Cancer vaccine composition and method of using the cancer vaccine composition for the prevention and / or treatment of cancer.

Abstract

The present invention is based in part on a cancer vaccine composition comprising PTEN- and p53-deficient cancer cells in which the TGFβ-Smad / p63 signaling pathway is activated, and methods of using the cancer vaccine composition for the prevention and / or treatment of cancer. In one aspect, provided herein is a cancer vaccine comprising cancer cells, wherein the cancer cells are (1) PTEN-deficient, (2) p53-deficient, and (3) modified to activate the TGFβ-Smad / p63 signaling pathway.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 876416, filed on 19 July 2019, the entire contents of which are incorporated herein by reference.

[0002] Statement regarding rights This invention was made possible with U.S. National Institutes of Health grants P50 CA168504, CA233810, CA187918, and R35 CA210057. The U.S. Government has certain rights to this invention. [Background technology]

[0003] Transforming growth factor β (TGFβ) is a pluripotent cytokine that plays a crucial role in regulating embryonic development, cell metabolism, tumor growth, and immune system homeostasis (David and Massague, Nat. Rev. Mol. Cell. Biol., 2018, Vol. 19: pp. 419-435). When TGFβ binds to its receptor located on the cell membrane, it regulates the expression of its downstream genes in a manner that may be Smad-dependent or Smad-independent. TGFβ controls cancer development and progression in a stage and cell background-dependent manner (Morikawa et al., (2016) Cold Spring Harb. Perspect. Biol., Vol. 8: a021873; Prunier et al., (2019) Trends Cancer, Vol. 5: pp. 66-78; Seoane and Gomis, (2017) Cold Spring Harb. Perspect. Biol., Vol. 9: a022277). TGFβ suppresses tumorigenesis by inducing cell proliferation arrest and apoptosis in pre-malignant cells. Suppression of the TGFβ signaling pathway promotes tumorigenesis in various mouse models (Cammareri et al., (2016) Nat. Commun., Vol. 7: 12493; Yu et al., (2014) Oncogene, Vol. 33: 1538-1547; Cohen et al., (2009) Cancer Res., Vol. 69: 3415-3424). Loss-of-function mutations in the TGFβ signaling pathway are also commonly found in various human cancers (Levy and Hill, (2006) Cytokine Growth Factor Rev., Vol. 17: 41-58). However, in late-stage cancers, TGFβ promotes tumor metastasis and drug resistance. On the other hand, due to the accumulation of oncogenic mutations, cancer cells themselves overcome the growth arrest and apoptosis induced by TGFβ. TGFβ induces epithelial-mesenchymal transition (EMT) in cancer cells, enhances the stem cell properties of those cancer cells, increases angiogenesis, and promotes drug resistance (Ahmadi et al., J. Cell Physiol., 2018, Vol. 234: pp. 12173-12187).On the other hand, TGFβ promotes the differentiation of CD4+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages, thereby suppressing the host's antitumor immunity, which supports cancer growth and metastasis (Dahmani and Delisle, Cancers (Basel), Vol. 10: p. 194, 2018). Because the TGFβ signaling pathway can act as both a tumor suppressor and a cancer promoter, the ability to utilize the TGFβ signaling pathway for desired therapeutic purposes is a crucial issue. Therefore, identifying anticancer therapies based on a deeper understanding of the role of the TGFβ signaling pathway in cancer is highly necessary in this field. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] David and Massague, (2018) Nat. Rev. Mol. Cell. Biol., Volume 19: 419-435 [Non-Patent Document 2] Morikawa et al., (2016) Cold Spring Harb. Perspect. Biol., Vol. 8: a021873. [Non-Patent Document 3] Prunier et al., (2019) Trends Cancer, Vol. 5: pp. 66-78. [Overview of the Initiative] [Means for solving the problem]

[0005] This invention is at least partially based on the discovery that PTEN and p53-deficient tumor cells having activated TGFβ-Smad / p63 signaling (e.g., treated with at least one TGFβ superfamily protein) fail to form tumors in a T cell-dependent manner in immune-normal hosts. Administration of these tumor cells also protects the host from recurrent and metastatic tumor lesions. This cancer vaccine, produced using these tumor cells, is advantageous in overcoming the robust obstacles in this field, such as the lack of tumor-specific antigen presentation, tumor heterogeneity, and reduced immune cell infiltration, by inducing a broad immune response. These actions have been shown to involve at least partially the activation of the Smad / p63 transcription complex within tumor cells, which controls the expression of multiple pathways that promote immune responses and ultimately facilitate cytotoxic T cell activation and immunological memory.

[0006] In one embodiment, a cancer vaccine comprising cancer cells is provided herein, wherein the cancer cells are (1) PTEN-deficient, (2) p53-deficient, and (3) modified to activate the TGFβ-Smad / p63 signaling pathway.

[0007] In another embodiment, a method is provided herein for preventing the development of cancer, delaying the onset of cancer, preventing the recurrence of cancer, and / or treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of a cancer vaccine comprising cancer cells that are (1) PTEN-deficient, (2) p53-deficient, and (3) modified to activate the TGFβ-Smad / p63 signaling pathway, wherein the subject is in some cases suffering from cancer. In one embodiment, the cancer cells are derived from cancer of the same type as the cancer treated with the cancer vaccine. In another embodiment, the cancer cells are derived from cancer of a different type than the cancer treated with the cancer vaccine. In yet another embodiment, the cancer treated with the cancer vaccine is characterized by loss of PTEN, p53, and / or p110, and the cancer may further express Myc. In yet another embodiment, the cancer treated with the cancer vaccine has functional PTEN and / or p53, and the cancer may have the Kras activating mutation G12D. In another embodiment, the cancer vaccine is homogeneous or heterogeneous to the subject. In yet another embodiment, the cancer vaccine is autologous, compatible allogeneic, incompatible allogeneic, or genetically related to the subject. In yet another embodiment, the cancer treated with the cancer vaccine is selected from the group consisting of breast cancer, ovarian cancer, or brain cancer, for example, a breast tumor, an ovarian tumor, or a brain tumor.

[0008] Numerous embodiments applicable to any aspect of the present invention described herein are further provided. For example, in one embodiment, the TGFβ-Smad / p63 signaling pathway is activated by contacting the cancer cells with at least one TGFβ superfamily protein. In another embodiment, the at least one TGFβ superfamily protein is LAP, TGFβ1, TGFβ2, TGFβ3, TGFβ5, activin A, activin AB, activin AC, activin B, activin C, C17ORF99, INHBA, INHBB, inhibin, inhibin A, inhibin B, BMP-1 / PCP, BMP-2, BMP-2 / BMP-6 heterodimer, BMP-2 / BMP-7 heterodimer, BMP The group is selected from -2a, BMP-3, BMP-3b / GDF-10, BMP-4, BMP-4 / BMP-7 heterodimer, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-15 / GDF-9B, decapentaplesic / DPP, artemin, GDNF, neutrinoline, percephin, lefty A, lefty B, MIS / AMH, nodal, and SCUBE3. In yet another embodiment, the at least one TGFβ superfamily protein is selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3. In yet another embodiment, the cancer cells are brought into contact with the TGFβ superfamily protein in vitro, in vivo, and / or ex vivo. For example, the cancer cells may be brought into contact with the TGFβ superfamily protein in vitro or ex vivo. In another embodiment, the cancer cells are administered to a subject, and the TGFβ superfamily protein is administered to the subject to bring the cancer cells into contact with the subject in vivo. In yet another embodiment, the TGFβ superfamily protein is administered before, after, or simultaneously with the administration of the cancer cells.In yet another embodiment, the TGFβ-Smad / p63 signaling pathway is activated by increasing the copy number, quantity, and / or activity of at least one biomarker listed in Table 1 and / or decreasing the copy number, quantity, and / or activity of at least one biomarker listed in Table 2 in the cancer cells. For example, the copy number, quantity, and / or activity of at least one biomarker listed in Table 1 can be increased by contacting the cancer cells with a nucleic acid molecule encoding at least one biomarker or a fragment thereof listed in Table 1, a polypeptide of at least one biomarker or a fragment thereof listed in Table 1, or a small molecule that binds to at least one biomarker listed in Table 1. In yet another embodiment, the TGFβ-Smad / p63 signaling pathway is activated by increasing the nuclear localization of Smad2. In yet another embodiment, the TGFβ-Smad / p63 signaling pathway is activated by increasing the binding of p63 to Smad2 in the nucleus of the cancer cells. In yet another embodiment, the copy number, quantity, and / or activity of at least one biomarker listed in Table 2 are reduced by contacting the cancer cells with a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interference agent, antisense oligonucleotide, peptide inhibitor or peptide mimetic inhibitor, aptamer, antibody, and / or intracellular antibody.

[0009] In yet another embodiment, the cancer cells are derived from a solid tumor or a hematological cancer. In yet another embodiment, the cancer cells are derived from a cancer cell line. In yet another embodiment, the cancer cells are derived from primary cancer cells. In yet another embodiment, the cancer cells are breast cancer cells. In yet another embodiment, the cancer cells are derived from triple-negative breast cancer (TNBC).

[0010] In yet another embodiment, activation of the TGFβ-Smad / p63 signaling pathway induces epithelial-mesenchymal transition (EMT) in the cancer cells. In yet another embodiment, activation of the TGFβ-Smad / p63 signaling pathway increases the expression levels of ICOSL, PYCARD, SFN, PERP, RIPK3, CASP9, and / or SESN1 in the cancer cells. In yet another embodiment, activation of the TGFβ-Smad / p63 signaling pathway decreases the expression levels of KSR1, KSR1, EIF4EBP1, ITGA5, EMILIN1, CD200, and / or CSF1 in the cancer cells. In yet another embodiment, the cancer cells can activate co-cultured dendritic cells (DCs) in vitro. In yet another embodiment, the cancer cells can upregulate CD40, CD80, CD86, CD103, CD8, HLA-DR, MHC-II, and / or IL1-β in the co-cultured dendritic cells in vitro. In another embodiment, the cancer cells can activate co-cultured T cells in vitro in the presence of DCs. In yet another embodiment, the cancer cells can increase the secretion of TNFα and / or IFNγ by the co-cultured T cells in vitro in the presence of DCs. In yet another embodiment, the cancer cells do not form tumors in immunocompetent subjects. In another embodiment, the cancer vaccine induces cytotoxic T cell-mediated anti-tumor immunity. In yet another embodiment, the cancer vaccine increases CD4+ T cells and CD8+ T cells in the blood and / or tumor microenvironment. In yet another embodiment, the cancer vaccine increases TNFα and INFγ-secreting CD4+ T cells and CD8+ T cells in the blood and / or tumor microenvironment. In another embodiment, the cancer vaccine increases the expression of Icos, Klrc1, Il2rb, Pik3cd, H2-D1, Ccl8, Ifng, Icosl, Il2ra, Cxcr3, Ccr7, Cxcl10, Cd74, H2-Ab1, Hspa1b, Cd45, Lifr, and / or Tnf in tumor tissue. In yet another embodiment, the cancer vaccine increases the amount of tumor-infiltrating dendritic cells.In yet another embodiment, the cancer vaccine upregulates CD80, CD103, and / or MHC-II in tumor-associated DCs. In yet another embodiment, the cancer vaccine reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing cancer cells. In yet another embodiment, the cancer vaccine reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing cancer cells at the initial immunization site. In yet another embodiment, the cancer vaccine reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing cancer cells in tissue distal to the immunization site. In yet another embodiment, the cancer vaccine induces a tumor-specific memory T cell response. In yet another embodiment, the cancer vaccine induces a CD4+ central memory T cell (T) response in the spleen and / or lymph nodes. CM ) and / or CD4+ effector memory T cells (T EM ) increases the percentage. In yet another embodiment, the cancer vaccine increases the spleen CD8+T CM It increases the percentage of cells. In another embodiment, the cancer vaccine increases the CD8+T in the spleen and / or lymph nodes. EM The percentage of cells is increased. In yet another embodiment, the cancer vaccine increases the amount of tumor-infiltrating CD4+ T cells and / or CD8+ T cells. In yet another embodiment, the cancer vaccine increases the amount of tumor-infiltrating CD4+ T CM Cells and / or CD4+T EM To increase the number of cells. In another embodiment, the cancer vaccine is tumor-infiltrating CD8+T CM Cells and / or CD8+T EM The number of cells is increased. In yet another embodiment, the cancer cells do not replicate. In yet another embodiment, the cancer cells do not replicate due to radiation. In yet another embodiment, the radiation is a sublethal dose.

[0011] In yet another embodiment, the cancer vaccine is administered to a subject in combination with immunotherapy and / or cancer therapy, and the immunotherapy and / or cancer therapy may be administered before, after, or concurrently with the cancer vaccine, if desired. In yet another embodiment, the immunotherapy is cell-based. In yet another embodiment, the immunotherapy comprises a cancer vaccine and / or a virus. In yet another embodiment, the immunotherapy inhibits immune checkpoints. In yet another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, and A2aR. In yet another embodiment, the immune checkpoint is PD1, PD-L1, or CD47. In yet another embodiment, the cancer therapy is selected from the group consisting of radiotherapy, radiosensitizers, and chemotherapeutic agents.

[0012] In yet another embodiment, a method is provided herein for evaluating the efficacy of a cancer vaccine to treat a subject suffering from cancer, comprising: (a) detecting the number of proliferating cells in the cancer and / or the volume or size of the tumor containing the cancer cells in a subject sample at a first time point; (b) repeating step (a) at at least one subsequent time point after administration of the cancer vaccine; and (c) comparing the number of proliferating cells in the cancer and / or the volume or size of the tumor containing the cancer cells detected in steps (a) and (b), wherein the absence or significant reduction in the number of proliferating cells in the cancer and / or the volume or size of the tumor containing the cancer cells in the subsequent sample compared to the number and / or volume or size in the sample at the first time point indicates that the cancer in the subject has been treated by the cancer vaccine. In one embodiment, between the first time point and the subsequent time point, the subject has been treated, completed treatment, and / or is in a state of palliative care for the cancer. In another embodiment, the first sample and / or at least one subsequent sample are selected from the group consisting of ex vivo samples and in vivo samples. In yet another embodiment, the first sample and / or at least one subsequent sample is a portion of a single sample or a portion of a pooled sample obtained from the subject. In yet another embodiment, the sample includes cells, serum, peripheral lymphoid organs, and / or intratumoral tissue obtained from the subject. In yet another embodiment, the method described herein further includes determining responsiveness to the drug by evaluating at least one criterion selected from the group consisting of clinical efficacy rate, survival to death, complete pathological response, semi-quantitative pathological response measure, complete clinical remission, partial clinical remission, clinical disease stability, recurrence-free survival, metastasis-free survival, disease-free survival, reduction of circulating tumor cells, circulating marker response, and RECIST criteria. In yet another embodiment, the cancer vaccine is administered in a pharmaceutically acceptable formulation. In yet another embodiment, the administration step is performed in vivo, ex vivo, or in vitro.

