure.

[0107] TOP2A-mRNA peptide sequence (SEQ ID NO: 31)

[0108] MRVTAPRTLILLLSGALALTETWAGGSGGGSGGGIVESILNWVKFKAQVQLNKKCSAVKGGSGGGGSGGMQSLDKDIVALMVRRAYDIAGSTKDGGSGGGGSGGSTPNHKKWKVKYYKGLGTTSTSKEAKEYGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA

[0109] [Example 16 - Identification of MHC I Top2A epitope] We designed TOP2A MHC I-restricted epitopes using IEDB NetMHCpan EL 4.1, which returns epitopes along with their predicted binding affinity, using two metrics for the top 27 expressed HLA alleles covering >97% of the human population. As shown in Figure 25, regions with high affinity for more MHC-I alleles are indicated as “hot” zones. We further calculated other epitope-specific clinical checkpoint parameters, such as immunogenicity, antigenicity, allergenicity, and toxicity, using additional algorithms. The MHC-I epitopes ultimately selected by us are listed in Table 3, which reach 91% population coverage as calculated by the IEDB coverage tool (Figure 26).

[0110] [Table 3]

[0111] [Example 17 - Combination of Cyclin E2 and KIF15 vaccines with TOP2A vaccine] The goal is to develop a multi-antigen prophylactic vaccine that simultaneously targets multiple TNBC tumor antigens (TOP2A, cyclin E2, and KIF15) to overcome potential antigen-negative variant escape. Previous human ductal carcinoma in situ (DCIS) vaccine studies have shown that targeting a single antigen (HER2) can induce target antigen loss in tumor cells. Since antigen escape is the primary potential mechanism for evading immunoprevention when targeting a single antigen, targeting multiple antigens could be an effective strategy to limit antigen-negative tumor escape.

[0112] In recent years, the applicants have explored two additional tumor antigens: cyclin E2 and KIF15. Overexpression of cyclin E2 has been detected in several human cancers, including breast cancer, leukemia, lung cancer, ovarian cancer, and bladder cancer. A cyclin E2 MHC class I-restricted peptide vaccine has also been identified and shown to induce peptide-specific cytotoxic T lymphocytes that target leukemia. KIF15 (kinesin family member 15) is overexpressed in multiple cancer types, including breast cancer, pancreatic cancer, hepatocellular carcinoma, and lung cancer.

[0113] Overexpression of KIF15 in breast cancer is associated with tumor size, lymph node metastasis, advanced TNM stage, and unfavorable prognosis. Both cyclin E2 and KIF15 are overexpressed in human TNBCs derived from the TCGA RNA-seq dataset (Figure 27A) and are overexpressed at the protein level using immunohistochemistry of TNBCs derived from C3(1) / Tag mice (Figures 27B-27E).

[0114] The applicants developed both cyclin E2 and KIF15 MHC-II vaccines using a multi-scoring system to identify immunogenic epitopes (Table 4). Two highly homologous interspecies cyclin E2-specific Th1 MHC class II-restricted epitopes (p298 & p334) induced type I immunity in mice (Figure 28A). Following cyclin E2 vaccination, mammary tumor growth was significantly inhibited in the C3(1) / Tag-RARE syngeneic mouse model (Figure 28B). For KIF15, one 93.3% homologous interspecies KIF15-specific Th1 MHC class II-restricted epitope (p129) induced type I immunity in mice (Figure 28A and Table 4). Following KIF15 vaccination, mammary tumor growth was significantly inhibited in the C3 / (1)Tag mouse model (Figure 28C). A multi-antigen prophylactic vaccine targeting Top2A, cyclin E2, and KIF15 was shown to be more effective than targeting cyclin E2 or KIF15 alone (and as effective as Top2A vaccine alone) in the C3(1) / Tag mouse model (Figure 29). This multi-antigen prophylactic vaccine should also help overcome the antigen-negative variant escape observed with single-antigen vaccines. mRNA vaccines containing the epitopes of TOP2A, cyclin E2, or KIF15 are currently under development and will be tested for their efficacy and immune response in relevant preclinical animal models.

[0115] [Table 4]

[0116] [Example 18 - Remarkable efficacy of a TOP2A-targeting vaccine for immunoprevention of triple-negative breast cancer] Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis. TOP2A is a key enzyme in DNA replication and a therapeutic target for breast cancer and other cancers. Using a combined scoring system, we identified TOP2A-specific Th1-promoting epitopes with optimal binding affinity to MHC II. The multipeptide TOP2A vaccine induced a robust immune response in immunized mice, as demonstrated by the significant production of Th1 cytokines from spleen cells of immunized animals stimulated in vitro with TOP2A peptides. The antitumor efficacy of the TOP2A vaccine was demonstrated in a syngeneic TNBC mouse model, where graft preprophylactic vaccination was associated with significantly reduced tumor growth compared to adjuvant controls. In a genetically engineered mouse (GEM) model of TNBC, vaccinated animals demonstrated a significant reduction in tumor incidence and mean tumor volume compared to adjuvant controls. Finally, the applicants tested the TCR sequences in CD4 tumor-infiltrating lymphocytes (TILs) derived from vaccinated mice and found that the TILs contained TCR sequences specific to the three vaccine peptides. These data indicate that the applicants' newly developed multipeptide TOP2A vaccine is highly immunogenic, induces TILs with vaccine-specific TCRs, and is highly effective in preventing and inhibiting TNBC development and progression in vivo.

[0117] [preface] Breast cancer is the leading cause of cancer in women, with an estimated 281,550 new cases and 43,600 deaths reported in the United States in 2023. 1 Approximately 20% of breast cancers are classified as triple-negative breast cancer (TNBC) because they do not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This includes basal cell-like and, more recently, claudin hypo-nucleus as molecular subtypes. 2Triple-negative breast cancer (TNBC) is a subtype of breast cancer observed in women with BRCA-1 germline mutations. In the sporadic setting, TNBC is also associated with African American ethnicity, younger age, higher grade and mitotic index, and advanced stage at diagnosis 3 Specific risk factors correlated with TNBC include reproductive factors (pregnancy and multiple births) and obesity. In TNBC patients, the 5-year survival rate is much lower than that of other types of breast cancer, including estrogen receptor (ER) + progesterone receptor (PR) + human epidermal growth factor receptor 2 (HER2) - luminal A and HER2 + subtypes. There are highly effective targeted therapies for luminal A tumors, such as hormonal agents (tamoxifen, aromatase inhibitors), while HER2 4、5 breast cancer is treated with anti-HER2 antibodies or small molecule HER2-kinase inhibitors + In contrast, early and intermediate stage TNBC is routinely treated with standard cytotoxic chemotherapy, which produces strong initial shrinkage in most patients; however, resistance to chemotherapy subsequently develops in most patients. Current challenges in the treatment of TNBC require new approaches to improve clinical outcomes. 6 Cancer vaccines studied to date have been mainly tested in late-stage cancers, providing limited efficacy, most likely due to the immunosuppressive tumor microenvironment inherent in advanced malignant lesions. Immunosuppression can involve multiple mediators, including regulatory T cells (Tregs), inhibitory macrophages, and other suppressor factors. One way to potentially use cancer vaccines more effectively is in a preventive or early-interference setting. If an antigen is expressed early in carcinogenesis, the vaccine can have utility in prevention. Some reports have now provided proof-of-concept to support the use of peptide vaccines targeting overexpressed self-antigens for cancer immunoprevention.

[0118] 7~10 ​Molecular analysis of tumors identified many genes overexpressed in breast cancer that can be utilized as tumor antigens and potential vaccine candidates. Currently, the most common tumor antigens used in cancer immunotherapy are upregulated autoproteins such as HER2. Vaccination with peptides targeting overexpressed HER2 in humans has been shown to be effective and well-tolerated. 11、12 Mutant epitopes are recognized by the immune system as exogenous "neoantigens" and induce a type 1 immune response, while epitopes derived from non-mutant autoantigens are more likely to activate T helper 2 (Th2) cytokines such as interleukin (IL)-10 and IL-4, which can inhibit the proliferation and function of cytotoxic T lymphocytes (CTLs). In recent years, attempts have been made to specifically identify Th1-selective epitopes derived from non-mutant autoantigens that can induce a Th1 response, induce uncompetitive type 1 immunity when used in vaccines, and be effective in preventing cancer growth in preclinical models. If Th2-inducible epitopes derived from the same protein are included in the vaccine, immunized Th2 cells can suppress the Th1-mediated antitumor effect, making it inevitable to remove Th2 epitopes from cancer. 12、13 .