[0013] As described above, certain embodiments are applicable to any aspect of the present invention as described herein. For example, in one embodiment, the cancer vaccine prevents recurrent and metastatic tumor lesions. In another embodiment, the cancer vaccine is administered intratumorally or subcutaneously to the subject. In yet another embodiment, the subject is an animal model of the cancer, and in some cases, the animal model is a mouse model. In yet another embodiment, the subject is a mammal, and in some cases, the mammal is in a palliative state for cancer. In yet another embodiment, the mammal is a mouse or a human. For example, the mammal is a human. [Brief explanation of the drawing]

[0014] [Figure 1A] Figures 1A-1C show that TGFβ-treated PP (PPT) tumor cells do not form tumors in immunonormal mice. Figure 1A shows a workflow for investigating the role of TGFβ in a mouse model of TNBC derived from the simultaneous removal of p53 (encoded by Trp53 in mice) and Pten (referred to as PP). [Figure 1B] Figure 1B shows the expression levels of EMT markers detected by real-time PCR in PP cells and TGFβ-treated PP (PPT) cells. Data are shown as mean ± standard error of the mean. * represents P < 0.05, *** represents P < 0.001, and **** represents P < 0.0001. n=4 for each group. [Figure 1C] Figure 1C shows in vivo proliferation of PP cells and PPT cells (n=10 per group). PP tumor cells and TGFβ-treated PP(PPT) tumor cells were introduced into allogeneic, syngeneic FVB wild-type mice. [Figure 2A] Figures 2A and 2B show that PPT tumor cells formed tumors with longer latency in immunodeficient mice. Figure 2A shows the growth rates of PP tumors and PPT tumors in nude mice. n=10 per group. [Figure 2B] Figure 2B shows the growth rates of PP tumors and PPT tumors in SCID mice. n=10 per group. [Figure 3A] Figures 3A to 3I show that PPT tumor cell-induced antitumor immunity was T cell-dependent. Figure 3A shows the proliferation of PP cells and PPT cells in FVB wild-type mice (n=10 per group). [Figure 3B] Figure 3B shows the proliferation of PPT tumor cells in FVB wild-type mice treated with anti-CD3 antibody or anti-IgG antibody (n=10 per group). [Figure 3C] Figure 3C shows a schematic diagram of the workflow for analyzing local and systemic antitumor immune responses in allogeneic mice. [Figure 3D-I] Flow cytometry was used to detect CD45+CD3+CD4+ T cells (Figures 3D-3F) and CD45+CD3+CD8+ T cells (Figures 3G-3I) in the spleen, peripheral blood, and tumor-infiltrating tissue. The proportions of TNFα-secreting and IFN-γ-secreting CD4+ T cells (Figures 3E and 3F) and CD8+ T cells (Figures 3H and 3I) in the spleen, peripheral blood, and tumor microenvironment are shown. Data are presented as mean ± standard error of the mean. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001. n=5 for each group. [Figure 4A] Figures 4A–4I show that activated TGFβ-induced antitumor immunity in tumor cells was triggered through enhanced activation of DCs and T cells. Customized mouse transcriptome profiling was performed to compare gene expression profiles between 6-day-old tumor tissues from PP and PPT (Figures 4A–4C). Gene ontology (GO) enrichment and KEGG pathway analysis were performed on upregulated genes (rpmPPT >2 times compared to rpmPP). Figure 4A shows the relevant GO vocabulary / KEGG pathways. [Figure 4B]Figure 4B shows the expression of several key targets derived from transcriptome data validated by real-time PCR. Data are shown as mean ± standard error of the mean. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001. n=5 for each group. [Figure 4C] Figure 4C shows the relevant gene interaction network that positively regulates antitumor immunity. [Figure 4D] Figures 4D and 4E show the percentage of tumor-infiltrating CD45+CD11C+DCs in 6-day-old PP and PPT tumor tissue (Figure 4D) as analyzed by flow cytometry. Expression of MHC-II, CD80, and CD103 in DCs was gated (Figure 4E). n=5 for each group. [Figure 4E] Same as above. [Figure 4F] Figure 4F shows a schematic diagram of the workflow for analyzing the effects of PP and PPT on the activation of DCs and T cells. [Figure 4G] Figure 4G shows the detection of DC activation markers by flow cytometry. n=6 for each group. **** represents P<0.0001. "Compatible allogeneic" immature DCs collected from the bone marrow of healthy allogeneic FVB mice were cultured with PP cells or PPT cells. [Figure 4H] Figures 4H and 4I show the determination of CD4+ T cell (Figure 4H) and CD8+ T cell (Figure 4I) activation by flow cytometry. n=6 per group. **** indicates P<0.0001. T cells and DCs were co-cultured overnight with or without tumor cells. [Figure 4I] Same as above. [Figure 5A] Figures 5A to 5D show that dendritic cells are required for T cell activation by PPT tumor cells. Figures 5A and 5B show MHC-II expression in CD45+ and CD45- cells in 6-day-old PP and PPT tumor tissue as analyzed by flow cytometry. n=5 for each group. **** represents P<0.0001. [Figure 5B] Same as above. [Figure 5C] Figures 5C and 5D show the expression of TNFα and IFN-γ in CD4+ T cells (Figure 5C) and CD8+ T cells (Figure 5D) as detected by flow cytometry. n=3 per group. T cells isolated from unsensitized mice were cultured overnight with PP cells or PPT cells. [Figure 5D] Same as above. [Figure 6A] Figures 6A–6C illustrate TGFβ-induced Smad2 / p63 complex-mediated antitumor immunity. Figure 6A shows the Smad-related transcription factor network in PPT cells as calculated based on customized mouse transcriptome profiling. Node size and color represent reads per million (rpm) of the displayed gene. "Smads" represents the complex of Smad2, Smad3, and Smad4. [Figure 6B] Figure 6B shows the proliferation of PPT-scramble tumors or PPT-shTrp63 tumors in allogeneic, syngeneic mice. n=10 per group. [Figure 6C] Figure 6C shows the expression of MHC-II, CD80, and CD103 in dendritic cells (DCs) as detected by flow cytometry. n=4 per group. "Compatible allogeneic" immature DCs collected from the bone marrow of healthy allogeneic FVB mice were co-cultured with PPT-scrambled cells or PPT-shTrp63 cells. [Figure 7A] Figures 7A-7D show that TGFβ induces Smad2 / p63 complex formation in PPT cells. Figure 7A shows the expression of p63 protein in PP cells and PPT cells. [Figure 7B] Figures 7B and 7C show the intracellular localization of Smad2 and p63 as analyzed by confocal microscopy (Figure 7B) and Western blotting (Figure 7C). [Figure 7C] Same as above. [Figure 7D]Figure 7D shows the protein-protein interactions for Smad2 and p63 as analyzed by co-immunoprecipitation assay. [Figure 8A] Figures 8A–8D show that PP cells were reprogrammed by TGFβ via the p63 / Smad2 signaling pathway. By comparing the transcriptomes of control, p63 knockdown PPT cells, and Smad2 knockdown PPT cells, we determined the genes that are co-upregulated (Figure 8A) and co-downregulated (Figure 8B) by Smad or p63 knockdown. The relevant GO vocabulary and KEGG pathway (bottom row) are also shown. The relevant targets that are co-upregulated (Figure 8C) and co-downregulated (Figure 8D) by p63 knockdown or Smad2 knockdown in PPT cells are shown by heatmaps. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 9A] Figures 9A to 9F show that TGFβ activated p63-dependent antitumor immunity in human breast cancer cells. Figure 9A shows the expression levels of the p63 protein in human breast cancer cell lines. [Figure 9B] Figure 9B shows immature human dendritic cells (DCs) cultured with either MCF7 human breast cancer cells or HCC1954 human breast cancer cells, as shown. Both MCF7T and HCC1954T cells were treated with TGFβ. [Figure 9C] Figures 9C to 9E show the expression of CD80, CD86, and CD103 in DCs by flow cytometry. n=4 per group. * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F]Figure 9F shows the relationship between the TP63-Smad signature (high for PYCARD, RIPK3, CASP9, SESN1, and TP63, and low for KSR1, EIF4EBP1, ITGA5, and EMILIN1) and patient survival using the Curtis breast cancer dataset. **** represents P<0.0001. [Figure 10A] Figures 10A and 10B show that PP tumor cells failed to proliferate when co-injected with PPT tumor cells into allogeneic mice. A mixture of PP and PPT cells (1:1) was introduced into allogeneic mice. Tumor growth (Figure 10A, n=10 per group) and long-term survival rate (Figure 10B, n=5 per group) are shown. [Figure 10B] Same as above. [Figure 11A] Figures 11A to 11D show that an immunological memory response is induced by immunization using TGFβ-activated tumor cells. Spleens and lymph nodes were collected at weeks 1, 2, and 6 after injection of PPT cells. The proportions of CD45+CD3+CD4+FOXP3-CD44+KLRG1-CD62L-central memory T cells (CD4+TCM cells) (Figure 11A), CD45+CD3+CD4+FOXP3-CD44+KLRG1+CD62L-effector memory T cells (CD4+TEM cells) (Figure 11B), CD45+CD3+CD8+FOXP3-CD44+KLRG1-CD62L-central memory T cells (CD8+TCM cells) (Figure 11C), and CD45+CD3+CD8+FOXP3-CD44+KLRG1+CD62L-effector memory T cells (CD8+TEM cells) (Figure 11D) were analyzed by flow cytometry. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001. Each group consists of n=5 mice. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 12A]Figures 12A to 12G show that an immune memory response against the parent tumor is induced by immunization using TGFβ-activated tumor cells. Figure 12A shows a schematic diagram of the workflow for determining the effectiveness of PPT immunization against PP tumor rejection. [Figure 12B] Figures 12B to 12E show the transplantation of PP cells or PP tumor fragments into control mice and PPT-immunized mice. Tumor growth curves (Figures 12B and 12D, n=10 per group) and long-term survival rates (Figures 12C and 12E, n=5 per group) are shown for the mice. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 12F] Figures 12F and 12G show that PP tumor cells were introduced into PPT-immunized mice or control mice via tail vein injection. Lung metastatic nodules were examined after 4 weeks. Each group consisted of 5 mice, and **** represents P<0.0001. [Figure 12G] Same as above. [Figure 13A] Figures 13A to 13D show that PP tumor loading induces a memory T cell response in the tumor microenvironment (TME) within PPT-immunized mice. Figure 13A shows the workflow for determining memory in the TME. [Figure 13B-D]Figure 13B shows the proportion of tumor-infiltrating CD4+ and CD8+ T cells among CD45+ leukocytes in PP tumors transplanted into PPT-immunized mice or control mice. Figure 13C shows the proportion of CD45+CD3+CD4+FOXP3-CD44+KLRG1-CD62L+ central memory T cells (CD4+TCM cells) and CD45+CD3+CD4+FOXP3-CD44+KLRG1+CD62L- effector memory T cells (CD4+TEM cells). Figure 13D shows the proportion of CD45+CD3+CD8+FOXP3-CD44+KLRG1-CD62L+ central memory T cells (CD8+TCM cells) and CD45+CD3+CD8+FOXP3-CD44+KLRG1+CD62L- effector memory T cells (CD8+TEM cells). Analysis was performed by flow cytometry. * represents P<0.05, *** represents P<0.001, and **** represents P<0.0001. n=6 for each group. [Figure 14A] Figures 14A to 14C show that the vaccine effect of PPT cells is not attenuated by radiation. Mice were immunized with PBS, PP cells, or PPT cells irradiated with 100 Gy of gamma rays. Four weeks after inoculation, PP tumor fragments were transplanted into the third fat body of the mice shown. PP tumor growth (Figure 14B, n=10 for each group) and mouse survival rates (Figure 14C, n=5 per group) are shown. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 15A]Figures 15A to 15H illustrate the potential of PPT cells as an allogeneic vaccine against various types of cancer. The indicated tumor cell lines were introduced into PBS-inoculated or PPT cell-inoculated mice. The proliferation of PPA tumors (Figure 15A; a mouse mammary cancer model characterized by triple deletion of p53, PTEN, and P110α), C260 tumors (Figure 15C; a mouse ovarian cancer model with double deletion of p53 / PTEN and high Myc expression), D658 tumors (Figure 15E; a Kras mutant recurrent mammary cancer cell line created from a PIK3CAH1047R mouse model of mammary cancer), and d333 tumors (Figure 15G; a brain tumor derived from p53 and PTEN double-deficient mice) is shown. n=10 for each group. The survival rates of mice transplanted with the indicated tumors are also shown in Figures 15B, 15D, 15F, and 15H. n=5 per group. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 15G] Same as above. [Figure 15H] Same as above. [Figure 16] Figure 16 is a schematic diagram of the TGFβ-Smad signaling pathway and molecular events, adapted from Zhang et al., (2013) J. Cell Sci., Vol. 126: pp. 4809-4813. [Figure 17] Figure 17 shows that TGFβ activation in tumor cells induces dendritic cell binding and subsequent T cell activation, leading to an anti-tumor immune response. In p63-positive tumor cells, TGFβ induces nuclear localization of Smad, promoting the formation of a p63-Smad transcriptional complex that upregulates multiple immune regulatory pathways and downregulates several major oncogenic signaling pathways. This induces anti-tumor immunity through the activation of dendritic cells (DCs) and T cells. [Figure 18] Figure 18 is a schematic diagram of a typical embodiment of a vaccine platform included within the scope of the present invention. [Figure 19] Figure 19 shows the gate setting strategies for T cell populations. Flow cytometry gate setting for CD4+ T cells, CD8+ T cells, and CD4+ regulatory T cells in the spleen, lymph nodes, blood, and tumors is shown. Representative plots derived from splenic cells are shown. [Figure 20-1] Figure 20 shows the gate setting strategies for memory T cell populations. Flow cytometry gate setting is shown for CD4+ central memory T cells (CD4+TCM), CD4+ effector memory T cells (CD4+TEM), CD8+ central memory T cells (CD8+TCM), and CD8+ effector memory T cells (CD8+TEM) in the spleen, lymph nodes, blood, and tumors. Representative plots derived from splenic cells are shown. [Figure 20-2] Same as above. [Figure 21] Figure 21 shows a gate setting strategy for tumor-infiltrating dendritic cells. Flow cytometry gate setting for tumor-infiltrating dendritic cells (DCs) is shown to examine the expression of MHCII, CD80, and CD103. [Modes for carrying out the invention]

[0015] For any figure that displays a bar histogram, curve, or other data in relation to its caption, the bars, curves, or other data shown from left to right in each display correspond in order to the top-to-bottom frame of the figure's caption.

[0016] This specification demonstrates that PTEN and p53-deficient tumor cells possessing activated TGFβ-Smad / p63 signaling (e.g., treated with at least one TGFβ superfamily protein) fail to form tumors in a T-cell-dependent manner in immune-normal hosts. For example, in vitro treatment of tumor cells derived from an allogeneic mouse mammary tumor model resulting from simultaneous p53 and Pten deficiency with TGFβ completely suppressed the tumorigenic potential of these cells in a T-cell-dependent manner in immune-normal mice. These cells were also shown to induce robust anti-tumor immunity via dendritic cell (DC) binding and activation, followed by T cell activation against target tumor cells. Furthermore, p63 was found to be an important cofactor of TGFβ / Smad-mediated transcription in response to TGFβ stimulation. For example, activation of the TGFβ-Smad / p63 axis upregulated transcriptional production that induces activation of multiple immune pathways, and these effects abolished upon deficiency of either p63 or Smad2. Furthermore, administration of tumor cells possessing activated TGFβ-Smad / p63 signaling protects the host from recurrent and metastatic tumor lesions through the induction of a long-term memory T cell response. Survival rates in breast cancer patients were also found to correlate very well with the TGFβ-Smad / p63 signature. These results reveal a novel molecular switch underlying the conflicting effects of TGFβ in tumorigenesis and provide strategies for developing effective tumor vaccines through TGFβ-based reprogramming. Thus, compositions and methods for preventing and / or treating cancer are provided, using a cancer vaccine comprising cancer cells that are (1) Pten-deficient, (2) p53-deficient, and (3) modified to activate the TGFβ-Smad / p63 signaling pathway. Methods for evaluating the efficacy of said cancer vaccine for preventing and / or treating cancer are also provided.

[0017] I. Definition The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one or more elements.

[0018] The term "administering" is intended to include the route of administration through which the drug performs its intended function. Examples of usable routes of administration for the treatment of the body include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral route, inhalation route, and transdermal route. The injection may be a bolus injection or a continuous infusion. To protect the drug from natural conditions that could adversely affect its ability to perform its intended function, the drug may be coated or placed in a selected material depending on the route of administration. The drug may be administered alone or in combination with a pharmaceutically acceptable carrier. The drug may be administered as a prodrug, which is converted to its active form in vivo.

[0019] The terms “change in quantity” or “change in level” refer to an increased or decreased copy number (e.g., germline and / or somatic) of the biomarker nucleic acid compared to the expression level or copy number in a control sample, or an increased or decreased expression level in a cancer sample, for example. The term “change in quantity” of a biomarker also includes an increased or decreased protein level of the biomarker protein in a sample, for example, a cancer sample, compared to the corresponding protein level in a normal control sample. Furthermore, changes in the quantity of a biomarker protein may be determined by detecting post-translational modifications of the marker, such as the methylation status, which can affect the expression or activity of the biomarker protein.

[0020] If the amount of a biomarker in a sample is greater than the standard error of the assay used to evaluate the quantity, preferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more than that amount, then the amount of the biomarker is "significantly" greater or less than the normal amount of that biomarker. Alternatively, if the amount of the biomarker in the sample is at least about twice, preferably at least about three times, four times, or five times greater or less than the normal amount of that biomarker, then the amount of the biomarker may be considered "significantly" greater or less. Such "significance" may also apply to any other measurement parameters described herein, such as expression, inhibition, cytotoxicity, and cell proliferation.

[0021] The term "change in expression level" of a biomarker refers to the expression level or copy number of that biomarker in a test sample, for example, a sample from a patient with cancer, which is greater or less than the standard error of the assay used to evaluate expression or copy number, and is preferably at least twice, more preferably three, four, five, or ten times or more, the average expression level or copy number of that biomarker in a control sample (for example, a sample from a healthy subject without pre-existing conditions) or several control samples. This change in expression level is preferably greater than or less than the standard error of the assay used to evaluate expression or copy number, and is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or greater than the average expression level or copy number of the biomarker in several control samples. In some embodiments, the level of the biomarker refers to the level of the biomarker itself, the level of a modified biomarker (e.g., a phosphorylated biomarker), or the level of the biomarker compared to another measurement variable such as a control (e.g., the level of a phosphorylated biomarker compared to a non-phosphorylated biomarker).

[0022] The term "change in biomarker activity" refers to a disease state in which the activity of the biomarker is increased or decreased in a cancer sample compared to the activity of the biomarker in a normal control sample. This change in biomarker activity may result from, for example, changes in the expression of the biomarker, changes in the protein level of the biomarker, changes in the structure of the biomarker, or changes in interactions with other proteins involved in the same or different pathways as the biomarker, or changes in interactions with transcription activators or repressors.

[0023] The term "structural alteration" of a biomarker refers to the presence of mutations or allele mutations within the biomarker nucleic acid or biomarker protein compared to a normal or wild-type gene or protein, such as mutations that affect the expression or activity of the biomarker nucleic acid or biomarker protein. Mutations include, but are not limited to, substitutions, deletions, or additions. The mutation may be located in the coding region or non-coding region of the biomarker nucleic acid.

[0024] Unless otherwise specified herein, the terms “antibody” and “antibody (plural)” broadly encompass native antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric antibodies, humanized antibodies, and multispecific antibodies, as well as all fragments and derivatives of the aforementioned antibodies that have at least an antigen-binding site. Antibody derivatives may include proteins or chemical portions bound to antibodies.