[0119] In this study, the applicants identified genes highly expressed in malignant tissue using the Cancer Genome Atlas (TCGA) database and transcriptomics and / or proteomics analyses of normal, versus, and malignant human breast tissue. The applicants found that topoisomerase 2 alpha (TOP2A) is highly expressed in human TNBC (Figures 35A–35F). TOP2A is a key enzyme known to be involved in DNA replication and cancer cell proliferation, and is a direct or indirect target of several cytotoxic anticancer agents (e.g., anthracyclines and etoposides). Recent studies have suggested that TOP2A has potential applications in breast cancer detection and management. 14The applicants constructed a TOP2A multipeptide vaccine and evaluated its immunogenicity and prophylactic efficacy against TNBC in a C3(1) / Tag mouse model, which is widely used due to its genetic similarity to the human basal subtype of TNBC. 15、16 Both C3(1) / Tag TNBC cells and breast cancer cell lines (derived from C3(1) / Tag breast tumors) implanted in the mammary fat body have been reported to spontaneously metastasize to the lungs and liver. 17 Using single-cell RNA sequencing (scRNAseq) analysis, the applicants demonstrate that TOP2A multipeptide vaccination induces antitumor CD4+ Th1 cells and cytotoxic CD8+ T cells in mouse mammary tumor and lymph node tissue samples. Furthermore, the applicants found that the TOP2A vaccine induces a potent TOP2A-specific memory immune response that rejects secondary tumor exposure. Finally, the applicants tested TCR sequences from CD4 tumor-infiltrating lymphocytes (TILs) in tumors derived from vaccinated mice and detected TILs with TCR sequences for all immunizing peptides. In summary, the applicants' data demonstrate that the multipeptide TOP2A vaccine is highly immunogenic and effective in the prevention of TNBC, warranting further investigation.

[0120] [result] (Genetic expression analysis of TOP2A) The TOP2A gene was found to be overexpressed in various mouse mammary tumor cell lines as well as in human TNBC and lung tumors (Figures 35A-35F). In mice, the TOP2A gene was overexpressed in mouse mammary tumor cell lines M27 (benign mammary tumor cell line), M6 (malignant mammary tumor cell line), and M6C (metastatic mammary tumor cell line), with the M28 mammary cell line used as a normal control (Figure 35A). The TOP2A gene was also highly overexpressed in TCGA TNBC samples (12 normal mammary tissues and 137 tumor samples from TNBC patients) and only limitedly expressed in normal mammary tissue (Figure 35B). The applicants also performed expression analysis of TNBC in African Americans (AA), who are at high risk for TNBC. Similar to the general TNBC population, TOP2A was significantly overexpressed in TNBC derived from the AA subpopulation (Figure 35C). Representative images of TOP2A IHC staining are shown in Figures 35D-35F. TOP2A was highly expressed in ductal carcinoma in situ (DCIS) and malignant tumor tissue from C3(1) / Tag mice (Figures 35E and 35F). However, TOP2A was not detected in normal mammary gland tissue from wild-type mice (Figure 35D). These data suggest that the TOP2A gene is overexpressed in TNBC isolated from both mice and humans.

[0121] (Identification of Th1 TOP2A epitopes for vaccine design) Using a multi-scoring system that combines multiple MHC class II peptide binding algorithms, the applicants identified and selected three Th1-promoting vaccine candidate peptides: p232 (p232-p246), p410 (p410-p425), and p604 (p605-p621) (Figure 30A). The immunogenicity of the TOP2A peptide in C3(1) / Tag-REAR mice was assessed by the IFN-γ ELISpot assay (Figure 30C). Splenocytes from mice vaccinated with TOP2A demonstrated a strong immune response on average for IFN-γ secreting cells with approximately 400 spots per well (SPW), compared to fewer than 10 SPW for the negative control HIV peptide. Furthermore, the applicants tested the immunogenicity of all three combined peptides (combo). Interestingly, mice vaccinated with the combo showed a significantly stronger immune response than mice vaccinated with a single peptide. Of the three TOP2A peptides, two mouse peptide sequences (p232-246 and p605-621) showed 100% sequence identity with the human TOP2A sequence, while the third peptide sequence (p410-425) showed 93% similarity. These peptides, p232, p410, and p604, were selected for formulation into a multipeptide TOP2A vaccine for prophylactic efficacy studies.

[0122] (TOP2A vaccination prevented TNBC development in genetically modified mouse models.) To test the tumor-preventive effects of TOP2A vaccines in a more clinically relevant model, the applicants used a C3(1) / Tag transgenic mouse model that develops invasive mammary tumors that share important molecular and biological characteristics with human basal cell-like TNBCs. 17The experimental design for this experiment is shown in Figure 30D. Briefly, mice were vaccinated every two weeks for four consecutive weeks, and then monthly at 14 and 18 weeks of age. The applicants measured palpable tumor volume at the end of the experiment (20 weeks). As shown in Figure 30E, TOP2A vaccination significantly reduced tumor volume compared to the adjuvant control (CpG only), with an average tumor size of 736 mm in the adjuvant control. 3 , versus 11 mm in vaccinated mice 3 (p<0.05). Notably, the vaccine completely prevented mammary tumor growth in 8 out of 11 mice vaccinated with TOP2A, while the whole adjuvant control group developed tumors. The applicant confirmed the immunogenicity of the TOP2A peptide by IFN-γ ELISpot assay (Figures 36A and 36B). The applicant also confirmed the tumor area of ​​1 mm² in animals that developed tumors (3 mice vaccinated with TOP2A and 12 CpG control mice). 2 When the number of CD4+ and CD8+ TILs per cell was assessed, TOP2A vaccination was observed to significantly increase CD4+ and CD8+ TILs compared to the adjuvant control (Figure 30F, g; p<0.01). Close monitoring of all animals in all groups by the applicants revealed that body weight, serum ALT, and AST levels did not change significantly at 20 weeks of age (Figures 30I-30K). These results demonstrate that TOP2A vaccination can effectively prevent TNBC development in this genetically engineered mouse model and suggest that vaccine-induced tumor antigen-specific T cell responses play a crucial role in the immunopreventive effect of the vaccine.

[0123] (TOP2A vaccination induced a Th1 cytokine response.) To assess Th1 and Th2 cytokine production in response to the TOP2A vaccine, we measured the production of 12 cytokines from in vitro peptide-stimulated splenocytes isolated from vaccinated C3(1) / Tag GEM mice (Figure 30H). The most abundant cytokines detected in response to TOP2A peptide pool stimulation were the Th1 cytokines IL-2 and IFN-γ, which increased approximately 7-fold and 3-fold, respectively, compared to the adjuvant control. In contrast, there was no notable increase in Th2 cytokine production (IL-4, IL-5, and IL-13) compared to the adjuvant control. Interestingly, the TOP2A vaccine stimulated not only Th1 cytokine production (IL-2 and IFN-γ) but also IL-23, which promotes the differentiation of Th17 lymphocytes. These data suggest that Th1 and Th17 immune responses are predominantly induced by the TOP2A-specific peptide vaccine.

[0124] (TOP2A multipeptide vaccination slowed the growth of syngeneic mouse TNBC tumors.) The applicants then investigated the antitumor efficacy of TOP2A vaccination in the syngeneic TNBC mouse model, C3(1) / Tag-REAR mice, inoculated with M6 tumor cells. As described in Figure 31A, 7-week-old C3(1) / Tag-REAR mice received their first vaccination with TOP2A peptide and CpG oligodeoxynucleotide (CpG ODN) adjuvant, followed by three more vaccinations at one-week intervals. One week after the last vaccination, M6 cells were implanted into the #4 mammary fat body of the C3(1) / Tag-REAR mice. Additional vaccinations were administered at four-week intervals until the experimental endpoint. Notably, TOP2A vaccination significantly reduced tumor volume compared to CpG-only treated control mice (Figure 31B; p<0.05). The mean tumor size at the experimental endpoint was 757.2 mm in the control group. 3 , versus, 413 mm in vaccinated animals 3The TOP2A vaccine also reduced tumor weight by nearly 40% (Figure 31C, p<0.05). Mice vaccinated with TOP2A were also tested for the presence of a systemic immune response. Splenocytes collected from TOP2A-vaccinated mice were stained for intracellular markers (granzyme B, IFN-γ, and TNFα) and analyzed by flow cytometry. A significant increase in CD4+ and CD8+ T cells expressing granzyme B, IFN-γ, and TNFα was observed in the spleens of TOP2A-vaccinated animals compared to controls treated with CpG alone (Figures 31D-31I). In summary, these results indicate that the TOP2A peptide vaccine induced both a focal and systemic type 1 immune response.