[0025] Furthermore, intracellular antibodies are well-known antigen-binding molecules that possess antibody characteristics but can be expressed intracellularly to bind to and / or inhibit target intracellular organisms (Chen et al., Human Gene Ther., Vol. 5: pp. 595-601, 1994). Methods for applying antibodies to target (e.g., inhibit) intracellular regions, such as the use of single-chain antibodies (scFv), modification of the immunoglobulin VL domain for hyperstability, modification of antibodies to counteract reductive intracellular environments, generation of fusion proteins to increase intracellular stability, and / or regulation of intracellular localization, are well known in this field. It is also possible to introduce and express intracellular antibodies in one or more types of cells, tissues, or organs of a multicellular organism for purposes such as prevention and / or treatment (e.g., as gene therapy) (see at least PCT International Publication No. 08 / 020079, International Publication No. 94 / 02610, International Publication No. 95 / 22618, and International Publication No. 03 / 014960, U.S. Patent No. 7004940, Cattaneo and Biocca, (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag), Kontermann, (2004) Journal of Methods, Vol. 34: pp. 163-170, Cohen et al., (1998) Journal of Oncogene, Vol. 17: pp. 2445-2456, Auf der Maur et al., (2001) FEBS See Lett., Vol. 508: pp. 407-412, and Shaki-Loewenstein et al., (2005) J. Immunol. Meth., Vol. 303: pp. 19-39.

[0026] As used herein, the term “antibody” also includes the “antigen-binding portion” (or simply “antibody portion”) of an antibody. As used herein, the term “antigen-binding portion” refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or a fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term "antigen-binding region" of an antibody include (i) the Fab fragment, a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain; (ii) the F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) the Fd fragment, consisting of the VH domain and CH1 domain; (iv) the Fv fragment, consisting of the VL domain and VH domain of the single arm of the antibody; (v) the dAb fragment, consisting of the VH domain (Ward et al., Nature, Vol. 341, pp. 544-546, 1989); and (vi) the isolation complementarity-determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these domains can be linked using synthetic linkers that enable the creation of a single protein chain in which the VL and VH regions pair up to form a monovalent polypeptide (known as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science, Vol. 242: pp. 423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. USA, Vol. 85: pp. 5879-5883, and Osbourn et al., 1998, Nature Biotechnology, Vol. 16: p. 778). Such single-chain antibodies are also included within the scope of the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a particular scFv can be conjugated to a human immunoglobulin constant region cDNA sequence or genomic sequence to generate an expression vector encoding a complete IgG polypeptide or other isotype.VH and VL can also be used to generate Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other types of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used to pair them with complementary domains on another chain, forming two antigen-binding sites (see, for example, Holliger et al., (1993) Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 6444-6448, and Poljak et al., (1994) Structure, Vol. 2: pp. 1121-1123).

[0027] Furthermore, an antibody or its antigen-binding moiety may be a larger immunoadhesive polypeptide portion formed by covalent or non-covalent bonding of the antibody or antibody moiety to one or more other proteins or peptides. Examples of such immunoadhesive polypeptides include the use of the streptavidin core region for the production of tetrameric scFv polypeptides (Kipriyanov et al., (1995) Human Antibodies and Hybridomas, Vol. 6: pp. 93-101), and the use of cysteine ​​residues, biomarker peptides, and C-terminal polyhistidine tags for the production of divalent and biotinylated scFv polypeptides (Kipriyanov et al., (1994) Mol. Immunol., Vol. 31: pp. 1047-1058). Antibody moieties, such as the Fab fragment and the F(ab')2 fragment, can be prepared from the whole antibody using conventional techniques, for example, by papain digestion or pepsin digestion of the whole antibody, respectively. Furthermore, antibodies, antibody moieties, and immunoadhesive polypeptides can be obtained using standard recombinant DNA techniques as described herein.

[0028] Antibodies may be polyclonal or monoclonal, and may be heterogeneous, homogeneous, or homogeneous, or modified thereof (e.g., humanized, chimeric, etc.). Antibodies may be entirely human antibodies. The antibodies of the present invention preferably bind specifically or substantially specifically to biomarker polypeptides or fragments thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a group of antibody polypeptides containing only one antigen-binding site capable of immunoreacting with a specific epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a group of antibody polypeptides containing multiple antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity to the specific antigen with which they immunoreact.

[0029] The antibody may be “humanized,” and such antibody may include an antibody produced by a non-human cell having a variable region and a constant region that have been modified to more closely resemble an antibody that would have been produced by a human cell. For example, by modifying the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibody of the present invention may include, for example, amino acid residues in the CDR that are not encoded by human germline immunoglobulin sequences (for example, mutations introduced by in vitro random mutagenesis or site-directed mutagenesis or in vivo somatic mutation). As used herein, the term “humanized antibody” also includes antibodies in which a CDR sequence derived from the germline of another mammalian species has been transplanted into a human framework sequence.

[0030] The term "biomarker" refers to a measurable substance of the present invention that is judged to predict the effectiveness of cancer therapy. Biomarkers may include, but are not limited to, nucleic acids (e.g., genomic nucleic acids and / or transcribed nucleic acid products) and proteins. Many biomarkers are also useful as therapeutic targets.

[0031] A “blocking” antibody or antibody “antagonist” is an antibody that inhibits or suppresses at least one biological activity of the antigen to which it binds. In certain embodiments, the blocking antibody or antagonist antibody or fragment thereof described herein substantially or completely inhibits a given biological activity of the antigen.

[0032] The term "body fluids" refers to liquids excreted or secreted from the body, as well as liquids that are not normally excreted or secreted (for example, amniotic fluid, aqueous humor, blood, blood and plasma, cerebrospinal fluid, earwax and earwax, Cowper's gland fluid or preejaculatory fluid, chyle, oozing fluid, feces, Skene's gland fluid, interstitial fluid, intracellular fluid, lymph, menstrual blood, milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal fluid, vitreous fluid, vomit).

[0033] The terms "cancer," "tumor," or "overgrowth" refer to the presence of cells that have characteristics typical of cancerous cells, such as unrestrained growth, immortality, metastatic ability, rapid growth rate, and certain distinctive morphological features.

[0034] Cancer cells often take the form of tumors, but such cells can exist independently within animals, or they may be non-tumorogenic cancer cells such as leukemia cells. As used herein, the term "cancer" includes pre-malignant and malignant cancers. Cancers include, but are not limited to, B-cell carcinomas, e.g., multiple myeloma; Waldenström macroglobulinemia, e.g., heavy chain diseases such as alpha-chain disease, gamma-chain disease, and μ-chain disease; benign monoclonal gammaglobulinemia and immunocellular amyloidosis; melanoma; breast cancer; lung cancer; bronchial cancer; colorectal cancer; prostate cancer; pancreatic cancer; gastric cancer; ovarian cancer; urinary tract or bladder cancer; brain cancer or central nervous system cancer; peripheral nervous system cancer; esophageal cancer; cervical cancer; uterine cancer or endometrial cancer; oral or pharyngeal cancer; liver cancer; kidney cancer; testicular cancer; bile duct cancer; small intestine cancer or appendiceal cancer; salivary gland cancer; thyroid cancer; adrenal cancer; osteosarcoma; chondrosarcoma; and cancers of hematological tissues. Other non-limiting examples of the types of cancer to which the methods included in the scope of this invention are applicable include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, intravascular sarcoma, lymphangiosarcoma, intralymphatic sarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminomas, fetal cancer, Wilms' tumor, cervical cancer, bone cancer, brain tumor, and testicular cancer. These include lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial, and this cancer includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In yet another embodiment, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. The characteristics of the epithelial cancer can be described in various ways, including, but are not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0035] The term "coding region" refers to a region of a nucleotide sequence that contains codons, which are translated into amino acid residues, while the term "non-coding region" refers to a region of a nucleotide sequence that is not translated into amino acid residues (e.g., the 5' untranslated region and the 3' untranslated region).

[0036] The term "complementary" refers to broad sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that an adenine residue in a first nucleic acid region can form a specific hydrogen bond ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region if that residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid chain can base pair with a residue in a second nucleic acid chain that is antiparallel to the first chain if that residue is guanine. A first nucleic acid region is complementary to a second nucleic acid region of the same or different nucleic acid if, when the two regions are aligned antiparallel, at least one nucleotide residue in the first region can base pair with a residue in the second region. It is preferable that, when the first and second regions are aligned antiparallel by the first region comprising a first portion and the second region comprising a second portion, at least about 50%, preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues in the first portion can base pair with nucleotide residues in the second portion. It is more preferable that all nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

[0037] As used herein, the terms “co-administration therapy” and “combination therapy” refer to the administration of two or more therapeutic substances. The different drugs constituting the combination therapy may be administered simultaneously with, before, or after the administration of one or more therapeutic agents.

[0038] The term “control” refers to any reference standard that is appropriate for comparison to the expression products in the test sample. In one embodiment, the control includes obtaining a “control sample” in which the levels of expression products are detected and the levels of those expression products are compared to the levels of the expression products in the test sample. Such a control sample may include, but is not limited to, any suitable sample, which may be a sample from a control cancer patient with a known outcome (which may be a preserved sample or a result of a previous sample measurement), normal tissue or cells isolated from a subject such as a healthy subject or a cancer patient, primary cultured cells / tissues isolated from a subject such as a healthy subject or a cancer patient, adjacent normal cells / tissues obtained from the same organ or body part of a cancer patient, tissue or cell samples isolated from a healthy subject, or primary cells / primary tissues obtained from a preservation institution. In another preferred embodiment, the control may include a reference standard expression level from any suitable source, such as a housekeeping gene, a range of expression levels in normal tissue (or other previously analyzed control samples), a previously determined range of expression levels in a group of patient samples, or a group of patients having a particular outcome (e.g., survival for one, two, three, or four years) or receiving a particular treatment (e.g., standard cancer treatment). Those skilled in the art will understand that such control samples or reference standard expression levels can be used in combination as controls in the method of the present invention. In one embodiment, the control may include a normal cell / tissue sample or a non-cancerous cell / tissue sample. In another preferred embodiment, the control may include a group of patients, such as a group of cancer patients' expression levels, or a group of cancer patients receiving a particular treatment, or a group of patients having a certain outcome compared to another. In the former example, the expression level of each patient may be specified to a certain percentile expression level or may be expressed as being higher or lower than the average or mean of the reference standard expression level. In another preferred embodiment, the controls may include normal cells, cells from patients being treated with combination chemotherapy, and cells from patients with benign cancer.In another embodiment, the control may also include measured values, such as the average expression level of a particular gene in the same population compared to the expression level of a housekeeping gene in that population. Such populations may include healthy subjects, cancer patients who have not received any treatment (i.e., treatment-naïve), cancer patients receiving standard treatment, or patients with benign cancer. In another preferred embodiment, the control includes a transformation of the ratio of expression product levels, which may include, but are not limited to, determining the ratio of expression product levels of two genes in the test sample and comparing that ratio to any appropriate ratio of the same two genes in a reference standard sample, determining the expression product levels of two or more genes in the test sample and determining the difference in expression product levels in any appropriate control, and determining the expression product levels of two or more genes in the test sample, normalizing the expression of the genes to the expression of a housekeeping gene in the test sample, and comparing it to any appropriate control. In a particularly preferred embodiment, the control includes a control sample of the same series and / or type as the test sample. In another embodiment, the control may include the levels of the expression product aggregated as percentiles in a series of patient samples, such as all cancer patients, or percentiles based on those samples. In one embodiment, for example, the level of the expression product as a control is defined by whether the level of the expression product is high or low compared to a specific percentile, which is used as a criterion for predicting the outcome. In another preferred embodiment, the level of the expression product as a control is defined using the level of the expression product from cancer control patients with known outcomes, and the level of the expression product from the test sample is compared to that control expression product level as a criterion for predicting the outcome. As demonstrated by the following data, the method of the present invention is not limited to using a specific cut point when comparing the level of the expression product in the test sample to a control.

[0039] The "copy number" of a biomarker nucleic acid refers to the number of DNA sequences in cells encoding a particular gene product (e.g., germline and / or somatic cells). For a given gene, mammals typically have two copies of that gene. However, copy number can increase due to gene amplification or duplication, or decrease due to deletion. For example, germline copy number changes include changes at one or more genomic loci that cannot be explained by the copy number of the normal complement of the germline copy in the control (e.g., the normal copy number of germline DNA in the same species from which a specific germline DNA and its corresponding copy number were determined). Somatic cell copy number changes include changes at one or more genomic loci that cannot be explained by the copy number of the control germline DNA (e.g., the copy number of germline DNA in the same subject from which somatic cell DNA and its corresponding copy number were determined).

[0040] The term "immune cells" refers to cells that play a role in the immune response. Immune cells are cells of hematopoietic origin and include lymphocytes such as B cells and T cells, as well as natural killer cells, and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0041] Macrophages (and their precursor, monocytes) are the "gluttons" of the immune system. These cells, though appearing in various forms such as microglia, Kupffer cells, and osteoclasts, are present in all tissues of the body, where they engulf apoptotic cells and pathogens and produce immune effector molecules. During tissue injury or infection, monocytes are rapidly recruited to the tissue, where they differentiate into tissue macrophages. Macrophages are remarkably plastic, capable of altering their functional phenotype in response to environmental cues. While they play a central role in protecting the host through their ability to eliminate pathogens and direct other immune cells, they also contribute to the development of inflammatory and degenerative diseases. Macrophages that promote inflammation are called M1 macrophages, while those that suppress inflammation and promote tissue repair are called M2 macrophages. M1 macrophages are activated by LPS and IFN-γ, secreting high levels of IL-12 and low levels of IL-10. M2 is a phenotype of commensal tissue macrophages that can be further increased by IL-4. M2 macrophages produce high levels of IL-10 and TGFβ, and low levels of IL-12. Paraneoplastic macrophages are mainly M2 phenotype macrophages and appear to actively promote tumor growth.

[0042] Myeloid-derived immunosuppressive cells (MDSCs) are an essential part of the myeloid cell lineage, a heterogeneous population composed of myeloid progenitor cells and precursor cells of granulocytes, macrophages, and dendritic cells. MDSCs are defined by their myeloid origin, immaturity, and ability to potently suppress T cell responses. In healthy individuals, MDSCs regulate immune responses and tissue repair, and their population rapidly proliferates during inflammation, infection, and cancer. MDSCs are one of the major components of the tumor microenvironment. A key characteristic of these cells is their potent immunosuppressive activity. MDSCs originate in the bone marrow and migrate to peripheral lymphoid organs and tumors in tumor-bearing hosts, contributing to the formation of the tumor microenvironment. This process is regulated by a set of specific chemokines, many of which are upregulated in cancer. Hypoxic conditions appear to play a crucial role in the differentiation and function of MDSCs in tumors. Currently, therapeutic strategies targeting MDSCs are being developed to promote anti-tumor immune responses or to suppress immune responses in the context of autoimmune diseases or transplant rejection.

[0043] Dendritic cells (DCs) are specialized antigen-presenting cells located in the skin, mucous membranes, and lymphoid tissues. Their primary function is to process antigens and present the processed antigens to T cells, thereby promoting immunity to foreign antigens and tolerance to self-antigens. Dendritic cells also secrete cytokines to regulate the immune response.

[0044] Conventional T cells, also known as Tconv or Teff, have effector functions (e.g., cytokine secretion, cytotoxic activity, suppression of self-recognition, etc.) that increase the immune response by expressing one or more T cell receptors. Tcon or Teff is generally defined as any T cell population that is not Treg, and this population includes, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated into the Th1 or Th2 lineages. In some embodiments, Teff is a small population of non-Treg T cells. In some embodiments, Teff is CD4+Teff or CD8+Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T lymphocytes. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. A "naive Tcon" is a CD4 + T cell that has differentiated in the bone marrow and successfully undergone positive and negative central selection processes in the thymus but has not yet been activated by exposure to an antigen. Naive Tcon is characterized by surface expression of L-selectin (CD62L), absence of activation markers such as CD25, CD44, or CD69, and absence of memory markers such as CD45RO. Thus, naove Tcon is considered to be in a quiescent state and non-dividing and requires interleukin-7 (IL-7) and interleukin-15 (IL-15) for constant survival (see at least International Publication No. WO 2010 / 101870). In the context of suppression of the immune response, the presence and activity of such cells are undesirable. Unlike Treg, Tcon is not anergic and proliferates in response to antigen-based T cell receptor activation (Lechler et al., (2001) Philos. Trans. R. Soc. Lond. Biol. Sci., Vol. 356: 625-637). In tumors, exhausted cells may exhibit anergic characteristics.

[0045] The term "immunotherapy" or "immunotherapy" refers to any treatment that uses a specific part of the target immune system to fight a disease such as cancer. For this purpose, one or more drugs are administered, or not administered, to stimulate (or suppress) the target's own immune system. Immunotherapy designed to induce or increase the immune response is called "activating immunotherapy." Immunotherapy designed to decrease or suppress the immune response is called "suppressive immunotherapy." Any drug thought to have an effect on the immune system against transplanted genetically modified cancer cells can be assayed to determine whether the drug is immunotherapy and whether a given genetic modification has an effect on regulating the immune response. In some embodiments, the immunotherapy is cancer cell-specific. In some embodiments, immunotherapy can be "non-targeted," meaning the administration of drugs that do not selectively interact with immune system cells but still regulate immune system function. Typical examples of non-targeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiotherapy.