[0125] (C3(1) / Tag mice vaccinated with TOP2A were protected from secondary exposure to M6 cells.) The applicants investigated the long-term immunological memory-protective effect of the TOP2A vaccine against secondary tumor cell development. The first "exposure" involved spontaneous tumor development in C3 / Tag mice (see Figure 32A for experimental design). Secondary tumor exposure involved transplantation of syngeneic M6 tumor cells into 22-week-old (4 weeks after the final vaccination) C3 / Tag mice. Notably, TOP2A-vaccinated mice did not develop spontaneous tumors, and vaccinated mice had significantly smaller M6 TNBC tumors at the experimental endpoint (Figures 32B and 32C). The mean tumor size in vaccinated mice was 42 mm compared to adjuvant controls. 3 , versus, 229.9mm 3 (Figure 32B; p<0.001). In mice vaccinated with TOP2A, tumor weight was also significantly reduced by almost 85% (Figure 32C). As shown in Figure 32D, the number of CD4+ and CD8+ TILs was also significantly increased in mice vaccinated with TOP2A. These results suggest that the TOP2A vaccine helps to induce long-term T cell memory and resist tumor recurrence.

[0126] (Single-cell gene expression landscape in breast tumors and lymph node tissue) The applicants used the Seurat package to perform fine clustering of sequenced single cells derived from mouse mammary tumors and lymph node tissue. 18、19 Single-cell gene expression data were aligned and projected in two dimensions using Uniform Manifold Projection (UMAP). 20 The gene expression patterns of canonical markers were analyzed to characterize different types of immune cell populations in tumor samples. Six immune cell populations were detected in breast tumor samples, including CD8+ T cells, CD4+ T cells, CD4 / CD8 double-negative T cells (DNTs), dendritic cells (DCs), macrophages, and neutrophils (Figures 37A and 37B). Single-cell expression data from lymph node tissue were also projected by UMAP, and the identified immune cell populations included CD8+ T cells, CD4+ T cells, DNTs, DCs, and macrophages (Figures 37C and 37D).

[0127] (TOP2A vaccine treatment increased the proportion of tumor-specific cytotoxic CD8+ T cells in mouse mammary tumors and lymph node tissue.) To determine the efficacy of TOP2A vaccine treatment in different T cell subsets, we performed deep clustering of CD8+ T cells, CD4+ T cells, and DNT cells derived from mouse mammary tumors in 3 mice with tumors (8 mice without tumors) out of 11 mice. The location of CD8+ T cells was identified using canonical markers (Figure 33A). Unsupervised clustering of CD8+ T cells using the TILPRED program (https: / / github.com / carmonalab / TILPRED) identified four distinct CD8+ T cell subsets with separate transcriptomics profiles (Figures 33B and 33C). 21The CD8 subset included effector memory (EM)-like, exhausted, memory-like, and naive CD8+ T cells. In cancer, EM-like and exhausted CD8+ cells are involved in antitumor and tumor-promoting functions, respectively. TOP2A vaccination significantly increased the abundance of antitumor EM-like CD8+ T cells in the mammary tumor microenvironment (TME) (Figure 33D). In contrast, exhausted CD8+ T cells were reduced by TOP2A vaccination treatment. These data suggest that TOP2A vaccination treatment improves the overall composition of beneficial antitumor CD8+ T cells in mammary tumors. In lymph node samples from mice with tumors, the applicants observed similar effects of TOP2A vaccination treatment on CD8+ T cell subsets. CD8+ T cells from lymph node samples were plotted (Figure 33E), and CD8+ T cell subsets were identified using corresponding markers (Figures 33F and 33G). TOP2A vaccine treatment increased the abundance of EM-like CD8+ T cells and significantly reduced the proportion of exhausted CD8+ T cells in the lymph nodes (Figure 33H).

[0128] (TOP2A vaccine treatment increased the abundance of CD4+ Th1 cells in mouse mammary tumors and lymph node tissue.) Functional CD4+ cells can play a major role in the antitumor response induced by TOP2A vaccine treatment. We analyzed CD4+ T cells derived from breast tumors and identified three types of CD4+ T cell subsets, including CD4 Th1, CD4 Th17, and Treg cells (Figures 33I–33K). CD4+ Th1 cells overexpressed the marker genes perforin, IFNγ, and TNF; CD4 Th17 T cells overexpressed IL17a and Rorc; and Treg cells overexpressed Foxp3 (Figure 33J). TOP2A vaccine treatment significantly increased the proportion of CD4+ Th1 and Th17 cells in breast tumors while decreasing the abundance of Treg cells (Figure 33I), suggesting that the antitumor function of CD4+ T cells was significantly enhanced by TOP2A vaccine treatment. In lymph node samples, four types of CD4+ T cell subsets were identified: CD4 CM (central memory T cells), CD4 Th1, Treg, and CD4 HSP (CD4+ T cells overexpressing Hspa1a) (Figures 33M-33O). Similar to breast tumor samples, the applicants observed an increased frequency of CD4+ Th1 cells after TOP2A vaccination (Figure 33I). CD4 CM cells were also increased, while immunosuppressive CD4 HSP cells were decreased by TOP2A vaccination treatment (Figure 33P). 22 Our results suggest that CD4+ Th1 cells are involved in the (rated) antitumor response generated by the TOP2A vaccine.

[0129] (TOP2A vaccine treatment also affects other immune cells in mouse tumors and lymph node tissue.) The applicants examined tumor and lymph node tissue for changes in other immune cell populations after TOP2A vaccine treatment. For DNT cells (CD4 / CD8 double-negative T cells) in mouse mammary tumors and lymph node tissue, the cells were divided into three subsets: helper, cytotoxic, and native DNT cells (Figures 38A-38D and 39A-39D). 23TOP2A vaccine treatment significantly increased the abundance of cytotoxic DNT cells, while the treatment decreased the proportion of native DNT cells in mouse mammary tumor tissue (Figure 38D). The percentage of helper DNT cells was not altered by TOP2A vaccination (Figure 38D). In lymph node samples, TOP2A vaccine treatment increased the percentage of helper DNT cells but did not alter the percentage of cytotoxic DNT cells (Figure 39D). These results suggest that TOP2A vaccine treatment specifically increases the abundance of cytotoxic DNT cells in mouse mammary tumors. Macrophages were subdivided into M1 and M2 subsets according to their marker gene expression in both mammary tumor and lymph node tissue (Figures 40A-40D and 41A-41D). Following TOP2A vaccination, the abundance of antitumor M1 macrophages increased and protumor-promoting M2 macrophages decreased in both mammary tumors and lymph node tissue (Figures 40D and 41D). These data suggest that macrophages are also involved in the antitumor response induced by TOP2A vaccination. Two subsets of DCs were detected in the treated mouse tissues based on analysis of corresponding marker genes: conventional DCs (cDCs) and plasmacytoid DCs (pDCs) (Figures 42A-42D and 43A-43D). 24 TOP2A vaccine treatment significantly increased the proportion of cDCs in breast tumor tissue but not in lymph nodes. cDCs are known to play a crucial role in antitumor immunity. 25 Finally, the applicants detected neutrophils in breast tumor samples. The neutrophils can be divided into stage I and stage II subsets, representing progenitor and mature neutrophils, respectively. 26 The applicants identified both neutrophil subsets in breast tumor tissue and found that TOP2A vaccine treatment did not alter the proportions of these two subsets (Figures 44A-44C).

[0130] (TCR clonal analysis revealed the emergence of new T cell clones as a result of TOP2A vaccination.) To explore the potential mechanisms underlying the antitumor response induced by TOP2A vaccine treatment, we analyzed scTCRseq data from control and TOP2A-vaccinated T cells. We sequenced 201 CD4+ T cells from tumor tissue derived from TOP2A-vaccinated mice and 57 CD4+ T cells from tumor tissue derived from CpG-control mice. The top five most frequent TCR clone types (frequency >= 4) constituted 15.5% of the total CD4+ TCR clone types in the TOP2A-vaccinated group; these clone types were not found in the CpG-control group, suggesting their involvement in the TOP2A vaccine-generating CD4+ T cell immune response (Figure 34A). Almost all TCR clone types in the vaccinated mice were unique to CpG controls (Figures 34A-34E). To predict the likelihood of TCR-peptide binding for TOP2A vaccine peptides, the applicants used ERGO (pEptide tcR matchinG predictiOn) software, a highly specific and comprehensive TCR-peptide binding predictor (https: / / github.com / louzounlab / ERGO). 27For the first TOP2A peptide - KDIVALMVRRAYDIA (SEQ ID NO: 1), the top four TCR clone types with the highest binding scores are CAAKPINYGNEKITF_CASSIWVGPSQNTLYF (2 cells, clonal frequency: 1%), CAVYQGGRALIF_CASSQRGIWENTGQLYF (6 cells, clonal frequency: 3%), NA_CASSGLGGDTQYF (2 cells, clonal frequency: 1%), and CALGDPGNTRKLIF_CASSLGGTGQLYF (9 cells, clonal frequency: 4.5%) (Figure 34C). For the second TOP2A peptide, ILNWVKFKAQVQLNKK (SEQ IS NO. 2), the top four clonal types with the highest binding scores were NA_CTCSVSYNSPLYF (2 cells, clonal frequency: 1%), NA_CASSHTNSDYTF (4 cells, clonal frequency: 2%), CAAKPINYG-NEKITF_CASSIWVGPSQNTLYF (2 cells, clonal frequency: 1%), and CALGDPGNTRKLIF_CASSLGGTGQLYF (9 cells, clonal frequency: 4.5%) (Figure 34D). For the third TOP2A peptide, KKWKVKYYKGLGTSTSK (SEQ ID NO: 3), the top four clonal types with the highest binding scores are NA_CASSHTNSDYTF (4 cells, clonal frequency: 2%), CAVRDSNYQ-LIW_CASSMGDNYAEQFF (2 cells, clonal frequency: 1%), NA_CASSGLGGDTQYF (2 cells, clonal frequency: 1%), and CAVYQGGRALIF_CASSQRGIWENTGQLYF (6 cells, clonal frequency: 3%) (Figure 34E). Notably, the top two most frequent TCR clone types, CALGDPGNTRKLIF_CASSLGGTGQLYF and CAVYQGGRALIF_CASSQRGIWENTGQLYF, both showed high binding affinity to the TOP2A peptide KDIVALMVRRAYDIA (SEQ ID NO: 1) (Figure 34C), suggesting that this TOP2A peptide plays a crucial role in the TOP2A vaccine-induced antitumor T cell response.

[0131] [Consideration] In this study, the applicants developed a multipeptide vaccine targeting TOP2A and demonstrated that this multipeptide TOP2A vaccine was remarkably effective in prophylactic efficacy studies using C3(1) / Tag transgenic mice. Top2A vaccination increased the percentage of CD4+ and CD8+ tumor-infiltrating lymphocytes, as well as functionally activated CD4+ and CD8+ cells in the spleen of vaccinated mice. TOP2A vaccination showed no toxicity in any key organs of vaccinated C3(1) / Tag mice, suggesting that the TOP2A vaccine is generally safe and does not induce clear autoimmunity. In addition, the applicants showed that a long-term memory response occurred in TOP2A-vaccinated C3(1) / Tag mice, due to their rejection of secondary exposure to M6 mammary cancer cells.

[0132] TOP2A is an enzyme that regulates the topological state of DNA. It catalyzes double-strand DNA breaks and promotes gene transcription during mitosis. 28 TOP2A is overexpressed in several malignant lesions, including breast cancer, ovarian cancer, and prostate cancer. TOP2A is directly associated with tumor cell proliferation and invasiveness in breast cancer, and its expression has been reported to be amplified in TNBC patients with high-risk factors such as large tumor size, high-grade tumors, and lymph node invasion. 14、29、30 In this specification, the applicants identified overexpression of the TOP2A gene in both mouse mammary tumor cell lines and human TNBC cancer samples (Figures 35A-35F). The applicants also confirmed TOP2A expression in TNBC derived from AA women. The incidence of TNBC has been reported to be higher in AA women compared to European American women, possibly due to a combination of factors including an increased likelihood of developing the disease and a poorer prognosis. 31、32 .

[0133] The potential usefulness of any specific animal model of cancer depends on whether it is reasonably equivalent to the corresponding human disease. This is easier to achieve when the cancer is driven by a clearly defined mutation or amplification, for example, pancreatic cancer (KRAS mutation), colon cancer (mutation in the APC or WNT pathway), squamous cell carcinoma (p53 mutation), or HER2-positive breast cancer (HER2 amplification). In contrast, sporadic TNBCs in humans do not have a single driving mutation, but the vast majority of tumors have p53 mutations and RB loss. 15、16 Tumors derived from the C3(1) / Tag mouse model similarly exhibit loss of p53 and RB function. Breast cancer in the C3(1) / Tag mouse model develops through atypical ductal hyperplasia and DCIS, beginning around 10 weeks of age, and progresses to mammary adenocarcinoma after 12 weeks of age. 33 Most importantly, tumors derived from this model appear to be similar to human TNBCs when compared through RNA expression and genomic modifications (amplification and deletion). 15、16 Therefore, this model appears to be a relatively superior model of human TNBC and responds to certain cytotoxic agents that have demonstrated usefulness against human TNBC. In this study, TOP2A vaccination was initiated before cancer development in C3(1) / Tag mice (Figure 30D), and the vaccine effectively inhibited tumor volume compared to adjuvant controls (Figure 30E). Interestingly, tumor incidence differed significantly between the adjuvant control and TOP2A vaccine groups. In TOP2A-vaccinated mice, tumors developed in only 27% of mice, while all adjuvant-treated controls developed cancer. The applicants did not observe any TOP2A vaccine-related toxicity in major organs, including the brain, kidneys, liver, lungs, and bone marrow (data not shown). Similarly, no toxicity was observed after vaccination of mice with vaccines targeting EGFR, HER2, or IGFBP-2 peptides in humans. 9、10、34、35 。

[0134] Peptides derived from any protein expressed within a given cell can be processed intracellularly in the ER and TAP and ultimately presented on the cell surface, but are usually ignored by the immune system, because the majority of these T cells are deleted from the T cell repertoire. However, in the case of overexpressed autoproteins, this is known to drive T cell responses from the small percentage of "low" affinity (latent) T cell epitopes present in the repertoire. Furthermore, when some tumor cells die, overexpressed proteins can be taken up by APCs and presented to both CD4 and CD8 T cells. CD4 T cells can differentiate into various Th subsets that can induce, modulate, and maintain an immune response against tumor antigens. Such CD4 T cell subsets include Th1, Th2, and regulatory T cells (Treg). The Th1 subset produces IFN-γ, TNF-α, and IL-2, which regulate cellular immunity and play a crucial role in the anti-tumor immune response. 10 The TOP2A vaccine used in this study predominantly induced a Th1 cell response without inducing a strong Th2 response. Type I cytokines secreted by Th1 cells, such as IFN-γ, can upregulate MHC class I expression on the APC membrane along with tumor cells, which may facilitate tumor recognition by CD8+ T cells. 36 Furthermore, Th1 cells can facilitate the cross-presentation of MHC class I-binding peptide antigens to CD8+ T cells. 10、37These mechanisms may be involved in generating a Th1 response associated with the TOP2A vaccine and, ultimately, facilitating the generation of a CD8+ T cell response. It is also possible that the Top2A MHC II peptide may induce an immune response for CD8+ T cells in vaccinated mice via cross-priming. Furthermore, our MHC class-II peptide contains two class-I peptide epitopes: P235 (VALMVRRAY) (SEQ ID NO: 32) within class-II peptide P232 and P414 (VKFKAQVQL) (SEQ ID NO: 33) within class-II peptide P410. Cytotoxic CD8+ T cells play a crucial role in the anti-tumor immune response by directly killing tumor cells. 38 Our research demonstrated that the TOP2A vaccine can stimulate both localized and systemic antigen-specific CD4+ and CD8+ T cell responses. We also found a significant increase in IL-23 secretion from splenocytes isolated from mice vaccinated with TOP2A. IL-23 is important and necessary for the differentiation of Th17 lymphocytes. 39 IL-23 is known to play a role in antitumor activity, so this point deserves attention. 40、41 .