[0046] Immunotherapy is a form of targeted therapy that may include the use of cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are viruses that can infect and lyse cancer cells but do not harm normal cells, which makes them potentially useful in cancer therapy. Replication of oncolytic viruses promotes the destruction of tumor cells and amplifies viral titers at the tumor site. Oncolytic viruses can also act as vectors for anti-oncogenes, specifically delivering these anti-oncogenes to the tumor site. The aforementioned immunotherapy may include passive immunity for short-term protection of the host, which is achieved by administering pre-formed antibodies against cancer antigens or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens that are optionally bound to chemotherapeutic agents or toxins). For example, anti-VEGF inhibitors and anti-mTOR inhibitors are known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on the use of cytotoxic lymphocyte-recognizing epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc., can be used to selectively regulate biomolecules associated with the development, progression, and / or pathogenesis of tumors or cancer.

[0047] Immunotherapy may include passive immunization for short-term protection of the host, which is achieved by administering pre-formed antibodies against cancer antigens or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens that are optionally bound to chemotherapeutic agents or toxins). Immunotherapy may also focus on utilizing cytotoxic lymphocyte-recognizing epitopes of cancer cell lines. Alternatively, biomolecules associated with the development, progression, and / or pathogenesis of tumors or cancers can be selectively modulated using antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc.

[0048] In some embodiments, immunotherapy includes inhibitors of one or more immune checkpoints. The term “immune checkpoint” refers to a group of molecules on the cell surface of CD4+ T cells and / or CD8+ T cells that fine-tune the immune response by downregulating or inhibiting the antitumor immune response. Immune checkpoint proteins are well known in the art, and these proteins include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophyllin, and A2aR (see, for example, International Publication No. 2012 / 177624). The term further encompasses biologically active proteins, as well as full-length immune checkpoint proteins and nucleic acids encoding fragments of these biologically active proteins. In some embodiments, this term further encompasses any fragments that conform to the description of complementarity presented herein. In one embodiment, the immune checkpoint is PD-1.

[0049] "Immune checkpoint suppression therapy" refers to the use of drugs that inhibit immune checkpoint nucleic acids and / or proteins. To more effectively treat cancer, the immune response can be upregulated by inhibiting one or more immune checkpoints, thereby disrupting or, in other cases, neutralizing inhibitory signaling. Examples of drugs useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptide mimes, natural ligands, and derivatives of natural ligands that can bind and / or inactivate or inhibit immune checkpoint proteins or their fragments, as well as RNA interference, antisense, and nucleic acid aptamers that can downregulate the expression and / or activity of immune checkpoint nucleic acids or their fragments. Examples of drugs that upregulate the immune response include antibodies against immune checkpoint proteins that interfere with the interaction between one or more immune checkpoint proteins and their innate receptors, inactivated immune checkpoint proteins (e.g., dominant-negative polypeptides), small molecules or peptides that interfere with the interaction between one or more immune checkpoint proteins and their innate receptors, fusion proteins that bind to the innate receptors (e.g., extracellular components of immune checkpoint inhibitory proteins fused to the Fc portion of antibodies or immunoglobulins), and nucleic acid molecules that interfere with the transcription or translation of immune checkpoint nucleic acids. Such drugs can upregulate the immune response by directly interfering with the interaction between one or more immune checkpoints and their innate receptors (e.g., antibodies) to prevent inhibitory signaling. Alternatively, drugs can upregulate the immune response by indirectly interfering with the interaction between one or more immune checkpoint proteins and their innate receptors to prevent inhibitory signaling. For example, soluble immune checkpoint protein ligands, such as stabilized extracellular domains, can bind to the receptor to indirectly reduce the effective concentration of the receptor bound to the appropriate ligand. In one embodiment, an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-PD-L2 antibody are used alone or in combination to inhibit an immune checkpoint.These embodiments can also be applied to specific therapies targeting specific immune checkpoints, such as the PD-1 pathway (for example, anti-PD-1 pathway therapies, also known as PD-1 pathway inhibitor therapies).

[0050] The term "immune response" includes T cell-mediated immune responses and / or B cell-mediated immune responses. Examples of immune responses include T cell responses, such as cytokine production and cellular cytotoxicity. The term immune response also includes immune responses indirectly mediated by T cell activation, such as antibody production (humoral response) and cytokine response cells, such as macrophage activation.

[0051] The term "immunotherapy" may include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to produce an immune response against a tumor or cancer in its target. Various immunotherapy agents are useful in the compositions and methods described herein.

[0052] The term “inhibit” includes, for example, a reduction, decrease, limitation, and / or interference of a particular action, function, and / or interaction. In some embodiments, an interaction between two molecules is “inhibited” if that interaction is reduced, interfered with, disrupted, or destabilized.

[0053] In some embodiments, cancer is “inhibited” if at least one symptom of the cancer is reduced, terminated, delayed, or prevented. As used herein, cancer is also “inhibited” if the recurrence or metastasis of the cancer is suppressed, delayed, delayed, or prevented.

[0054] The term "interaction," when referring to the interaction between two molecules, refers to the physical contact (e.g., binding) between those molecules. It is common for such interactions to result in the activation (producing biological effects) of one or both of those molecules.

[0055] "Isolated protein" refers to a protein that, when isolated from cells or produced by recombinant DNA technology, is substantially free of other proteins, other cellular material, isolation media, and culture media, or a protein that, when chemically synthesized, is substantially free of chemical precursors or other chemicals. "Isolated" or "purified" proteins or their biologically active portions are substantially free of cellular material or other contaminating proteins originating from the cells or tissues from which their antibodies, polypeptides, peptides, or fusion proteins are derived, or, when chemically synthesized, are substantially free of chemical precursors or other chemicals. The phrase "substantially free of cellular material" includes preparations in which the protein is isolated from the cellular components of the cells from which the biomarker polypeptide or fragment thereof is isolated or produced by recombinant technology. In one embodiment, the phrase “substantially free of cellular material” includes preparations of biomarker proteins or fragments thereof having less than about 30% (on a dry weight basis) of non-biomarker proteins (also referred herein as “contamination proteins”), more preferably less than about 20% of non-biomarker proteins, even more preferably less than about 10% of non-biomarker proteins, and most preferably less than about 5% of non-biomarker proteins. When antibodies, polypeptides, peptides, or fusion proteins, or fragments thereof, such as their biologically active fragments, are produced by recombinant technology, it is also preferable that the biologically active fragments are substantially free of culture medium, i.e., the culture medium constitutes less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%.

[0056] As used herein, the term "isotype" refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgM, IgG1, IgG2C, etc.).

[0057] "Normal" level of biomarker expression is the expression level of that biomarker in cells of a non-cancer subject, such as a human patient. "Overexpression" or "significantly high level of expression" of a biomarker refers to an expression level in the test sample that is greater than the standard error of the assay used to evaluate expression, and is preferably at least 10% higher than the expression activity or expression level of that biomarker in a control sample (e.g., a sample from a healthy subject without the disease associated with that biomarker), preferably at least 1.2 times, 1.3 times, or 10% higher than the average expression level of that biomarker in several control samples. It is more preferable that the ratio is 0.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 10.5 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, or higher. "Significantly low levels of expression" of a biomarker is defined as being at least 10% lower than the expression level of that biomarker in a control sample (e.g., a sample from a healthy subject without the disease associated with that biomarker), preferably at least 10% lower than the mean expression level of that biomarker in several control samples, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 times lower. This refers to expression levels in test samples that are 2.1 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 10.5 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, or lower.

[0058] "Overexpression" or "significantly high level of expression" of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay used to evaluate expression, and is preferably at least 10% higher than the expression activity or expression level of the biomarker in a control sample (e.g., a sample from a healthy subject who does not have the disease associated with the biomarker), preferably at least 1.2 times, 1.3 times, or 1. It is more preferable that the ratio is 0.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 10.5 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, or higher. "Significantly low levels of expression" of a biomarker is defined as being at least 10% lower than the expression level of that biomarker in a control sample (e.g., a sample from a healthy subject without the disease associated with that biomarker), preferably at least 10% lower than the mean expression level of that biomarker in several control samples, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 times lower. This refers to expression levels in test samples that are 2.1 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 10.5 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, or lower.

[0059] The term “predict” includes using the status of biomarker nucleic acids and / or biomarker proteins, e.g., increased or decreased tumor activity, appearance, expression, growth, sedation, recurrence, or resistance, before, during, or after treatment, to determine a possible response of cancer to a cancer vaccine alone or in combination with immunotherapy and / or cancer therapy. The use of such biomarkers for prediction includes, for example, (1) an increase or decrease in copy number in a proportion of the assayed human cancer type or cancer sample, such as more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 100%, or more than that (e.g., FISH, FISH plus SKY, at least J. (1) The biomarker may be confirmed by: (2) the absolute or relative presence or absence of the biomarker in a biological sample, such as a sample from a subject with cancer, such as human tissue, whole blood, serum, plasma, scraped buccal mucosa, saliva, cerebrospinal fluid, urine, feces, or bone marrow; or (3) the absolute or relative presence or absence of the biomarker in a small clinical population of cancer patients (for example, a small population of patients who respond to cancer vaccines alone or in combination with immunotherapy and / or cancer therapy, or who develop resistance to such therapies).

[0060] Terms such as "prevent," "prevention," "prevention," and "preventive measures" refer to reducing the probability of developing the target disease, disorder, or symptom, and do not include individuals who do not currently have the target disease, disorder, or symptom but are at risk of developing it or are prone to developing it.

[0061] The terms “cancer response,” “response to immunotherapy,” or “response to modifiers of T-cell intercellular cytotoxicity / immunotherapy combination therapy” refer to any response of hyperproliferative impairment (e.g., cancer) to modifiers of T-cell intercellular cytotoxicity and anticancer drugs such as immunotherapy, preferably referring to changes in tumor burden and / or tumor volume after neoadjuvant therapy or the initiation of adjuvant therapy. Hyperproliferative impairment responses may be evaluated, for example, in terms of efficacy or in the context of neoadjuvant therapy or adjuvant therapy, and the size of the tumor after systemic intervention may be compared to the initial size and dimensions measured by CT, PET, mammography, ultrasound, or palpation. Responses can also be evaluated by calipas measurement or pathological examination of the tumor after biopsy or surgical resection. Responses can be recorded quantitatively, such as the percentage change in tumor volume, or qualitatively, such as “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical disease stability” (cSD), “clinical disease progression” (cPD), or other qualitative criteria. The evaluation of the hyperproliferative disorder response can be performed neoadjuvant therapy or early after the initiation of adjuvant therapy, for example, several hours, several days, several weeks, or preferably several months later. Typical endpoints for response evaluation are completion of neoadjuvant chemotherapy or surgical removal of residual tumor cells and / or tumor bed. This is typically three months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the treatment procedures described herein can be determined by evaluating the clinical benefit rate (CBR). This clinical benefit rate is evaluated by determining the sum of the percentages of patients in complete remission (CR), partial remission (PR), and disease stable (SD) at least six months after the completion of treatment. A simplified notation for this formula is CBR at 6 months = CR + PR + SD. In some embodiments, the CBR of a particular cancer treatment plan is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or higher.Other criteria for evaluating the response to cancer therapy relate to “survival,” which includes all of the following: “survival to death” (which may be death from any cause or tumor-related death), also known as overall survival (the term recurrence includes both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease includes cancer and cancer-associated diseases). The survival periods can be calculated by referring to a predetermined start point (e.g., diagnosis or initiation of treatment) and an end point (e.g., death, recurrence, or metastasis). Furthermore, criteria for the effectiveness of treatment can be expanded to include the response to chemotherapy, survival probability, probability of metastasis within a given timeframe, and probability of tumor recurrence. For example, a specific cancer treatment plan can be implemented for a target population to determine an appropriate threshold, and its outcome can be correlated with biomarker measurements determined before the implementation of any cancer therapy. The evaluation items may include the pathological response to treatments administered in the context of neoadjuvant therapy. Alternatively, evaluation criteria such as overall survival and disease-free survival may be monitored over a period of time for subjects after cancer therapy for which biomarker measurements are known. In certain embodiments, the dose administered is a standard dose known in the art for cancer therapy. The period for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds correlated with cancer therapy outcomes can be determined using methods well known in the art, such as those described in the examples.

[0062] The term "resistance" refers to acquired or innate resistance (i.e., being unresponsive to treatment or having a suppressed or limited response) in a cancer sample or mammal to cancer therapy, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or higher percentages, such as 2x, 3x, 4x, 5x, 10x, 15x, 20x, or higher, or any percentage within the range including both ends. The reduction in response can be assessed by comparison with the same cancer sample or mammal before the resistance was acquired, or by comparison with a different cancer sample or mammal known not to be resistant to the treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance may be mediated by P-glycoprotein or other mechanisms, or it may arise when mammals are infected with multidrug-resistant microorganisms or mixed microorganisms. Determining resistance to treatment is routine in the art and within the scope of the skills of experienced physicians, and can be evaluated, for example, by cell proliferation assays and cell death assays, which are described as “sensitive” herein. In some embodiments, the term “reversing resistance” means that when a primary cancer therapy (e.g., chemotherapy or radiotherapy) alone cannot produce a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor, combining that primary cancer therapy (e.g., chemotherapy or radiotherapy) with a second agent can produce a significant reduction in tumor volume with a certain level of statistical significance (e.g., p<0.05) compared to the tumor volume of an untreated tumor in that situation. This generally applies to tumor volume measurements performed when the untreated tumor is logarithmically growing.

[0063] The terms “response” or “responsiveness” refer to a cancer response in the sense of a reduction in tumor size or suppression of tumor growth. These terms may also refer to an improvement in prognosis, such as reflected by an increase in time to recurrence, which is the time to the first recurrence excluding a second primary cancer as the first event or death without evidence of recurrence, or an increase in overall survival, which is the time from treatment to death from any cause. Responding or having a response means that there is a beneficial endpoint achieved when exposed to a stimulus, or that negative or adverse symptoms are minimized, mitigated, or attenuated upon exposure to the stimulus. It should be understood that assessing the likelihood of a tumor or subject exhibiting a favorable response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a favorable response (i.e., exhibit a lack of response or be unresponsive).

[0064] As used herein, “RNA interferant” is defined as any agent that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interferants include, but are not limited to, RNA molecules or fragments thereof that are homologous to the target biomarker gene of the present invention, short interfering RNA (siRNA), and nucleic acid molecules including small molecules that interfere with or inhibit the expression of a target biomarker nucleic acid by RNA interference (RNAi).

[0065] RNA interference (RNAi) is an evolutionarily conserved process in which the expression or introduction of RNA having a sequence identical or very similar to that of a target biomarker nucleic acid leads to sequence-specific degradation or sequence-specific post-transcriptional silencing (PTGS) of messenger RNA (mRNA) transcribed from the target gene (see Coburn and Cullen, (2002) J. Virol., Vol. 76: pp. 9225), thereby inhibiting the expression of the target biomarker nucleic acid. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plant, invertebrate, and mammalian cells. In nature, RNAi is initiated by Dicer, a dsRNA-specific endonuclease, which facilitates the processing cleavage of long dsRNA into double-stranded fragments called siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves the target mRNA. RNAi can also be initiated by the introduction of nucleic acid molecules, such as synthetic siRNAs or RNA interference agents, to inhibit or repress the expression of the target biomarker nucleic acid. As used herein, “inhibition of target biomarker nucleic acid expression” or “inhibition of marker gene expression” includes any reduction in the expression or protein activity or level of the target biomarker nucleic acid or the protein encoded by the target biomarker nucleic acid. Such reduction may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression of the target biomarker nucleic acid or the activity or level of the protein encoded by the target biomarker nucleic acid that is not targeted by the RNA interference agent.

[0066] In addition to RNAi, genome editing can be used to modify the copy number or gene sequence of a target biomarker, for example, to introduce sequential or inducible knockout or mutation of the target biomarker. For example, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g., for the creation of non-functional or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes may be expressed. For example, a vector containing only this guide RNA may be administered to animals or cells genetically modified with the Cas9 enzyme. Similar strategies (e.g., designer zinc fingers, activator-like effectors (TALEs), or homing meganucleases) may be used. Such systems are well known in the art (for example, U.S. Patent No. 8,697,359, by Sander and Joung, (2014) Nat. Biotech., Vol. 32: pp. 347-355; Hale et al., (2009) Cell, Vol. 139: pp. 945-956; Karginov and Hannon, (2010) Mol. Cell, Vol. 37: p. 7; U.S. Patent Application Publication No. 2014 / 0087426 and No. 2012 / 0178169, by Boch et al., (2011) Nat. Biotech., Vol. 29: pp. 135-136, Boch et al., (2009) Science, Vol. 326: pp. 1509-1512, Moscow and Bogdanove, (2009) Science, Vol. 326: p. 1501, Weber et al., (2011) PLoS One, Vol. 6: p. 19722, Li et al., (2011) Nucl. Acids Res., Vol. 39: pp. 6315-6325, Zhang et al., (2011) Nat. Biotech., Vol. 29: pp. 149-153, Miller et al., (2011) Nat. Biotech., Vol. 29: pp. 143-148, Lin et al., (2014) Nucl. Acids See Res., Vol. 42: e47). Such genetic strategies can be implemented using constitutive or inducible expression systems according to methods well known in the art.

[0067] Piwi-interacting RNAs (piRNAs) are the largest class of small non-coding RNAs. piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes are associated with both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly during spermatogenesis. piRNAs differ from microRNAs (miRNAs) in size (26–31 nt rather than 21–24 nt), lack of sequence conservation, and increased complexity. However, like other short-chain RNAs, piRNAs are thought to be involved in gene silencing, specifically transposon silencing. The majority of piRNAs are antisense to transposon sequences, suggesting that transposons are targets of piRNAs. In mammals, piRNA activity in transposon silencing appears to be most important during embryonic development, and in *C. elegans* and humans, piRNAs are required for spermatogenesis. piRNAs play a role in RNA silencing through the formation of RNA-induced silencing complexes (RISCs).