[0135] In summary, the applicants hereby demonstrate that a vaccine based on TOP2A multipeptides possesses prophylactic activity in a TNBC mouse model. TOP2A vaccination induced a Th1 immune response that significantly reduced TNBC tumorigenesis in both syngeneic and GEM models. Meanwhile, no toxicity was observed in the vaccinated mice. The applicants designed 15-17 amino acid peptides with 100% sequence identity between human and mouse TOP2A, enabling these peptides to be directly translated into clinical studies. Overall, the applicants' data demonstrate that the multipeptide TOP2A vaccine is highly immunogenic and has the potential to be effective in immunopreventing TNBC in humans.

[0136] [method] (mouse) The transgenic C3(1) / Tag mice and C3(1) / Tag-REAR (abbreviation for rearrangement) mice were generously donated by Dr. Jeffery E. Green. 17 FVB / N wild-type mice were purchased from Jackson Laboratory. Only F2 C3(1) / Tag or C3(1) / Tag-REAR generation mice were used for all experiments. Mice were acclimatized for one week after arrival at the facility. When the experiment reached its endpoint, all mice were euthanized by CO2 inhalation in their cages followed by neck dislocation. Mice were maintained and bred at the Biomedical Resources Center of the Medical College of Wisconsin (MCW), Milwaukee, WI, and the Houston Methodist Research Institute, Houston, TX. All procedures were approved by the Institutional Animal Care Committee (IACUC).

[0137] (cell line) All M27 (weakly tumor-genic / benign tumor), M6 (malignant tumor), M6C (metastatic tumor), and M28 (normal control) cells, derived from C3(1) / Tag mice, were provided by Dr. Jeffery E. Green. 42 The cell lines were maintained in high-glucose DMEM medium (Gibco) supplemented with 5% fetal bovine serum, penicillin / streptomycin, and sodium pyruvate (Invitrogen).

[0138] (Immunohistochemistry (IHC)) IHC staining was performed by the Histology Core of the Pediatric Institute at MCW. Leica Bond Immunostainer Max (model #10897664) and Leica Bond Immunostainer RX system (model #11784892) were used for human tissue microarrays (TMAs) and mouse samples, respectively. Mouse mammary gland samples from C3(1) / Tag or C3(1) / Tag-REAR mice were formalin-fixed and paraffin-embedded (Sakura Tissue Tek VIP5). Four 4 μm sections were used for IHC staining. The number of CD4+ and CD8+ tumor-infiltrating T lymphocytes (TILs) was determined by using CD4 antibody (Invitrogen 14-9766-82) and CD8 antibody (Invitrogen 14-0808-82) to measure tumor area per 1 mm². 2 It was determined to be the winning cell.

[0139] (ELISpot assay) Cell suspensions derived from the entire spleen were filtered through a 70 μm cell filter (BD) and subjected to erythrocyte lysis using ACK lysis buffer. 3.0 × 10 5 Cells were coated with anti-interferon-γ (IFN-γ) detection antibody and seeded into individual wells of a MAIPS4510 multiscreen 96-well plate containing medium with peptide, concanavalin A (positive control), HIV peptide (negative control), or antigen-free (negative control). After 72 hours of incubation, the plate was washed and incubated overnight at 4°C with secondary antibody (BD). The wells were then washed with PBS and HRP streptavidin was added. After 1 hour of incubation, the plate was developed using AEC substrate for 5–25 minutes. The plate was imaged and the number of spots quantified using an automated plate reader system (CTL Technologies).

[0140] (Scoring system for predicting MHC class II-binding epitopes) Using a combined scoring system published by Dr. Disis and his colleagues, they identified selected antigen-specific MHC epitopes with optimal binding affinity. 34In short, to identify antigen-specific MHC class II epitopes with optimal binding affinity and randomness across multiple alleles, the following algorithms were used for prediction: NetMHCIIpan (https: / / services.healthtech.dtu.dk / service.php?NetMHCIIpan-4.0, Technical University of Denmark, Lyngby, Denmark) and Rankpep (http: / / imed.med.ucm.es / Tools / rankpep.html, University of Comptense Madrid, Harvard, Madrid, Spain). For each available MHC class II allele, 20 peptide sequences were initially selected based simply on their ranking in binding affinity predicted by each algorithm. The sequences are approximately 15 amino acids long. A score was assigned to each individual amino acid for each selected peptide, with 1 being the amino acid in the peptide sequence with the highest rank for predicted binding affinity. The scoring of individual amino acids accounted for multi-peptide duplication occurring within and between algorithms. The score (S) for each amino acid was summed across multiple MHC class II alleles derived from two different algorithms. Next, the number (N) of MHC class II alleles predicted to have high affinity binding for each amino acid was counted. The final score for each amino acid was calculated by multiplying S and N. To facilitate the identification of the most potentially immunogenic segments of the protein, each amino acid was assigned a color (from dark red to light blue) based on its final score percentile, with dark red representing the highest score at ≥75% and light blue representing the lowest score at <10%. The color stratification is as follows: dark red = ≥75% of the highest score; red = 50-75% of the highest score; orange = 40-50% of the highest score; yellow = 30-40% of the highest score; green = 20-30% of the highest score; blue = ≤20% of the highest score. Therefore, dark red corresponds to sequences in which multiple peptides obtained high scores both within and across algorithms. The light blue color represents the sequence with the least potential immunogenicity for any predicted highly bound peptide.

[0141] (Vaccine preparation and immunization) Mice were administered a total of 150 μg of vaccine, each containing 50 μg of individual peptides. Three different TOP2A peptides were purchased from Genemed Synthesis and diluted in phosphate-buffered saline (PBS) to 50 μl / mouse. The peptides and an equal amount of adjuvant CpG (class B CpG oligonucleotide; mouse TLR9 ligand, catalog number tlrl-1826, InvivoGen) were added to bring the total vaccine volume to 100 μl / mouse. 50 μg of CpG was used per mouse. Mice were subcutaneously injected with the TOP2A vaccine according to the timelines shown in Figures 30D, 31A, and 32A.

[0142] (in vivo oncogenicity assay) In a syngeneic model, the loss of one of the original multiple copies of the C3(1) / Tag antigen transgene, which results in a spontaneous cancer-free phenotype, generated C3(1) / Tag-REAR mice from the C3(1) / Tag established line. 17 M6 cells derived from C3(1) / Tag transgenic mammary tumors were implanted into the mammary fat pads of C3(1) / Tag-REAR mice. The M6 ​​cells were washed and 1 × 10⁶ cells were added to 100 μl PBS. 6 The cells were resuspended in PBS at a density of 100 cells and injected into the #4 mammary fat pad of female C3(1) / Tag-REAR mice. After implantation, the tumor diameter was measured using calipers, and the tumor volume was calculated using the following formula: maximum diameter × (minimum diameter). 2 ×0.4. In the spontaneous GEM model, C3(1) / Tag mice were treated with the TOP2A peptide vaccine according to the experimental design in Figure 30D. C3(1) / Tag mice were euthanized at 20 weeks of age to estimate tumor development. Tumor volume was measured using calipers and calculated using the following formula: maximum diameter × (minimum diameter) 2 ×0.4.

[0143] (Cytokine analysis) The Mouse Th1 / Th2 / Th17 Cytokines Multi-AnalyteELISArray™ Kit (Qiagen) was used for cytokine analysis. The cytokines represented by this array are IL2, IL4, IL5, IL6, IL10, IL12, IL13, IL17A, IL23, IFN-γ, TNFα, and TGFβ1. Splenocytes from various mouse groups were stimulated with various peptides for 72 hours, and the culture supernatant was collected and assayed according to the manufacturer's instructions.