[0068] An "aptamer" is an oligonucleotide or peptide molecule that binds to a specific target molecule. "Nucleic acid aptamers" are nucleic acids designed through repeated in vitro selection to bind to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, or through equivalent SELEX (Systematic Evolution of Ligands by Exponential Enrichment). "Peptide aptamers" are artificial proteins selected or designed to bind to a specific target molecule. These proteins consist of one or more peptide loops made up of variable sequences presented by a protein scaffold. These aptamers are typically isolated from combinatorial libraries and then often improved through direct mutagenesis or multiple mutagenesis and selection of variable regions. An evolved form of peptide aptamer, the "affimer protein," is a very stable small protein designed to present a peptide loop that provides a high-affinity binding surface to a specific target protein. Affimer proteins are low molecular weight proteins of 12-14 kDa derived from the cystatin family of cysteine ​​protease inhibitors. Aptamers offer molecular recognition properties comparable to those of antibodies, which are commonly used biomolecules, making them useful in biotechnology and therapeutic applications. Aptamers offer advantages over antibodies in addition to fine recognition because they can be fabricated entirely in vitro, are easily synthesized chemically, have desirable storage properties, and induce little to no immunogenicity in therapeutic applications.

[0069] As used herein, the term “intracellular immunoglobulin molecule” refers to complete immunoglobulins that are the same as naturally secreted immunoglobulins but remain inside the cell after synthesis. “Intracellular immunoglobulin fragment” refers to any fragment, including single-chain fragments, of an intracellular immunoglobulin molecule. Therefore, intracellular immunoglobulin molecules or their fragments are not secreted or expressed on the outer surface of cells. Single-chain intracellular immunoglobulin fragments are referred to herein as “single-chain immunoglobulin.” As used herein, the term “intracellular immunoglobulin molecule or its fragment” is understood to encompass “intracellular immunoglobulin,” “single-chain intracellular immunoglobulin” (or its fragment), “intracellular immunoglobulin fragment,” “intracellular antibody” (or its fragment), and “intracellular antibody” (or its fragment). Therefore, the terms “intracellular immunoglobulin,” “intracellular Ig,” “intracellular antibody,” and “intracellular antibody” may be used interchangeably herein and are all encompassed by the general definition of “intracellular immunoglobulin molecule or its fragment.” In some embodiments, the intracellular immunoglobulin molecule or fragment thereof of the present invention may comprise two or more subunit polypeptides, e.g., a “first intracellular immunoglobulin subunit polypeptide” and a “second intracellular immunoglobulin subunit polypeptide.” However, in other embodiments, the intracellular immunoglobulin may be a “single-chain intracellular immunoglobulin” comprising only a single polypeptide. As used herein, “single-chain intracellular immunoglobulin” is defined as any single fragment having a desired activity, e.g., intracellular binding to an antigen. Thus, single-chain intracellular immunoglobulins include single-chain intracellular immunoglobulins comprising heavy-chain variable regions and light-chain variable regions that act together to bind to an antigen, as well as single-chain intracellular immunoglobulins having only a single variable region that binds to an antigen, e.g., a “camelized” heavy-chain variable region as described herein. Intracellular immunoglobulins or Ig fragments may be expressed substantially anywhere within a cell, e.g., in the cytoplasm, on the inner surface of the cell membrane, or in intracellular compartments (also called cell compartments or cell divisions) such as the cell nucleus, Golgi apparatus, endoplasmic reticulum, endosomes, and mitochondria.Other cellular compartments include those described herein and those well known in the art.

[0070] The term “sample” used for detecting or determining the presence or level of at least one biomarker typically refers to whole blood, plasma, serum, saliva, urine, feces (e.g., stool), tears, and any other bodily fluids (e.g., bodily fluids as described in the definition of “bodily fluids” above), or tissue samples such as bone marrow samples and bone samples (e.g., biopsy samples), or surgically excised tissue. In certain cases, the method of the present invention further comprises obtaining the sample from an individual before detecting or determining the presence or level of at least one marker in the sample.

[0071] The term "sensitize" means altering cancer cells or tumor cells so that the associated cancer is more effectively treated by cancer therapies (e.g., immune checkpoint suppression therapy, chemotherapy, and / or radiation therapy). In some embodiments, normal cells are not affected to the extent that they would be excessively damaged by the treatment. Increased or decreased sensitivity to treatment is measured according to methods known in the art for the specific treatments and methods described herein, including cell proliferation assays (Tanigawa N, Kern DH, Kikasa Y, Morton DL, Cancer Res, 1982, Vol. 42: pp. 2159-2164), cell death assays (Weisenthal LM, Shoemaker RH, Marsden JA, Dill PL, Baker JA, Moran EM, Cancer Res, 1984, Vol. 94: pp. 161-173, Weisenthal LM, Lippman ME, Cancer Treat Rep, 1985, Vol. 69: pp. 615-632, Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds., Drug Resistance in Leukemia and Lymphoma, Langhorne, Pennsylvania, Harwood) This includes, but is not limited to, the work of Weisenthal LM in Academic Publishers, 1993, pp. 415–432, and Contrib Gynecol Obstet, 1994, Vol. 19: pp. 82–90. The aforementioned susceptibility or resistance can also be assessed in animals by measuring tumor size over a period of time, for example, 6 months in humans.A composition or method enhances the response to treatment if the increase in treatment sensitivity or decrease in treatment resistance compared to the absence of such a composition or method is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or higher, for example, 2x, 3x, 4x, 5x, 10x, 15x, 20x, or higher, or any rate in the range including both ends. Determining sensitivity or resistance to treatment is routine in the art and within the scope of the skill of an experienced physician. It should be understood that any method described herein for enhancing the efficacy of cancer therapy is equally applicable to methods for enhancing overgrowth cells or, in other cases, cancerous cells (e.g., resistant cells) to that cancer therapy.

[0072] Short interfering RNA (siRNA), also known as "small interfering RNA" in this specification, is defined as a factor that functions to inhibit the expression of a target biomarker nucleic acid, for example, by RNAi. siRNA can be chemically synthesized, produced by in vitro transcription, or produced in host cells. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule about 15 to about 40 nucleotides long, preferably about 15 to about 28 nucleotides long, more preferably about 19 to about 25 nucleotides long, more preferably about 19, 20, 21, or 22 nucleotides long, and each strand may contain 3' and / or 5' overhangs of about 0, 1, 2, 3, 4, or 5 nucleotides in length. The length of the overhangs is independent between the two strands, i.e., the length of the overhang of one strand is independent of the length of the overhang of the second strand. Preferably, the siRNA can promote RNA interference via degradation of the target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0073] In another embodiment, the siRNA is a short hairpin (also called stem-loop) RNA (shRNA). In one embodiment, these shRNAs consist of a short antisense strand (e.g., 19-25 nucleotides), followed by a 5-9 nucleotide loop, and a similar sense strand. Alternatively, this sense strand may precede the nucleotide loop structure, followed by the antisense strand. These shRNAs may be contained within plasmids, retroviruses, and lentiviruses and may be expressed from, for example, the pol III U6 promoter or another promoter (see, for example, Stewart et al., RNA (2003), April; Vol. 9 (No. 4): pp. 493-501, as incorporated herein by reference).

[0074] RNA interfering agents, such as siRNA molecules, may be administered to patients with cancer or those at risk of developing cancer to inhibit the expression of biomarker genes that are overexpressed in the cancer, thereby treating, preventing, or suppressing the target cancer.

[0075] The term "small molecule" is a term used in the art and includes molecules with a molecular weight of less than approximately 1000 or less than approximately 500. In one embodiment, a small molecule does not exclusively contain peptide bonds. In another embodiment, a small molecule is not an oligomer. Examples of small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptide mimes, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al., Science (1998), Vol. 282: p. 63), and natural product extract libraries. In another embodiment, the compounds are low-molecular-weight organic non-peptide compounds. In other embodiments, a small molecule is not a biosynthetic molecule.

[0076] The term "specific binding" refers to an antibody that binds to a given antigen. Typically, this antibody is determined by surface plasmon resonance (SPR) using a BIACORE® assay instrument, where the antigen of interest is used as the analyte and this antibody is used as the ligand, and the specific binding is approximately 10. -7 Affinity (K) less than MD ), for example, about 10 -8 Less than M, 10 -9 Less than M, or 10 -10 The antibody binds with an affinity less than M or even higher, and binds to the predetermined antigen with an affinity at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, or 10.0 times or more than the affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The terms "antigen-recognizing antibody" and "antigen-specific antibody" are used interchangeably in this specification with the term "antibody that specifically binds to an antigen." Selective binding is a relative term referring to the ability of an antibody to distinguish binding to one antigen from binding to another antigen.

[0077] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from cancer, such as brain cancer, lung cancer, ovarian cancer, pancreatic cancer, liver cancer, breast cancer, prostate cancer, colorectal cancer, melanoma, multiple myeloma, etc. The term "subject" is interchangeable with "patient."

[0078] The term “survival” includes all of the following: “survival” (also known as overall survival, which may be death from any cause or tumor-related death), “recurrence-free survival” (the term recurrence includes both local and distant recurrence), “metastasis-free survival,” and “disease-free survival” (the term disease includes cancer and cancer-associated diseases). The survival can be calculated by referring to a predetermined starting point (e.g., diagnosis or initiation of treatment) and an ending point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for the effectiveness of treatment can be expanded to include the response to chemotherapy, the probability of survival, the probability of metastasis within a given period, and the probability of tumor recurrence.

[0079] The term "synergistic effect" refers to the possibility that the combined effect of two or more anticancer drugs (for example, a cancer vaccine combined with immunotherapy) may be greater than the combined effect of each anticancer drug / treatment individually.

[0080] The term "T cell" refers to CD4 + T cells and CD8 + This includes T cells. The term T cell includes both T helper 1 T cells and T helper 2 T cells. The term antigen-presenting cells include specialized antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0081] The term "therapeutic effect" refers to a topical or systemic effect in animals, specifically mammals, and more specifically humans, caused by a pharmacologically active substance. Therefore, this term means any substance intended for use in the diagnosis, treatment, alleviation, cure, or prevention of disease in animals or humans, or for the enhancement of desirable physical or mental development and condition. The term "therapeutic effective dose" means the amount of such substance that produces some desirable topical or systemic effect at a reasonable risk-benefit ratio applicable to any treatment. In certain embodiments, the therapeutic effective dose of a compound depends on its therapeutic index, solubility, etc. For example, a particular compound discovered by the method of the present invention may be administered in an amount sufficient to produce a reasonable risk-benefit ratio applicable to such treatment.

[0082] As used herein, the terms “therapeutic dose” and “effective dose” mean the amount of a compound, material, or composition containing the compound of the present invention that is effective in producing some desirable therapeutic effect in at least a small population of animal cells at a reasonable risk-benefit ratio applicable to any medical treatment. The toxicity and therapeutic efficacy of the compound in question shall be determined by standard pharmaceutical methods, e.g., LD, in cell cultures or experimental animals. 50 and ED 50This can be determined by a method for determining the therapeutic index. Compositions exhibiting a high therapeutic index are preferred. In some embodiments, LD 50 The lethal dose is measurable and can be reduced by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, with the administration of the drug compared to not administering the drug. Similarly, ED 50 (i.e., the concentration that achieves maximum suppression of half of the symptoms) is measurable, and the effect can be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, with the drug compared to not administering the drug. Similarly, IC 50 (i.e., the concentration that achieves the maximum cytotoxic effect or cell proliferation inhibitory effect on cancer cells) can be measured, and compared to not administering the drug, the drug can increase by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. In some embodiments, cancer cell proliferation during the assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even up to 100%. In another embodiment, a reduction of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% of solid malignant tumors can be achieved.

[0083] The phrase "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins that have been isolated from chemical precursors or other chemicals involved in protein synthesis. In one embodiment, the phrase "substantially free of chemical precursors or other chemicals" includes preparations of antibodies, polypeptides, peptides, or fusion proteins having less than about 30% (on a dry weight basis) of chemical precursors or non-antibodies, polypeptides, peptides, or fusion proteins, more preferably less than about 20% of chemical precursors or non-antibodies, polypeptides, peptides, or fusion proteins, even more preferably less than about 10% of chemical precursors or non-antibodies, polypeptides, peptides, or fusion proteins, and most preferably less than about 5% of chemical precursors or non-antibodies, polypeptides, peptides, or fusion proteins.

[0084] A “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analogues of such RNA or cDNA) that is complementary or homologous to all or part of the mature mRNA produced by the transcription of a biomarker nucleic acid and, if any, the normal post-transcriptional processing (e.g., splicing) of that RNA transcript and the reverse transcription of that RNA transcript.

[0085] The term “host cell” refers to a cell into which nucleic acids included in the scope of the present invention, such as recombinant expression vectors included in the scope of the present invention, have been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to specific target cells but also to their offspring cells or cells that may be offspring. Such offspring may not actually be identical to the parent cells because certain changes may occur in the progeny due to either mutation or environmental influences, but they are still included within the scope of the term as used herein.

[0086] The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it is bound. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, which can to which additional DNA segments can be ligated to its viral genome. Certain vectors are capable of autonomous replication within the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into that cell and thereby replicate together with that host genome. Furthermore, certain vectors can express the gene to which they are linked in order to function. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Expression vectors commonly used in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably herein. However, the present invention includes other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0087] As used herein, the term “unresponsive” includes the refractive power of cancer cells to treatment or stimulation, such as the refractive power of therapeutic cells, such as immune cells, to stimulation via activating receptors or cytokines. Unresponsiveness may occur, for example, due to exposure to immunosuppressants or exposure to high doses of antigens. As used herein, the terms “unresponsive” or “tolerable” include the refractive power to activating receptor-mediated stimulation. Such refractive power is antigen-specific and generally persists after exposure to the tolerable antigen has ended. For example, T cell unresponsiveness (in contrast to unresponsiveness) is characterized by a lack of cytokine production, such as IL-2 production. T cell unresponsiveness occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal). Re-exposure of the cells to the same antigen under these conditions results in the inability to produce cytokines and therefore proliferation (even if re-exposure occurs in the presence of a co-stimulatory polypeptide). However, unresponsive T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell unresponsiveness can also be observed by the absence of IL-2 production by T lymphocytes, as assessed by ELISA or proliferation assays using indicator cell lines. Alternatively, a reporter gene construct can be used. For instance, unresponsive T cells are unable to initiate transcription of the IL-2 gene induced by a multimer AP1 sequence (Kang et al., Science, Vol. 257: pp. 1134, 1992) which can be found in a heterologous promoter or within the enhancer under the control of the 5' IL-2 gene enhancer.

[0088] The term "TGFβ-SMAD / p63 signaling pathway" refers to a branch of the TGFβ signaling pathway. The TGFβ signaling pathway is involved in many cellular processes, including but not limited to cell proliferation, cell differentiation, apoptosis, cellular homeostasis, and other cellular functions, in both adults and developing embryos. In some embodiments, TGFβ superfamily ligands (e.g., TGFβ1, TGFβ2, and / or TGFβ3) bind to type II receptors, which recruit and phosphorylate type I receptors. The type I receptors then bind to receptor-regulated SMADs (R-SMADs; e.g., SMAD1, SMAD2, SMAD3, SMAD5, or SMAD9), which in turn can bind to coSMADs (e.g., SMAD4). The R-SMAD / coSMAD complexes accumulate in the nucleus, where they act as transcription factors and are involved in regulating the expression of target genes. In the aforementioned sub-pathway of the "TGFβ-Smad / p63 signaling pathway," the R-SMAD / coSMAD complex further associates with p63 in the nucleus to regulate the expression of target genes. In one embodiment, R-SMAD is Smad2. Activation of the TGFβ-Smad / p63 signaling pathway can be evaluated by analyzing Smad2 phosphorylation, Smad2 nuclear translocation, Smad2 association with p63, and / or activation of TGFβ-Smad / p63 signature genes. TGFβ-Smad / p63 signatures may include, but are not limited to, the upregulation of ICOSL, PYCARD, SFN, PERP, RIPK3, and / or SESN1, and the downregulation of KSR1, EIF4EBP1, ITGA5, EMILIN1, CD200, and / or CSF1.

[0089] In some embodiments, TGFβ promotes the formation of a TGFBRII-TGFBR1 heterodimer on the cell plasma membrane upon binding to its receptor. Subsequently, cytoplasmic signaling molecules R-Smad (e.g., Smad2 and Smad3) are phosphorylated by activated TGFBRI. These activated R-Smads form complexes with Co-Smad (e.g., Smad4) and translocate into the cell nucleus. As demonstrated herein, in combination with p63 (or other p53 family members such as p53 or p73), this Smad / p63 transcriptional complex upregulates pro-inflammatory genes (e.g., Icosl, Nfkbib, Tnfaip3, Pik3r1, and Perp) and downregulates oncogenic genes (e.g., Cd200, Cxcl5, Csf1, Pdgfrb, Fgfr1, Vegfa). Therefore, tumor cells possessing an activated TGFβ-Smad / p63 signature present a potent "eat me" signal to the immune system, triggering an anti-tumor immune response by recruiting antigen-presenting cells (such as dendritic cells). Dendritic cells (DCs) take up tumor-specific antigens, promoting tumor-specific effector responses and memory T cell responses to provide the host with complete protection against the tumor. The TGFβ-Smad / p63 signaling pathway can be activated by modulating the signaling molecules involved in this pathway. In certain embodiments, the Smad superfamily (including Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, and Smad9) and the p53 superfamily (including p53, p63, and p73) are modulated to activate the TGFβ-Smad / p63 signaling pathway in compositions and methods included within the scope of the present invention.

[0090] The TGFβ-Smad / p63 signaling pathway can be activated by providing TGFβ superfamily ligands or agonists of the TGFβ signaling pathway. This signaling pathway may be controllable and / or at the Smad and p63 levels. Examples of agents useful for activating the TGFβ-Smad / p63 signaling pathway or other biomarkers described herein include small molecules, peptides, and nucleic acids that can upregulate the expression and / or activity of one or more biomarkers or fragments thereof listed in Table 1, and / or small molecules, peptides, and nucleic acids that can reduce the copy number, quantity, and / or activity of one or more biomarkers or fragments thereof listed in Table 2. Examples of agents useful for activating the TGFβ-Smad / p63 signaling pathway or other biomarkers described herein include TGFβ superfamily ligands.

[0091] In one embodiment, suitable agonists include native agonists of the TGFβ superfamily members or their fragments and variants. For example, soluble endoglins may be agonists of TGFβ signaling. See, for example, U.S. Patent No. 5,719,120, No. 5,830,847, and No. 6,015,693, which are incorporated herein by reference in whole. In another embodiment, suitable agonists may include inhibitors of native TGFβ antagonists. Several native modifiers that regulate TGFβ signaling have been identified. For example, the approach of TGFβ ligands to receptors is inhibited by soluble proteins LAP, decorin, and α2-macroglobulin, which bind to and capture the ligand (Balemans and Van Hul, Dev. Biol., 2002, Vol. 250: pp. 231-250). The approach of TGFβ ligands to receptors is also regulated by membrane-bound receptors. BAMBI acts as a decoy receptor and competes with type I receptors (Onichtchouk et al., Nature, Vol. 401, pp. 480-485, 1999), β-glycan (TGFβ type II receptor) increases the binding of TGFβ to type II receptors (Brown et al., Science, Vol. 283, pp. 2080-2082, Massague, Annu. Rev. Biochem., Vol. 67, pp. 753-791, del Re et al., J. Biol. Chem., Vol. 279, pp. 22765-22772, 2004), and endoglins increase the binding of TGFβ to ALK1 in endothelial cells (Marchuk, Curr. Opin., 1998). Hematol., Vol. 5: 332-338; Massague, (2000) Nat. Rev. Mol. Cell. Biol., Vol. 1: 169-178; Shi and Massague, (2003) Cell, Vol. 113: 685-700).Cripto, an EGF-CFC GPI-anchored membrane protein, acts as a co-receptor, interfering with activin signaling while increasing the binding of TGFβ ligands nodal, Vg1, and GDF1 to the activin receptor (Cheng et al., Genes Dev., 2003, Vol. 17: pp. 31-36; Shen and Schier, Trends Genet., 2000, Vol. 16: pp. 303-309). Suitable agonists include synthetic or human recombinant compounds. Molecules that can function as agonists include, but are not limited to, small molecules, antibodies (including Fab fragments, Fab'2 fragments, and their fragments or variants such as scFv), and peptide mimes.

[0092] As used herein, the term “TGFβ superfamily” refers to a large family of multifunctional proteins that regulate various cellular functions, including cell proliferation, migration, differentiation, and apoptosis. Currently, the TGFβ superfamily comprises more than 30 members, most notably activin, inhibin, transforming growth factor β (TGFβ), growth and differentiation factor (GDF), bone morphogenetic protein (BMP), and Müllerian duct inhibitor (MIS). All of these molecules are peptidic growth factors structurally related to TGFβ. All of these molecules share a common motif called the cysteine ​​knot, which consists of seven particularly conserved cysteine ​​residues organized into a robust structure (Massague, Annu. Rev. Biochem., 1998, Vol. 67: pp. 753-791). Unlike classical hormones, members of the TGFβ superfamily are multifunctional proteins whose effects are as dependent on the type and stage of the target cell as they are on the growth factors themselves.

[0093] Suitable TGFβ superfamily members for use in carrying out the present invention include any member of the TGFβ superfamily capable of activating the TGFβ-Smad / p63 signaling pathway. In one embodiment, the TGFβ superfamily members are members of the TGFβ family, including but not limited to LAP, TGFβ1, TGFβ2, TGFβ3, and TGFβ5. In another embodiment, the TGFβ superfamily members are members of the activin family, including but not limited to activin A, activin AB, activin AC, activin B, activin C, C17ORF99, INHBA, INHBB, inhibin, inhibin A, and inhibin B. In yet another embodiment, the TGFβ superfamily members are members of the BMP (bone morphogenetic protein) family, including, but not limited to, BMP-1 / PCP, BMP-2, BMP-2 / BMP-6 heterodimer, BMP-2 / BMP-7 heterodimer, BMP-2a, BMP-3, BMP-3b / GDF-10, BMP-4, BMP-4 / BMP-7 heterodimer, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-15 / GDF-9B, and decapentaplesic / DPP. In yet another embodiment, the TGFβ superfamily members are members of the GDNF family, including, but not limited to, artemin, GDNF, neurturin, and parcefin. Other TGFβ superfamily members include Lefty A, Lefty B, MIS / AMH, Nordal, and SCUBE3.

[0094] In certain embodiments, members of the TGFβ superfamily are members of the TGFβ family. TGFβ, an early member of the TGFβ family, has been shown to play a variety of roles ranging from embryonic pattern formation to the regulation of cell proliferation in adult tissues. Mammalian cells can produce three distinct TGFβ isoforms: TGFβ1, TGFβ2, and TGFβ3. These isoforms share the same basic structure (a homodimer consisting of 112 amino acids stabilized by intrachain and interchain disulfide bonds), and their amino acid sequences exhibit high homology (over 70%). However, each isoform is encoded by a different gene, and each isoform is expressed in a tissue-specific and developmentally regulated manner (Massague, Annu. Rev. Biochem., 1998, Vol. 67: pp. 753-791). TGFβ exerts its biological function through a signaling cascade that ultimately activates and / or represses the expression of a series of specific genes. Crosslinking studies have shown that TGFβ primarily binds to three types of high-affinity cell surface proteins called type I TGFβ receptors, type II TGFβ receptors, and type III TGFβ receptors (Massague and Like, J. Biol. Chem., Vol. 260, pp. 2636-2645, 1985; Cheifetz et al., J. Biol. Chem., Vol. 261, pp. 9972-9978, 1986). In some embodiments, TGFβ first binds to its type II receptor, and then signals by recruiting and activating its type I receptor. Subsequently, the activated type I receptor phosphorylates its intracellular signaling molecule, Smad protein (Heldin et al., Natutre, Vol. 390, pp. 465-471, 1997; Derynck et al., Cell, Vol. 95, pp. 737-740, 1998).

[0095] The term "TGFβ1" or "transforming growth factor β1" refers to the secreted ligand of the TGFβ superfamily protein. Ligands of this family bind to various TGFβ receptors, triggering the recruitment and activation of SMAD family transcription factors that regulate gene expression. The encoded preproprotein undergoes proteolytic processing to produce a latent associated peptide (LAP) and a mature peptide. This preproprotein is found in both the latent form, consisting of a mature peptide homodimer, a LAP homodimer, and a latent TGFβ-binding protein, and the active form, consisting solely of the mature peptide homodimer. This mature peptide can also form heterodimers with other TGFβ family members. Activation to the mature form involves several steps. After cleavage of the proprotein within the Golgi apparatus, the LAP and TGFβ1 chains remain non-covalently bound, keeping TGFβ1 inactive while it is stored in the extracellular matrix. Simultaneously, the LAP chain interacts with LTBP1, LRRC32 / GARP, and LRRC33 / NRROS, which are "environmental molecules" that regulate TGFβ1 activation and maintain it in a latent state while stored in the extracellular environment. TGFβ1 is dissociated from LAP by integrins. The binding of integrins to LAP stabilizes an alternative three-dimensional structure of LAP called a bowtie tail, causing a twist in the LAP chain, after which activated TGFβ1 is dissociated. Once activated after the dissociation of LAP, TGFβ1 acts by binding to TGFβ receptors, which then transmit signals. In preferred embodiments, the term "TGFβ1" refers to activated TGFβ1.

[0096] TGFβ1 regulates cell proliferation, differentiation, and growth, and can modulate the expression and activation of other growth factors, including interferon-γ and tumor necrosis factor-α. TGFβ1 plays a crucial role in bone remodeling. It acts as a potent stimulant of osteoblast formation, causing chemotaxis, proliferation, and differentiation of participating osteoblasts. TGFβ1 can promote differentiation into either T helper 17 cells (Th17) or regulatory T cells (Treg) in a concentration-dependent manner. At high concentrations, TGFβ1 induces FOXP3-mediated repression of RORCs and downregulation of IL-17 expression, supporting Treg cell development. At low concentrations, TGFβ1, in conjunction with IL-6 and IL-21, induces the expression of IL-17 and IL-23 receptors, supporting differentiation into Th17 cells. TGFβ1 stimulates sustained collagen production through the activation of CREB3L1 via regulatory intramembrane proteolysis (RIP). TGFβ1 activates SMAD2 / 3 by inducing its phosphorylation and subsequent nuclear translocation (Hwangbo et al., Oncogene, Vol. 35: pp. 389-401, 2016). TGFβ1 can also induce epithelial-mesenchymal transition (EMT) and cell migration of various cell types (Hwangbo et al., Oncogene, Vol. 35: pp. 389-401, 2016). TGFβ1 is frequently upregulated in tumor cells, and mutations in this gene lead to Kamuraci-Engelmann disease.

[0097] The term "TGFβ1" is considered to include its fragments, variants (e.g., allele variants), and derivatives. Representative human TGFβ1 cDNA sequences and human TGFβ1 protein sequences are well known in the art, and these protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). For example, one human TGFβ1 isoform is publicly known. The human TGFβ1 transcript (NM_000660.7) encodes the TGFβ1 proprotein preproprotein (NP_000651.3). The nucleic acid and polypeptide sequences of TGFβ1 orthologs in non-human organisms are well known, for example, chimpanzee TGFβ1 (XM_016936045.2 and XP_016791534.1, XM_512687.6 and XP_512687.2, and XM_009435655.3 and XP_009433930.1), canine T This product includes GFβ1 (NM_001003309.1 and NP_001003309.1), camel TGFβ1 (NM_001166068.1 and NP_001159540.1), mouse TGFβ1 (NM_011577.2 and NP_035707.1), and rat TGFβ1 (NM_021578.2 and NP_067589.1).

[0098] The terms "TGFβ2" or "transforming growth factor β2" refer to the secreted ligands of the TGFβ superfamily proteins. As described herein, ligands of this family bind to various TGFβ receptors and trigger the recruitment and activation of SMAD family transcription factors that regulate gene expression. The encoded preproprotein undergoes proteolytic processing to produce a latent associated peptide (LAP) and a mature peptide, and this preproprotein is found in both the latent form, which consists of a mature peptide homodimer, an LAP homodimer, and a latent TGFβ-binding protein, and the active form, which consists only of a mature peptide homodimer. This mature peptide can also form heterodimers with other TGFβ family members. Activation to the mature form involves several steps. After cleavage of the proprotein in the Golgi apparatus, the LAP chain and TGFβ2 chain remain non-covalently bound, keeping TGFβ2 inactive while it is stored in the extracellular matrix. Simultaneously, the LAP chain interacts with "environmental molecules" such as LTBP1 and LRRC32 / GARP, which regulate the activation of TGFβ2 and maintain it in a latent state while stored in the extracellular environment. Once activated after LAP dissociation, TGFβ2 acts by binding to TGFβ receptors, which then transmit signals. In preferred embodiments, the term "TGFβ2" refers to activated TGFβ2. Disruption of the TGFβ / SMAD pathway has been shown in various human cancers. TGFβ2 controls various processes, including angiogenesis and cardiac development (Boileau et al., (2012) Nat. Genet., Vol. 44: pp. 916-921; Lindsay et al., (2012) Nat. Genet., Vol. 44: pp. 922-927). Chromosomal translocations involving the TGFβ2 gene are associated with Peters anomalies, a congenital defect of the anterior chamber. Mutations in the TGFβ2 gene may be associated with Loeys-Dietz syndrome.

[0099] The term "TGFβ2" is considered to include its fragments, variants (e.g., allele variants), and derivatives. Representative human TGFβ2 cDNA sequences and human TGFβ2 protein sequences are well known in the art, and these protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). For example, two human TGFβ2 isoforms are known. TGFβ2 transcript variant 1 (NM_001135599.3) is the longest transcript and encodes the longer isoform 1 (NP_001129071.1). TGFβ2 transcript variant 2 (NM_003238.5) lacks an in-frame exon in the 5' coding region compared to variant 1. The resulting isoform 2 (NM_003238.5) is shorter than isoform 1. Both isoforms may undergo similar proteolytic processing. The nucleic acid and polypeptide sequences of TGFβ2 orthologs in non-human organisms are well known, for example, chimpanzee TGFβ2 (XM_001172158.6 and XP_001172158.1, and XM_514203.7 and XP_514203.2), monkey TGFβ2 (NM_001266518.1 and NP_001253447.1), and canine TGFβ2 (XM_005640824.2 and XP_005640881.1, XM_545713.6 and XP_545713.2). , and also including XM_853584.5 and XP_858677.1), camel TGFβ2 (NM_001113252.1 and NP_001106723.1), mouse TGFβ2 (NM_001329107.1 and NP_001316036.1, and NM_009367.4 and NP_033393.2), rat TGFβ2 (NM_031131.1 and NP_112393.1), and chicken TGFβ2 (NM_001031045.3 and NP_001026216.2).

[0100] The terms "TGFβ3" or "transforming growth factor β3" refer to the secreted ligands of the TGFβ superfamily proteins. As described herein, ligands of this family bind to various TGFβ receptors and trigger the recruitment and activation of SMAD family transcription factors that regulate gene expression. The preproprotein it encodes undergoes proteolytic processing to produce a latent associated peptide (LAP) and a mature peptide. This preproprotein is found in both the latent form, which consists of a mature peptide homodimer, an LAP homodimer, and a latent TGFβ-binding protein, and the active form, which consists only of a mature peptide homodimer. This mature peptide can also form heterodimers with other TGFβ family members. Activation of TGFβ3 into the mature form involves several steps. After cleavage of the proprotein in the Golgi apparatus, the LAP and TGFβ3 chains remain non-covalently bound, keeping TGFβ3 inactive while it is stored in the extracellular matrix. Simultaneously, the LAP chain interacts with "environmental molecules" such as LTBP1 and LRRC32 / GARP, which regulate the activation of TGFβ3 and maintain it in a latent state while stored in the extracellular environment. TGFβ3 is dissociated from LAP by integrins. Integrin binding causes a twist in the LAP chain, followed by the dissociation of activated TGFβ3. Once activated after LAP dissociation, TGFβ-3 acts by binding to TGFβ receptors, which then transmit signals. In preferred embodiments, the term "TGFβ3" refers to activated TGFβ3.

[0101] TGFβ3 is involved in embryonic development and cell differentiation and may play a role in wound healing. TGFβ3 is required for various processes, including secondary palatal development. Mutations in the TGFβ3 gene are a cause of aortic aneurysm and aortic dissection, as well as familial arrhythmogenic right ventricular dysplasia.

[0102] The term "TGFβ3" is considered to include its fragments, variants (e.g., allele variants), and derivatives. Representative human TGFβ3 cDNA sequences and human TGFβ3 protein sequences are well known in the art, and these protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). For example, three human TGFβ3 isoforms are known. TGFβ3 transcript variant 1 (NM_003239.4) is the longest transcript and encodes the longer isoform 1 (NP_003230.1). TGFβ3 transcript variant 2 (NM_001329939.1) differs from variant 1 in its 5'UTR and encodes the same isoform as variant 1 (NP_001316868.1). TGFβ3 transcript variant 3 (NM_001329938.2) lacks several exons, and its 3' terminal exon extends beyond the splicing site used in variant 1. This results in an earlier stop codon and a new 3'UTR compared to mutant 1. The encoded isoform 2 (NP_001316867.1) has a shorter C-terminus than isoform 1. Nucleic acid and polypeptide sequences of TGFβ3 orthologs in non-human organisms are well known, for example, chimpanzee TGFβ3 (XM_016926465.2 and XP_016781954.1, XM_016926464.2 and XP_016781953.1, XM_001161669.5 and XP_001161669.1, and XM_009428178.2 and XP_009426453.1), monkey TGFβ3 (NM_00125747 This includes 5.1 and NP_001244404.1), canine TGFβ3 (XM_849026.5 and XP_854119.2), camel TGFβ3 (NM_001101183.1 and NP_001094653.1), mouse TGFβ3 (NM_009368.3 and NP_033394.2), rat TGFβ3 (NM_013174.2 and NP_037306.1), and chicken TGFβ3 (NM_205454.1 and NP_990785.1).