[0144] (Flow cytometry) Cell pellets were incubated with the target surface marker at the recommended or dose-set concentration, incubated at 4°C for 30 minutes, and protected from light. After incubation, cells were washed and resuspended in FACS fixation buffer for either analysis or intracellular staining. To begin intracellular staining, cells were fixed with the Foxp3 / transcription factor staining buffer set (eBioscience) and stained with the target intracellular marker at the recommended or dose-set concentration, while protected from light at 4°C for at least 30 minutes. Samples were washed with permeabilization buffer and resuspended in FACS fixation buffer. Stained cells were fixed in 1% paraformaldehyde and permeabilized according to the manufacturer's instructions to assess the expression of intracellular targets granzyme B, IFN-γ, and TNF-α. Flow cytometry was performed using an LSR-II flow cytometer (BD). Data were analyzed using FlowJo software (Tree Star). FACS sequential gating / sorting strategies are provided in Figure 45.

[0145] (Single-cell RNA sequencing (scRNAseq) and TCR sequencing (scTCRseq)) For scRNAseq, randomly selected lymph node samples (one from the CpG control group and two from the TOP2A vaccine-treated group) and mammary tumor samples (one from the CpG control group and one from the TOP2A vaccine-treated group) from C3(1) / Tag mice were collected at the end of the study, chopped, and digested in mouse tumor dissociation buffer (Miltenyi Biotec, CA) at 37°C for 30 minutes to produce single-cell suspensions according to the manufacturer's instructions. The processed samples were directly stained with violet viability dye and APC anti-CD45, and CD45+ leukocytes were sorted using FACS. Next, the FACS-sorted CD45+ leukocytes were spun down at 300g for 5 minutes and manually counted using a Neubauer Chamber. Approximately 2.0 × 10⁶ cells were counted. 4 Load the individual cells onto a 10X Chromium controller according to the manufacturer's instructions, approximately 1 x 10 4 This resulted in the recovery of individual cells. For lymph node samples, a single-cell transcriptome library was generated using the Chromium Single Cell 3' v3 reagent kit (10x Genomics). For tumor samples, single-cell transcriptomes and single-cell TCR libraries were prepared using the 10x Chromium Single Cell 5' and VDJ library construction kits. All libraries were sequenced using the NextSeq 500 / 550 High Output kit v2 (150 cycles) (Illumina) according to the manufacturer's protocol.

[0146] (ScRNA-seq data analysis) Raw sequencing data was demultiplexed and converted to a gene-barcode matrix using Cell Ranger (version 2.2.0) mkfastq and the counting function (10x Genomics). The mouse reference genome mm10 was used for alignment. The data was further analyzed in R (version 3.4.0) with Seurat (version 3). The number of genes detected per cell, the number of unique molecular identifiers (UMIs), and the percentage of mitochondrial genes were plotted, outliers were removed, and doublets and dead cells were filtered out. Raw UMI counts were normalized and logarithmically transformed. Next, a combined analysis was performed to identify shared cell clusters present across different datasets. Principal component analysis was performed using variable genes, and the top 20 most statistically significant principal components were used for UMAP analysis.

[0147] (statistical analysis) All in vitro assays were replicated at least three times. Six to twelve mice were used per group for in vivo studies. Differences between the control group and each treatment group were assessed using a two-sided Student's t-test. A P-value < 0.05 was considered statistically significant.

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PubMed PMID: 29608179. ·Carmona SJ, Siddiqui I, Bilous M, Held W, Gfeller D. Deciphering the transcriptomic landscape of tumor-infiltrating CD8 lymphocytes in B16 melanoma tumors with single-cell RNA-Seq. Oncoimmunology. 2020;9(1):1737369. Epub 2020 / 04 / 22. Doi:10.1080 / 2162402X.2020.1737369. PubMed PMID: 32313720; PMCID: PMC7153840. ·Carey L.A., Perou C.M., Livasy C.A., Dressler L.G., Cowan D., Conway K., Karaca G., Troester M.A., Tse C.K., Edmiston S., et al. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA. 2006 Jun 7;295(21):2492-502. PMID: 16757721 ·Cecil, D.L., et al., Elimination of IL-10-inducing T-helper epitopes from an IGFBP-2 vaccine ensures potent antitumor activity. Cancer Res, 2014. 74(10): p. 2710-8 ·Disis, M.L., et al., Peptide-based, but not whole protein, vaccines elicit immunity to HER-2 / neu, oncogenic self-protein. 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[0149] [References for Example 18] 1. Siegel, R. L., Miller, K. D., Fuchs, H. E. & Jemal, A. Cancer statistics. CA Cancer J. Clin 73, 17-48 (2023). 2. Prat, A. et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 12, 68-86 (2010). 3. Jemal, A. et al. Global cancer statistics. CA A Cancer J. Clin 61, 69-90 (2011). 4. Onitilo, A. A., Engel, J. M., Greenlee, R. T. & Mukesh, B. N. Breast Cancer Subtypes Based on ER / PR and Her2 Expression: Comparison of Clinicopathologic Features and Survival. Clin Med Res. 7, 4-13 (2009). 5.Harbeck, N. & Gnant, G. Breast cancer. Lancet 389, 1134-1150 (2017). 6. Gao, J. J. & Swan, S. M. Luminal A Breast Cancer and Molecular Assays: A Review. Oncologist 23, 556-565 (2018). 7. Lollini, P. L., Cavallo, F., Nanni, P. & Forni, G. Vaccines for tumour prevention. Nat. Rev. Cancer 6, 204-216 (2006). 8. Disis, M. L. et al. A multiantigen vaccine targeting neu, IGFBP-2, and IGF-IR pre- vents tumor progression in mice with preinvasive breast disease. Cancer Prev. Res. 6, 1273-1282 (2013). 9. Ebben, J. D., Lubet, R. A., Gad, E., Disis, M. L. & You, M. Epidermal growth factor receptor derived peptide vaccination to prevent lung adenocarcinoma formation: An in vivo study in a murine model of EGFR mutant lung cancer. Mol. Carcinog. 55, 1517-1525 (2016). 10. Pan, J. et al. Immunoprevention of KRAS-driven lung adenocarcinoma by a multipeptide vaccine. Oncotarget 8, 82689-82699 (2017). 11. Schneble, E. J. et al. The HER2 peptide nelipepimut-S (E75) vaccine (NeuVax TM ) in breast cancer patients at risk for recurrence: correlation of immunologic data with clinical response. Immunotherapy 6, 519-531 (2014). 12. Cecil, D. L. et al. Elimination of IL-10-inducing T-helper epitopes from an IGFBP-2 vaccine ensures potent antitumor activity. Cancer Res. 74, 2710-2718 (2014). 13. Disis, M. L. et al. Peptide-based, but not whole protein, vaccines elicit immunity to HER-2 / neu, oncogenic self-protein. J. Immunol. 156, 3151-3158 (1996). 14. Klintman, M. et al. Changes in Expression of Genes Representing Key Biologic Processes After Neoadjuvant Chemotherapy in Breast Cancer, and Prognostic Implications in Residual Disease. Clin. Cancer Res. 15, 2405-2416 (2016). 15. Herschkowitz, J. I. et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Gen- ome Biol. 8, R76 (2007). 16. Herschkowitz, J. & Lubet, R. Mouse models of triple negative [basal-like / claudin low] breast cancer. Breast Dis. 32, 63-71 (2010). 17. Aprelikova, O. et al. Development and Preclinical Application of an Immuno- competent Transplant Model of Basal Breast Cancer with Lung, Liver and Brain Metastases. PloS One 12, e0155262 (2016). 18. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411-420 (2018). 19. Stuart, T. et al. Comprehensive Integration of Single-Cell Data. Cell 177, 1888-1902 (2019). 20. Bachelier, R. et al. Effect of bilateral oophorectomy on mammary tumor formation in BRCA1 mutant mice. Oncol. Rep. 14, 1117-11120 (2005). 21. Carmona, S. J., Siddiqui, I., Bilous, M., Held, W. & Gfeller, D. Deciphering the transcriptomic landscape of tumor-infiltrating CD8 lymphocytes in B16 mela- noma tumors with single-cell RNA-Seq. Oncoimmunology 9, 1737369 (2020). 22. Oh, D. et al. Intratumoral CD4+ T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder. Cancer. Cell 181, 1612-1625 (2020). 23. Yang, L. et al. Transcriptome landscape of double negative T cells by single-cell RNA sequencing. J. Autoimmun. 121, 102653 (2021). 24. Villar, J. & Segura, E. Decoding the Heterogeneity of Human Dendritic Cell Sub- sets. Trends Immunol. 41, 1062-1071 (2020). 25. Murphy, T. L. & Murphy, K. M. Dendritic cells in cancer immunology. Cell. Mol. Immunol. 19, 3-13 (2022). 26. Giladi, A. et al. Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis. Nat. Cell Biol. 20, 836-846 (2018). 27. Springer, I. et al. Prediction of Specific TCR-Peptide Binding From Large Dic- tionaries of TCR-Peptide Pairs. Front. Immunol. 11, 1803 (2020). 28. Pei, Y. F., Yin, X. M. & Liu, X. Q. TOP2A induces malignant character of pancreatic cancer through activating β-catenin signaling pathway. Biochim. Biophys. Acta Mol. Basis Dis. 1864, 197-207 (2018). 29. Zheng, H. et al. Quantum dot-based immunofiuorescent imaging and quantita- tive detection of TOP2A and prognostic value in triple-negative breast cancer. Int. J. Nanomed. 11, 5519-5529 (2016). 30. Nakagawa, M. Expression of p53, Ki-67, E-cadherin, N-cadherin and TOP2A in triple-negative breast cancer. Anticancer Res. 31, 2389-2393 (2011). 31. Carey, L. A. et al. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA. 295, 2492-2502 (2006). 32. Siddharth, S. & Sharma, D. Racial Disparity and Triple-Negative Breast Cancer in African American Women: A Multifaceted Affair between Obesity, Biology, and Socioeconomic Determinants. Cancers 10, 514 (2018). 33. Green, J. E. et al. The C3(1) / SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma. Oncogene 19, 1020-1027 (2000). 34. Park, K. H. et al. Insulin-like growth factor-binding protein-2 is a target for the immunomodulation of breast cancer. Cancer Res. 68, 8400-8409 (2008). 35. Mittendorf, E. A. et al. Vaccination with a HER2 / neu peptide induces intra- and inter-antigenic epitope spreading in patients with early stage breast cancer. Surgery 139, 407-418 (2006). 36. Zhou, F. Molecular mechanisms of viral immune evasion proteins to inhibit MHC class I antigen processing and presentation. Int. Rev. Immunol. 28, 76-393 (2009). 37. Matsuo, M. et al. IFN-gamma enables cross-presentation of exogenous protein antigen in human Langerhans cells by potentiating maturation. Proc. Natl Acad. Sci. USA 101, 14467-14472 (2004). 38. Martinez-Lostao, L., Anel, A. & Pardo, J. How do cytotoxic lymphocytes kill cancer cells? Clin. Cancer Res. 21, 5047-5056 (2015). 39. Toussirot, E. The IL23 / Th17 pathway as a therapeutic target in chronic infiam- matory diseases. Infiamm Allergy Drug Targets 11, 159-168 (2012). 40. Lo, CH et al. Antitumor and Antimetastatic Activity of IL-23. J. Immunol. 171, 600-607 (2003). 41. Ma, X. et al. Interleukin-23 Engineering Improves CAR T Cell Function in Solid Tumors. Nat. Biotechnol. 38, 448-459 (2020). 42. Holzer, R. et al. Development and characterization of a progressive series of mammary adenocarcinoma cell lines derived from the C3(1) / SV40 Large T-antigen transgenic mouse model. Breast Cancer Res. Treat 77, 65-76 (2003).