[0103] The term "Smad" refers to a family of receptor-activated signaling transcription factors that transmit signals from TGFβ family receptors. Members of the Smad family proteins are identified based on homology to the Drosophila gene Mothers Against DPP (mad), which encodes essential elements of the Drosophila dpp signaling pathway (Sekelsky et al., Genetics, Vol. 139: pp. 1347-1358, 1995; Newfeld et al., Development, Vol. 122: pp. 2099-2108, 1996). Smad proteins are generally characterized by highly conserved amino-terminal and carboxy-terminal domains separated by a high-proline linker. The amino-terminal domain (MH1 domain) mediates binding to DNA, while the carboxy-terminal domain (MH2 domain) binds to the aforementioned receptor.

[0104] At least eight Smad proteins have been identified and shown to be involved in signal responses induced by TGFβ family members (Kretzschmar and Massague, Current Opinion in Genetics and Development, Vol. 8: pp. 103-111, 1998). These Smads can be divided into three subgroups. One group (Smad1, Smad2, Smad3, Smad5, and Smad9) includes Smads that are direct substrates of TGFβ family receptor kinases. Another group (Smad4) includes Smads that are not direct substrates of the receptor but are involved in signal transduction through binding to receptor-activated Smads. A third group of Smads (Smad6 and Smad7) consists of proteins that inhibit the activation of the first two groups of Smads.

[0105] Smad proteins have specific roles in the pathways of various TGFβ family members. Among the Smad proteins identified for TGFβ family members, Smad2 and Smad3 are specific to TGFβ signaling (Heldin et al., Nature, Vol. 390, pp. 465-471, 1997). These activated Smad2 and Smad3 interact with a common intermediary, Smad4, translocate into the nucleus, and activate a series of specific genes within the nucleus (Heldin et al., Nature, Vol. 390, pp. 465-471, 1997). This TGFβ pathway activates Smad2 and / or Smad3 and similarly uses the inhibitory signaling proteins Smad6 and Smad7 to balance the net output of signaling.

[0106] Smad2 and Smad3 possess intrinsic transactivation activity as transcription factors (Zawel et al., (1998) Mol. Cell., Vol. 1: pp. 611-617), while studies have shown that they can activate the expression of specific genes through specific interactions with other nuclear factors (Derynck et al., (1998) Cell, Vol. 95: pp. 737-740). By activating specific Smad proteins, a specific TGFβ-mediated effect on a given cell type can be achieved, resulting in altered expression of specific genes. Smad2 is among the Smad proteins of particular interest (Nakao et al., (1997) J. Biol. Chem., Vol. 272: pp. 2896-2900).

[0107] The term "SMAD2" refers to SMAD family member 2, belonging to the SMAD family of proteins similar to the gene products of the Drosophila gene "Mother's Against Decapentaplesic" (Mad) and the Elegans nematode gene Sma. SMAD proteins are signaling and transcriptional modifiers that mediate multiple signaling pathways. SMAD2 mediates TGFβ signaling and thus regulates several cellular processes, including cell proliferation, apoptosis, and differentiation. SMAD2 is recruited to the TGFβ receptor via interaction with the SMAD anchor of the receptor-activating (SARA) protein. In response to TGFβ signaling, SMAD2 is phosphorylated by the TGFβ receptor. This phosphorylation induces dissociation of SMAD2 from SARA and binding to its family member, SMAD4. Binding to SMAD4 is crucial for SMAD2's translocation into the nucleus, where it binds to target promoters and forms transcriptional repression complexes with other cofactors (e.g., p63). SMAD2 binds to TRE sequences within the promoter regions of many TGFβ-regulated genes. SMAD2 can also be phosphorylated by activin type 1 receptor kinase, mediating signaling from activin. SMAD2 may act as a tumor suppressor in colorectal cancer. SMAD2 positively regulates PDPK1 kinase activity by stimulating dissociation from the 14-3-3 protein YWHAQ, which acts as a negative regulator. In one embodiment, the human SMAD2 protein has 467 amino acids and a molecular weight of 52306 Da.

[0108] The term "SMAD2" is considered to include its fragments, variants (e.g., allele variants), and derivatives. Representative human SMAD2 cDNA sequences and human SMAD2 protein sequences are well known in the art, and these protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). For example, three human SMAD2 isoforms are known. SMAD2 transcript variant 2 (NM_001003652.4) is the longest transcript and encodes the longer isoform 1 (NP_001003652.1). SMAD2 transcript variant 1 (NM_005901.6) uses an alternative exon (1b) in the 5'UTR compared to variant 2, but encodes the same isoform 1 (NP_005892.1). SMAD2 transcript variant 3 (NM_005901.6) lacks an in-frame exon in the 5' coding region compared to variant 2, resulting in isoform 2 (NP_001129409.1), which is shorter than isoform 1.The nucleic acid and polypeptide sequences of SMAD2 orthologs in non-human organisms are well known, for example, chimpanzee SMAD2 (XM_512121.7 and XP_512121.1, XM_001149646.5 and XP_001149646.1, XM_009433959.2 and XP_009432234.1, XM_016933662.1 and XP_016789151.1, XM_016933657.1 and XP_016789146.1, XM_ 016933659.1 and XP_016789148.1, XM_016933658.1 and XP_016789147.1, XM_009433960.3 and XP_009432235.1, and XM_016933663.1 and XP_016789152.1), Monkey SMAD2 (NM_001266803.1 and NP_001253732.1), Dog SMAD2 (XM_005622832.3 and XP_005622889.1, XM_0224 21406.1 and XP_022277114.1, XM_847706.5 and XP_852799.1, XM_005622830.3 and XP_005622887.1, XM_005622831.3 and XP_005622888.1, XM_861095.5 and XP_866188.1, and XM_022421405.1 and XP_022277113.1), Camel SMAD2 (NM_001046218.1 and NP_001039683.1) This includes mouse SMAD2 (NM_001252481.1 and NP_001239410.1, NM_001311070.1 and NP_001297999.1, and NM_010754.5 and NP_034884.2), rat SMAD2 (NM_001277450.1 and NP_001264379.1, and NM_019191.2 and NP_062064.1), and chicken SMAD2 (NM_204561.1 and NP_989892.1). Representative sequences of SMAD2 orthologs are shown in Table 1 below.

[0109] Suitable anti-SMAD2 antibodies for detecting the SMAD2 protein are well known in the art, including, for example, AM06653SU-N and AM31101PU-N (OriGene Technologies, Rockville, Maryland), AF3797, NB100-56462, NBP2-67376, and NBP2-44217 (Antibodies from Novus Biologicals, Littleton, Colorado), ab40855, ab63576, and ab202445 (Antibodies from AbCam, Cambridge, Massachusetts). Reagents for detecting SMAD2 expression are also well known. Furthermore, several siRNA constructs, shRNA constructs, and CRISPR constructs for reducing SMAD2 expression can be found in the product lists of the companies referenced above, including siRNA product numbers sc-38374 and sc-44338, CRISPR product number sc-400475, RNAi products SR320897, TG309255, TR309255, and TL309255, and CRISPR products KN404604 and KN516271 (Origene), as well as several CRISPR products from GenScript (Piscataway, New Jersey). It should be noted that this term may be further used to refer to any combination of characteristics described herein with respect to the SMAD2 molecule. For example, any combination of sequence composition, percentage specificity, sequence length, domain structure, functional activity, etc., can be used to describe the SMAD2 molecule included in the scope of the present invention.

[0110] The terms "p63" or "TP63" refer to members of the p53 family of transcription factors. Functional domains of p53 family proteins include the N-terminal transactivation domain, the central DNA-binding domain, and the oligomerization domain. Alternative splicing of the p63 gene and the use of alternative promoters result in multiple transcript variants encoding different isoforms with altered functional properties. These isoforms function in skin development and maintenance, adult stem cell / primordial cell regulation, cardiac development, and premature aging. Several isoforms have been shown to protect the germline by eliminating DNA-damaged oocytes or testicular germ cells. Mutations within the p63 gene are associated with ectodermal dysplasia and cleft lip and palate syndrome3 (EEC3), cleft hand and foot malformation4 (SHFM4), eyelid-syndromic ectodermal hypoplasia-cleft lip and palate, ADULT (Limb-Skin-Nail-Lacrimal-Tooth) syndrome, limb-breast syndrome, Lapp-Hodgkin syndrome (RHS), and cleft lip and palate8. P63 acts as a sequence-specific DNA-binding transcription activator or repressor. These isoforms contain a set of variable transactivating and self-regulating transactivating repressing domains, thus exhibiting isoform-specific activity. Isoform 2 activates RIPK4 transcription. P63, in conjunction with TP73 / p73, may be required for the initiation of p53 / TP53-dependent apoptosis in response to genotoxic substance damage and the presence of activated oncogenes. P63 is likely involved in Notch signaling by inducing JAG1 and JAG2. P63 plays a role in regulating epithelial morphogenesis. ΔN-type isoforms and TA * The ratio of P63 type isoforms influences the maintenance of the epithelial stem cell compartment and can control the initiation of epithelial stratification from undifferentiated embryonic ectoderm. P63 is required for limb formation from the ectodermal apex. P63 activates the transcription of the p21 promoter. In one embodiment, the human P63 protein has 680 amino acids and a molecular weight of 76785 Da.

[0111] The terms "p63" or "TP63" include its fragments, variants (e.g., allele variants), and derivatives. Representative human p63 cDNA sequences and human p63 protein sequences are well known in the art, and these protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). For example, 13 human XBP1 isoforms are known. p63 transcript variant 1 (NM_003722.5) is the longest transcript and encodes the longest isoform, p63 isoform 1 (NP_003713.3). p63 transcript variant 2 (NM_001114978.2) lacks an exon in the 3' coding region compared to variant 1, resulting in a frameshift. The resulting isoform (isoform 2, also known as TAp63β and TAβ, NP_001108450.1) is shorter than isoform 1 and has a distinctive C-terminus. p63 transcript variant 3 (NM_001114979.2) differs from variant 1 in the 3'UTR and coding region. The resulting isoform (isoform 3, also known as TAp63γ, TA-γ, and p51A; NP_001108451.1) is shorter than isoform 1 and has a distinctive C-terminus. p63 transcript variant 4 (NM_001114980.2) differs from variant 1 in the 5'UTR and coding region. The resulting isoform (isoform 4, also known as ΔNp63α, ΔN-α, P51delNα, CUSP, and p73H; NP_001108452.1) is shorter than isoform 1 and has a distinctive N-terminus. p63 transcript mutant 5 (NM_001114981.2) differs from mutant 1 in the 5'UTR and coding region, and also lacks an exon in the 3' coding region, resulting in a frameshift. The resulting isoform (isoform 5, also known as ΔNp63β, P51delNβ, and ΔNβ; NP_001108453.1) is shorter than isoform 1 and has unique N-terminus and C-terminus. p63 transcript mutant 6 (NM_001114982.2) differs from mutant 1 in the 5'UTR and coding region, as well as the 3'UTR and coding region. The resulting isoform (isoform 6, also known as ΔNp63γ, P51delNγ, and ΔN-γ; NP_001108454.1) is shorter than isoform 1 and has a distinctive N-terminus and C-terminus. p63 transcript mutant 7 (NM_001329144.2) lacks two exons in the 3' coding region compared to mutant 1, resulting in a frameshift. The encoded isoform (isoform 7, also known as TAp63Δ, TA-Δ, and P51Δ; NP_001316073.1) is shorter than isoform 1 and has a distinctive C-terminus. p63 transcript mutant 8 (NM_001329145.2) has several differences compared to mutant 1. As a result of these differences, an alternative start codon is used and a frameshift is introduced in the 3' coding region. The encoded isoform (isoform 8, also known as ΔN-Δ, NP_001316074.1) is shorter than isoform 1 and has a unique N-terminus and C-terminus. p63 transcript variant 9 (NM_001329146.2) lacks several 5' exons compared to variant 1 and uses an alternative start codon. The encoded isoform (isoform 9, also known as ΔNp73L, NP_001316075.1) is shorter than isoform 1 and has a unique N-terminus. p63 transcript variant 10 (NM_001329148.2) uses an alternative in-frame splicing site in the central coding region compared to variant 1. The encoded isoform (isoform 10, also known as p63-Δ, NP_001316077.1) is shorter than isoform 1. p63 transcript variant 11 (NM_001329149.2) has several differences compared to variant 1. These differences result in the use of an alternative start codon and the introduction of a frameshift in the 3' coding region. The encoded isoform (isoform 11) (NP_001316078.1) is shorter than isoform 1 and has distinctive N-terminus and C-terminus. p63 transcript variant 12 (NM_001329150.2) has several differences compared to mutant 1. As a result of these differences, an alternative start codon is used and a frameshift is introduced in the 3' coding region. The encoded isoform (isoform 12) (NP_001316079.1) is shorter than isoform 1 and has a distinctive N-terminus and C-terminus. p63 transcript mutant 13 (NM_001329964.1) uses an alternative promoter and is therefore different from mutant 1 in the 5'UTR and 5' coding region. This promoter and 5' terminal exon sequence are from the endogenous retrovirus LTR (PMID:21994760). The resulting isoform (isoform 13, also known as GTAp63, NP_001316893.1) is shorter than isoform 1 and has a distinctive N-terminus. The encoded protein is mainly expressed in testicular germ cells and eliminates germ cells with DNA damage. The nucleic acid and polypeptide sequences of p63 orthologs in non-human organisms are well known, for example, chimpanzee p63(XM_009447014.3 and XP_009445289.1, XM_001160376.5 and XP_001160376.1, XM_009447013.3 and XP_009445288). 1, XM_003310173.3 and XP_003310221.1, XM_001160425.5 and XP_001160425.1, XM_016942495.2 and XP_016797984.1, and XM_001160182.3 and XP_001160182.1), Saru p63 (XM_0288435 65.1 and XP_028699398.1, XM_015132502.2 and XP_014987988.1, XM_015132501.2 and XP_014987987.1, XM_001092093.3 and XP_001092093.1, XM_028843566.1 and XP_028699399.1, X M_028843567.1 and XP_028699400.1, XM_001091977.4 and XP_001091977.3, XM_015132503.2 and XP_014987989.1, and XM_015132504.2 and XP_014987990.2), Inu p63 (XM_022414176.1 and XP_022269884.1, XM_005639826.3 and XP_005639883.1, XM_856247.5 and XP_861340.3, XM_005639828.3 and XP_005639885.1, XM_005639827.2 and XP_005639884.1, XM_856275.3 and XP_861368.1, and XM_022414177.1 and (XP_022269885.1), Camel p63 (NM_001191337.1 and NP_001178266.1), Mouse p63 (NM_001127259.1 and NP_001120731.1, NM_001127260.1 and NP_001120732.1, NM_001127261.1 and NP_001120733.1, NM_001127262.1 and NP_00112 0734.1, NM_001127263.1 and NP_001120735.1, NM_001127264.1 and NP_001120736.1, NM_001127265.1 and NP_001120737.1, and NM_011641.2 and NP_035771.1), rat p63 (NM_001127339.1 and NP_001120811.1, NM_001127341 This includes .1 and NP_001120813.1, NM_001127342.1 and NP_001120814.1, NM_001127343.1 and NP_001120815.1, NM_001127344.1 and NP_001120816.1, and NM_019221.3 and NP_062094.1), and chicken p63 (NM_204351.1 and NP_989682.1). Representative sequences of p63 orthologs are shown in Table 1 below.

[0112] Suitable anti-p63 antibodies for detecting the p63 protein are well known in the art, including, for example, TA323790 and CF811064 (OriGene Technologies, Rockville, Maryland), AF1916 (Novus Biologicals antibody, Littleton, Colorado), ab124762, ab53039, and ab735, ab97865 (AbCam antibody, Cambridge, Massachusetts). Reagents for detecting p63 expression are also well known. Furthermore, several siRNA constructs, shRNA constructs, and CRISPR constructs for reducing p63 expression can be found in the product lists of the companies referenced above, including siRNA product numbers sc-36620 and sc-36621, RNAi products TR308688, TG308688, TL308688, and SR322466 from Santa Cruz Biotechnology, and CRISPR products KN208013 and KN208013BN (Origene), as well as several CRISPR products from GenScript (Piscataway, New Jersey). It should be noted that this term may be further used to refer to any combination of characteristics described herein with respect to the p63 molecule. For example, any combination of sequence composition, percentage specificity, sequence length, domain structure, functional activity, etc., can be used to describe the p63 molecule included in the scope of the present invention.