[0150] [Numbered clauses] This disclosure may also be described in the following numbered clauses.

[0151] Clause 1. A composition comprising one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0152] Clause 2. The composition according to Clause 1, comprising a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 1.

[0153] Clause 3. The composition according to Clause 1 or 2, comprising a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 2.

[0154] Clause 4. A composition according to any one of Clauses 1 to 3, comprising a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0155] Clause 5. The composition according to Clause 1, comprising or consisting of a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1, a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0156] Clause 6. The composition according to Clause 1, comprising or consisting of a polypeptide having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, and a polypeptide having the sequence of SEQ ID NO: 3.

[0157] The composition according to Clause 7, wherein a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1, a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3 are part of a single continuous polypeptide.

[0158] Composition according to clause 7, wherein the first linker is connected to a polypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 and a polypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2, and / or the second linker is connected to a polypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 and a polypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 3.

[0159] Composition according to clause 6, wherein the polypeptide having the sequence of SEQ ID NO: 1, the polypeptide having the sequence of SEQ ID NO: 2 and the polypeptide having the sequence of SEQ ID NO: 3 are part of one continuous polypeptide.

[0160] Composition according to clause 9, wherein the first linker is connected to a polypeptide having the sequence of SEQ ID NO: 1 and a polypeptide having the sequence of SEQ ID NO: 2, and / or the second linker is connected to a polypeptide having the sequence of SEQ ID NO: 2 and a polypeptide having the sequence of SEQ ID NO: 3.

[0161] Composition according to any one of clauses 1 to 10, further comprising a polypeptide having at least 80%, 85%, 90%, 95% or 99% identity to one or more of SEQ ID NOs: 20 - 27.

[0162] Composition according to any one of clauses 1 to 11, further comprising a polypeptide having at least 80%, 85%, 90%, 95% or 99% identity to one or more of SEQ ID NOs: 28 - 30.

[0163] Composition according to any one of clauses 1 to 12, further comprising an adjuvant.

[0164] Composition according to clause 13, wherein the adjuvant comprises a CpG oligodeoxynucleotide.

[0165] Clause 15. A composition comprising a polynucleotide encoding one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0166] Clause 16. The composition according to Clause 15, wherein the polynucleotide codes for a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1.

[0167] Clause 17. The composition according to Clause 15 or 16, wherein the polynucleotide encodes a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2.

[0168] Clause 18. A composition according to any one of Clauses 15 to 17, wherein the polynucleotide codes for a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0169] Clause 19. The composition according to Clause 15, wherein the polynucleotide codes for a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1, a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0170] Clause 20. The composition according to Clause 15, wherein the polynucleotide encodes a polypeptide having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, and a polypeptide having the sequence of SEQ ID NO: 3.

[0171] Clause 21. The composition according to Clause 19, wherein a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1, a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3 are part of a single continuous polypeptide.

[0172] Clause 22. The composition according to Clause 21, wherein the polynucleotide codes for a first linker connected to a polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 and a polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and / or a second linker connected to a polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 and a polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.

[0173] Clause 23. The composition according to Clause 20, wherein the polypeptide having the sequence of SEQ ID NO: 1, the polypeptide having the sequence of SEQ ID NO: 2, and the polypeptide having the sequence of SEQ ID NO: 3 are part of a single continuous polypeptide.

[0174] Clause 24. The composition according to Clause 23, wherein the polynucleotide codes for a first linker connected to a polypeptide having the sequence of SEQ ID NO: 1 and a polypeptide having the sequence of SEQ ID NO: 2, and / or a second linker connected to a polypeptide having the sequence of SEQ ID NO: 2 and a polypeptide having the sequence of SEQ ID NO: 3.

[0175] Clause 25. The composition according to any one of Clauses 15 to 24, wherein the polynucleotide further encodes a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with one or more of SEQ ID NOs. 20 to 27.

[0176] Clause 26. The composition according to any one of Clauses 15 to 25, wherein the polynucleotide further encodes a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with one or more of SEQ ID NOs. 28 to 30.

[0177] Clause 27. The composition according to any one of Clauses 15 to 26, wherein the polynucleotide is mRNA.

[0178] Clause 28. A composition according to any one of Clauses 15 to 27, wherein polynucleotides are present in lipid nanoparticles.

[0179] Clause 29. A method for treating a subject or a method for the prophylactic treatment of a subject, comprising the step of administering to a subject a composition described in any of Clauses 1 to 14 or any of Clauses 15 to 28.

[0180] Clause 30. The method described in Clause 29, wherein the subject has not been previously diagnosed with cancer or triple-negative breast cancer (TNBC), and / or the subject has not been previously treated for cancer or TNBC.

[0181] Clause 31. The method described in Clause 29, wherein the subject has been diagnosed with TNBC.

[0182] Clause 32. The method described in Clause 29, wherein the subject has been diagnosed with cancer and / or breast cancer.

[0183] Clause 33. The method according to Clause 32, wherein the breast cancer does not express one or more of the following: estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor 2 (HER2).

[0184] Clause 34. The method according to any of Clauses 29-33, wherein the subject exhibits one or more risk factors related to TNBC, and one or more risk factors are selected from pregnancy, multiple birth, and obesity.

[0185] Clause 35. The method according to any one of Clauses 29 to 34, wherein the step of administering comprises administering at least one dose of a therapeutically effective amount of the composition described in any one of Clauses 1 to 14 or any one of Clauses 15 to 28.

[0186] Clause 36. The method according to Clause 29, wherein the step of administering comprises administering at least two doses of a therapeutically effective amount of the composition described in any of Clauses 1 to 14 or any one of Clauses 15 to 28.

[0187] Clause 37. The method according to any one of Clauses 29 to 36, wherein the step of administering the composition results in at least a partial increase in the levels of one or more of IFN-γ, TNF-α, IL-2, or IL-23 in the subject compared to an untreated control.

[0188] Clause 38. The method described in Clause 29, provided that the subject has not been previously diagnosed with cancer or lung cancer, and / or has not been previously treated for cancer or lung cancer.

[0189] Article 39. The method described in Article 29, wherein the subject has been diagnosed with lung cancer.

[0190] Clause 40. The method described in Clause 29, wherein the subject has been diagnosed with cancer and / or lung cancer.

[0191] Clause 41. The composition according to Clause 1, wherein the polypeptide is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 31.

[0192] Clause 42. The composition according to Clause 15, wherein the polynucleotide codes for a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 31.

[0193] As described above, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described exemplary herein may be implemented as appropriate even if there are no elements (one or more) or limitations (one or more) not expressly disclosed herein. The terms and expressions used are for illustrative purposes only, not limitation, and in the use of such terms and expressions there is no intention to exclude any equivalents of the shown or described features or parts thereof, but it will be recognized that various modifications are possible within the scope of the invention. Thus, although the invention is illustrated by specific embodiments and optional features, it should be understood that modifications and / or variations of the concepts disclosed herein may be used by those skilled in the art, and such modifications and variations will be considered within the scope of the invention.

[0194] Any method described herein may be carried out in any preferred order, unless otherwise indicated herein or unless the context clearly contradicts it. The use of any examples provided herein is intended solely to illustrate the invention more clearly and does not limit the scope of the invention unless otherwise claimed. Nothing described herein should be construed as indicating that any unclaimed element is essential for carrying out the invention.

[0195] This specification makes numerous references to patent and non-patent documents. These references are incorporated herein by reference in their entirety. In the event of any inconsistency between the definitions of terms herein and those in the references, the terms shall be interpreted according to the definitions herein.

Claims

1. A composition comprising one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 1; at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 2; or at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No.

3.

2. The composition according to claim 1, comprising the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No.

1.

3. The composition according to claim 1, comprising the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No.

2.

4. The composition according to claim 1, comprising the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No.

3.

5. The composition according to claim 1, comprising or consisting of the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 1, the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 2, and the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No.

3.

6. The composition according to claim 1, comprising or consisting of a polypeptide having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, and a polypeptide having the sequence of SEQ ID NO:

3.

7. The composition according to claim 5, wherein the polypeptide identical to at least 80%, 85%, 90%, 95%, or 99% of Sequence ID No. 1, the polypeptide identical to at least 80%, 85%, 90%, 95%, or 99% of Sequence ID No. 2, and the polypeptide identical to at least 80%, 85%, 90%, 95%, or 99% of Sequence ID No. 3 are part of a single continuous polypeptide.

8. The composition according to claim 7, wherein a first linker is connected to the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 and the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and / or a second linker is connected to the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 and the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

3.

9. The composition according to claim 6, wherein the polypeptide having the sequence of SEQ ID NO: 1, the polypeptide having the sequence of SEQ ID NO: 2, and the polypeptide having the sequence of SEQ ID NO: 3 are part of a single continuous polypeptide.

10. The composition according to claim 9, wherein a first linker is connected to the polypeptide having the sequence of SEQ ID NO: 1 and the polypeptide having the sequence of SEQ ID NO: 2, and / or a second linker is connected to the polypeptide having the sequence of SEQ ID NO: 2 and the polypeptide having the sequence of SEQ ID NO:

3.

11. The composition according to claim 1, further comprising a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with one or more of Sequence IDs 20 to 27.

12. The composition according to claim 1, further comprising a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with one or more of Sequence IDs 28 to 30.

13. The composition according to claim 1, further comprising an adjuvant.

14. The composition according to claim 13, wherein the adjuvant comprises a CpG oligodeoxynucleotide.

15. A composition comprising a polynucleotide encoding one or more polypeptides that are at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1; at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2; or at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

3.

16. The composition according to claim 15, wherein the polynucleotide encodes the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

1.

17. The composition according to claim 15, wherein the polynucleotide encodes the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

2.

18. The composition according to claim 15, wherein the polynucleotide encodes the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

3.

19. The composition according to claim 15, wherein the polynucleotide encodes the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1, the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

3.

20. The composition according to claim 15, wherein the polynucleotide encodes a polypeptide having the sequence of SEQ ID NO: 1, a polypeptide having the sequence of SEQ ID NO: 2, and a polypeptide having the sequence of SEQ ID NO:

3.

21. The composition according to claim 19, wherein the polypeptide identical to at least 80%, 85%, 90%, 95%, or 99% of Sequence ID No. 1, the polypeptide identical to at least 80%, 85%, 90%, 95%, or 99% of Sequence ID No. 2, and the polypeptide identical to at least 80%, 85%, 90%, 95%, or 99% of Sequence ID No. 3 are part of a single continuous polypeptide.

22. The composition according to claim 21, wherein the polynucleotide codes for a first linker connected to the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 and the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2, and / or a second linker connected to the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 and the polypeptide which is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

3.

23. The composition according to claim 20, wherein the polypeptide having the sequence of SEQ ID NO: 1, the polypeptide having the sequence of SEQ ID NO: 2, and the polypeptide having the sequence of SEQ ID NO: 3 are part of a single continuous polypeptide.

24. The composition according to claim 23, wherein the polynucleotide codes for a first linker connected to the polypeptide having the sequence of SEQ ID NO: 1 and the polypeptide having the sequence of SEQ ID NO: 2, and / or a second linker connected to the polypeptide having the sequence of SEQ ID NO: 2 and the polypeptide having the sequence of SEQ ID NO:

3.

25. The composition according to claim 15, wherein the polynucleotide further encodes a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with one or more of the sequence numbers 20 to 27.

26. The composition according to claim 15, wherein the polynucleotide further encodes a polypeptide having at least 80%, 85%, 90%, 95%, or 99% identity with one or more of the sequence numbers 28 to 30.

27. The composition according to claim 15, wherein the polynucleotide is mRNA.

28. The composition according to claim 15, wherein the polynucleotide is present in lipid nanoparticles.

29. A method for treating a subject or a method for preventive treatment of the subject, comprising the step of administering the composition according to claim 1 or the composition according to claim 15 to the subject.

30. The method according to claim 29, wherein the subject has not been previously diagnosed with cancer or triple-negative breast cancer (TNBC), and / or the subject has not been previously treated for cancer or TNBC.

31. The method according to claim 29, wherein the subject has been diagnosed with TNBC.

32. The method according to claim 29, wherein the subject has been diagnosed with cancer and / or breast cancer.

33. The method according to claim 32, wherein the breast cancer does not express one or more of the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor 2 (HER2).

34. The method according to claim 29, wherein the subject represents one or more risk factors related to TNBC, and the one or more risk factors are selected from pregnancy, multiple birth and obesity.

35. The method according to claim 29, wherein the administration step includes administering at least one dose of the composition according to claim 1 or claim 15 in a therapeutically effective amount.

36. The method according to claim 29, wherein the administration step includes administering at least two doses of the composition according to any one of claims 1 to 14 or any one of claims 15 to 28 in a therapeutically effective amount.

37. The method according to claim 29, wherein the step of administering the composition results in at least a partial increase in the level of one or more of IFN-γ, TNF-α, IL-2, or IL-23 in the subject compared to an untreated control.

38. The method according to claim 29, wherein the subject has not been previously diagnosed with cancer or lung cancer, and / or the subject has not been previously treated for cancer or lung cancer.

39. The method according to claim 29, wherein the subject has been diagnosed with lung cancer.

40. The method according to claim 29, wherein the subject has been diagnosed with cancer and / or lung cancer.

41. The composition according to claim 1, wherein the polypeptide is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

31.

42. The composition according to claim 15, wherein the polynucleotide encodes a polypeptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 31.

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