[0113] The term "TP53" refers to the oncoprotein P53, a tumor suppressor protein containing a transcriptional activation domain, a DNA-binding domain, and an oligomerization domain. The encoded protein induces cell cycle arrest, apoptosis, senescence, DNA repair, or metabolic changes by regulating the expression of target genes in response to various cellular stresses. Mutations within this gene are associated with various human cancers, including hereditary cancers such as Li-Fraumeni syndrome. TP53 mutations are ubiquitous across multiple cancers. Deficiencies in tumor suppressors are most often mediated by large deletion events such as frameshift mutations or early stop codons. However, it has been found that many mutations observed in cancer in TP53 are single-nucleotide missense mutations. While these mutations are widely distributed throughout the gene, the majority are localized within the DNA-binding domain. There is no single hotspot within the DNA-binding domain, and the majority of mutations occur at amino acid positions 175, 245, 248, 273, and 282 (NM_000546). While a large proportion of cancer genome research focuses on somatic mutations, TP53 is also being considered in the germline. Germline TP53 mutations are characteristic of Li-Fraumeni syndrome, and numerous mutations (both germline and somatic) have been shown to have a prognostic impact on patient outcomes. TP53 acts as a tumor suppressor in many tumor types by inducing growth arrest or apoptosis depending on the physiological environment and cell type. TP53 is involved in cell cycle regulation as a transactivator that negatively controls the process by regulating a set of genes necessary for cell division. One of the genes activated is a cyclin-dependent kinase inhibitor. Apoptosis induction appears to occur either by stimulating the expression of BAX and FAS antigens or by suppressing Bcl-2 expression. In cooperation with mitochondrial PPIF, TP53 is involved in the activation of oxidative stress-induced necrosis, although this function is largely unrelated to transcription. TP53 induces the transcription of long-chain intergeneric noncoding RNA-p21 (lincRNA-p21) and lincRNA-Mkln1. lincRNA-p21 appears to be involved in TP53-dependent transcriptional repression that triggers apoptosis and thus has an effect on cell cycle regulation.TP53 is thought to be involved in Notch signaling cross-reactivity. In response to DNA damage, TP53 binds to the CAK complex, inhibiting CDK7 kinase activity and thus halting cell cycle progression. TP53 isoform 2 enhances the transactivation activity of isoform 1 from some, but not all, TP53-inducible promoters. TP53 isoform 4 inhibits transactivation activity and impairs isoform 1-mediated proliferation inhibition. TP53 isoform 7 inhibits isoform 1-mediated apoptosis. TP53 regulates the circadian clock by repressing CLOCK-ARNTL / BMAL1-mediated transcriptional activation of PER2 (Miki et al., Nat Commun, 2013, Vol. 4: p. 2444). In some embodiments, the human TP53 protein has 393 amino acids and a molecular weight of 43653 Da. Known coupling partners for TP53 include AXIN1, ING4, YWHAZ, HIPK1, HIPK2, WWOX, GRK5, ANKRD2, RFFL, RNF34, and TP53INP1.

[0114] The term "TP53" is considered to include its fragments, variants (e.g., allele variants), and derivatives. Representative human TP53 cDNA sequences and human TP53 protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, at least 12 different human TP53 isoforms are known. Human TP53 isoform a (NP_000537.3, NP_001119584.1) can be encoded by transcript variant 1 (NM_000546.5) and transcript variant 2 (NM_001126112.2). Human TP53 isoform b (NP_001119586.1) can be encoded by transcript variant 3 (NM_001126114.2). Human TP53 isoform c (NP_001119585.1) can be encoded by transcript variant 4 (NM_001126113.2). Human TP53 isoform d (NP_001119587.1) can be encoded by transcript variant 5 (NM_001126115.1). Human TP53 isoform e (NP_001119588.1) can be encoded by transcript variant 6 (NM_001126116.1). Human TP53 isoform f (NP_001119589.1) can be encoded by transcript variant 7 (NM_001126117.1). Human TP53 isoform g (NP_001119590.1, NP_001263689.1, and NP_001263690.1) can be encoded by transcript variant 8 (NM_001126118.1), transcript variant 1 (NM_001276760.1), and transcript variant 2 (NM_001276761.1). Human TP53 isoform h (NP_001263624.1) can be encoded by transcript variant 4 (NM_001276695.1). Human TP53 isoform i (NP_001263625.1) can be encoded by transcript variant 3 (NM_001276696.1). The human TP53 isoform j (NP_001263626.1) can be encoded by transcript variant 5 (NM_001276697.1).Human TP53 isoform k (NP_001263627.1) can be encoded by transcript variant 6 (NM_001276698.1). Human TP53 isoform l (NP_001263628.1) can be encoded by transcript variant 7 (NM_001276699.1). The nucleic acid and polypeptide sequences of TP53 orthologs in non-human organisms are well known, for example, chimpanzee TP53 (XM_001172077.5 and XP_001172077.2, and XM_016931470.2 and XP_016786959.2), monkey TP53 (NM_001047151.2 and NP_001040616.1), dog TP53 (NM_001003210.1 and NP_001003210.1), camel TP53 (NM_174201.2 and NP_776626.1), mouse TP53 (NM_001127233.1 and N This includes P_001120705.1, and NM_011640.3 and NP_035770.2), rat TP53 (NM_030989.3 and NP_112251.2), tropical clawed frog TP53 (NM_001001903.1 and NP_001001903.1), and zebrafish TP53 (NM_001271820.1 and NP_001258749.1, NM_001328587.1 and NP_001315516.1, NM_001328588.1 and NP_001315517.1, and NM_131327.2 and NP_571402.1). Representative sequences of TP53 orthologs are shown in Table 1 below.

[0115] Several anti-TP53 antibodies suitable for detecting the TP53 protein are well known in the art, including, for example, antibodies such as TA502925 and CF502924 (Origene), NB200-103 and NB200-171 (Novus Biologicals, Littleton, Colorado), ab26 and ab1101 (AbCam, Cambridge, Massachusetts), 700439 (ThermoFisher Scientific), and 33-856 (ProSci). Reagents for detecting TP53 are also well known. Several clinical tests for TP53 are available in the NIH Genetic Testing Registry (GTR®) (e.g., GTR test ID: GTR000517320.2 provided by Fulgent Clinical Diagnostics Laboratory (Temple City, California)). Furthermore, several siRNA constructs, shRNA constructs, and CRISPR constructs for reducing TP53 expression can be found in the product lists of the companies referenced above, including Santa Cruz Biotechnology's siRNA product numbers sc-29435 and sc-44218, CRISPR product numbers sc-416469, RNAi products SR322075 and TL320558V, and CRISPR product KN200003 (Origene), as well as several CRISPR products from GenScript (Piscataway, New Jersey). Chemical inhibitors of TP53, including cyclic biphytrin-α-hydrobromide, RITA (TOCRIS, Minnesota), etc., are also available. It should be noted that this term may be further used to refer to any combination of characteristics of the TP53 molecule described herein. For example, any combination of sequence composition, percentage specificity, sequence length, domain structure, functional activity, etc., can be used to describe the TP53 molecule that falls within the scope of the present invention. A known and clear correspondence exists between the amino acid sequence of a particular protein and the nucleotide sequence that can encode that protein, as defined by the genetic code (see below). Similarly, a known and clear correspondence exists between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0116] [Table 4]

[0117] An important and well-known feature of the genetic code is its redundancy, which allows for the use of one more nucleotide triplet codes (as exemplified above) for most of the amino acids used to construct proteins. Therefore, numerous different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they produce the same amino acid sequence in all organisms (although some organisms may translate one sequence more efficiently than others). Furthermore, sometimes methylation variants of purines or pyrimidines can be found within a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0118] Considering the foregoing, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to obtain a polypeptide amino acid sequence using the genetic code for translating that DNA or RNA into an amino acid sequence. Similarly, for the amino acid sequence of a polypeptide, the corresponding nucleotide sequence that can encode that polypeptide can be inferred from the genetic code (due to the redundancy of the genetic code, multiple nucleic acid sequences are generated for any given amino acid sequence). Therefore, the description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include the description and / or disclosure of the amino acid sequence encoded by that nucleotide sequence. Similarly, the description and / or disclosure herein of an amino acid sequence of a polypeptide should be considered to also include the description and / or disclosure of all possible nucleotide sequences that can encode that amino acid sequence.

[0119] Finally, the nucleic acid sequence information and amino acid sequence information of the gene loci and biomarkers included in the scope of the present invention, as well as related biomarkers (e.g., the biomarkers listed in Tables 1 and 2), are well known in the art and readily available from public databases such as the National Center for Biotechnology Information (NCBI). For example, the following are examples of nucleic acid sequences and amino acid sequences obtained from public sequence databases.

[0120] Table 1 [Table 1]

[0121] Sequence ID 1: Human Smad2 transcript mutant 2 mRNA sequence (NM_001003652.4, CDS: 127~1530) [ka] [ka] [ka]

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0122] Nucleic acid molecules containing a DNA-binding domain coding region having a nucleic acid sequence of any of the sequence numbers listed in Table 1, or a nucleic acid sequence having identity of at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher percentages of the full length, are included in Table 1. Such nucleic acid molecules may encode polypeptides having the function of full-length polypeptides as further described herein.

[0123] Polypeptide molecules containing a DNA-binding domain having an amino acid sequence of any of the sequence numbers listed in Table 1, or an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher percentage identity to the full length, are included in Table 1. Such polypeptides may have the functions of full-length polypeptides as further described herein.

[0124] Table 2 [Table 2]

[0125] Sequence ID 177: Human Smad6 cDNA sequence (NM_005585.5, CDS: 1024~2514) [ka] [ka] [ka] Sequence ID 178: Human Smad6 amino acid sequence (NP_005576.3) [ka] Sequence ID 179: Mouse Smad6 cDNA sequence (NM_008542.3, CDS: 1036~2523) [ka] [ka] [ka] Sequence ID 180: Mouse Smad6 amino acid sequence (NP_032568.3) [ka] [ka] Sequence ID 181: Human Smad7 transcript mutant 1 cDNA sequence (NM_005904.3, CDS:288~1568) [ka] [ka] [ka] Sequence ID 182: Human Smad7 isoform 1 amino acid sequence (NP_005895.1) [ka] Sequence ID 183: Human Smad7 transcript mutant 2 cDNA sequence (NM_001190821.1, CDS:288~1565) [ka] [ka] [ka] Sequence ID 184: Human Smad7 isoform 2 amino acid sequence (NP_001177750.1) [ka] Sequence ID 185: Human Smad7 transcript mutant 3 cDNA sequence (NM_001190822.2, CDS: 138~773) [ka] [ka] [ka] Sequence ID 186: Human Smad7 isoform 3 amino acid sequence (NP_001177751.1) [ka] Sequence ID 187: Human Smad7 transcript mutant 4 cDNA sequence (NM_001190823.1, CDS: 150~866) [ka] [ka] [ka] Sequence ID 188: Human Smad7 isoform 4 amino acid sequence (NP_001177752.1) [ka] Sequence ID 189: Mouse Smad7 cDNA sequence (NM_001042660.1, CDS: 1592~2872) [ka] [ka] [ka] [ka] Sequence ID 190: Mouse Smad7 amino acid sequence (NP_001036125.1) [ka]

[0126] Nucleic acid molecules containing a DNA-binding domain coding region having a nucleic acid sequence of any of the sequence numbers listed in Table 2, or a nucleic acid sequence having identity of at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher percentages of the full length, are included in Table 2. Such nucleic acid molecules may encode polypeptides having the function of full-length polypeptides as further described herein.

[0127] Polypeptide molecules containing a DNA-binding domain having an amino acid sequence of any of the sequence numbers listed in Table 2, or an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher percentage identity to the full length, are included in Table 2. Such polypeptides may have the functions of full-length polypeptides as further described herein.

[0128] II. Cancer Vaccines The present invention provides a cancer vaccine comprising cancer cells, wherein the cancer cells are (1) PTEN-deficient, (2) p53-deficient, and (3) modified to activate the TGFβ-Smad / p63 signaling pathway. The cancer cells may be derived from solid tumors or hematological malignancies (e.g., breast cancer). In certain embodiments, the breast cancer cells are triple-negative breast cancer (TNBC). In one embodiment, the cancer cells are derived from a subject. For example, the cancer cells may be derived from breast cancer resulting from co-deficiency of p53 and PTEN. In another embodiment, the cancer cells are derived from a cancer cell line. The cancer cells may be derived from any cancer cell line or primary cancer cells. For example, the cancer cells may be derived from a cell line selected from the group consisting of HCC1954 cells, SUM149 cells, BxPC-3 cells, T3M4 cells, 143B cells, A549 cells, H520 cells, H23 cells, HaCaT cells, H357 cells, H400 cells, Detroit cells, OKF6 cells, BICR6 cells, H103 cells, 5PT cells, JHU12 cells, JHU22 cells, HSC3 cells, SCC25 cells, and NTERT cells. The cancer cells may have various types of additional genetic mutations. The cancer cells may be derived from the subject treated with the cancer vaccine. The cancer cells may be derived from a different subject not treated with the cancer vaccine. The cancer cells may be derived from the same type of cancer as the cancer treated with the cancer vaccine. The cancer cells may be derived from a different type of cancer than the cancer treated with the cancer vaccine. The cancer cells may originate from a cancer having the same genetic mutation as the cancer treated with the cancer vaccine. Alternatively, the cancer cells may originate from a cancer having a different genetic mutation than the cancer treated with the cancer vaccine.

[0129] Isolation and purification of cancer cells In some embodiments, the cancer cells are derived from the subject. Isolation and purification of tumor cells from various tumor tissues, such as surgically resected tumor tissue, ascites, or malignant pleural effusion, is a common process for obtaining purified tumor cells. Cancer cells can be purified from fresh biopsy samples from cancer patients or animal tumor models. These biopsy samples often contain heterogeneous cell populations including normal tissue, blood, and cancer cells. The purified cancer cell composition is preferably able to contain all viable cancer cells in a percentage of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher, or any range between these, or any value above these. Numerous methods are available for purifying cancer cells from heterogeneous populations.

[0130] In one embodiment, laser microdissection is used for the isolation of cancer cells. The target cancer cells can be carefully excised from tissue sections prepared for microscopic observation. In this method, the tissue section is covered with a plastic film, and a focused near-infrared laser pulse is irradiated onto the area containing the selected cells. This causes small circles to dissolve within the plastic film, with the cells bound to them. These captured cells are then removed for further analysis. This technique is a good technique for separating and analyzing cells from various parts of a tumor, thereby comparing the similar and different characteristics of those cells. In recent years, this technique has been used for the analysis of pituitary cells derived from excised tissue as well as heterogeneous cultured populations of pituitary cells, thyroid cells, and carcinoid tumor cells, as well as for the analysis of individual cells found in various sarcomas.

[0131] In another embodiment, fluorescence-activated cell sorting (FACS), also known as flow cytometry, is used for sorting and analyzing various cell populations. Cells possessing a target cell marker or other specific markers are tagged with an antibody, or typically a mixture of antibodies that bind to those cell markers. Each antibody against a different marker is conjugated against a detectable molecule, particularly a fluorescent dye that can be distinguished from other fluorescent dyes bound to other antibodies. A stream of tagged or "stained" cells passes through a light source that excites its fluorochrome, and the presence of a specific labeled antibody is determined by the emission spectrum from those cells detected. Simultaneous detection of different fluorochromes, also known in the art as multicolor fluorescence cell sorting, can identify cells exhibiting different sets of cell markers and isolate them from other cells in the population. Other FACS parameters, including, but not limited to, side scatter (SSC), forward scatter (FSC), and live cell dye staining (e.g., staining with propidium iodide), enable cell selection based on size and viability. FACS sorting and analysis of HSCs and related lineage cells are well known in the art, for example, U.S. Patent No. 5,137809, U.S. Patent No. 5750397, and Specification No. 5840580, Specification No. 6465249, Manz et al., (202) Proc. Natl. Acad. Sci. USA, Vol. 99: pp. 11872-11877, and Akashi et al., (200) Nature, Vol. 404: pp. 193-197. General guidelines for fluorescence-activated cell selection are described, for example, in Shapiro, (2003) Practical Flow Cytometry, 4th edition, Wiley-Liss (2003), and Ormerod, (2000) Flow Cytometry: A Practical Approach, 3rd edition, Oxford University Press.

[0132] Another method for isolating useful cell populations involves solid or insoluble substrates to which antibodies or ligands interacting with specific cell surface markers are bound. In immunoadsorption, cells are brought into contact with a substrate containing the antibody (e.g., bead columns, flasks, magnetic particles, etc.), and any unbound cells are removed. Immunoadsorption can be scaled up to directly process large numbers of cells collected in clinical settings. Suitable substrates include, but are not limited to, plastics, cellulose, dextran, polyacrylamide, agarose, and others known in the art (e.g., Pharmacia Sepharose 6MB macrobeads). When solid substrates containing magnetic or paramagnetic beads are used, cells bound to those beads can be easily isolated by magnetic separators (see, e.g., Kato and Radbruch, (1993) Cytometry, Vol. 14: pp. 384-392). Cell separation by affinity chromatography typically involves passing a cell suspension through a support carrying a selection ligand immobilized on its surface. The ligand interacts with its specific target molecule on the cell and is captured on its matrix. The bound cell is released by adding an eluent to the column's running buffer, and the free cell is washed off the column and collected as a homogeneous population. As will be apparent to those skilled in the art, adsorption methods are not limited to techniques using specific antibodies, and nonspecific adsorption may be utilized. For example, adsorption to silica is a simple method for removing phagocytic cells from cell preparations. One of the most common applications of this technique is the isolation of circulating tumor cells (CTCs) from the blood of patients with breast cancer, NSC lung cancer, prostate cancer, and colon cancer using antibodies against EpCAM, a cell surface glycoprotein known to be frequently expressed in epithelial cancers.

[0133] FACS and most batch immunoadsorption methods can be applied to both positive and negative screening (see, for example, U.S. Patent No. 5,877,299). In positive screening, desired cells are labeled with an antibody and isolated from the remaining unlabeled / unwanted cells. In negative screening, unwanted cells are labeled and removed. Another type of negative screening that may be used is the use of antibody / complement treatment or immunotoxin to remove unwanted cells.

[0134] In yet another embodiment, microfluidics, one of the latest technologies, is used for cancer cell isolation. This method uses a microfluidics chip with a helical channel that can isolate circulating tumor cells (CTCs) from blood based on size. When a blood sample is pumped into the device and cells flow through the channel at high speed, inertia and centrifugal force cause smaller cells to flow along the outer wall and larger cells, including CTCs, to flow along the inner wall. Researchers have used this chip technology to isolate CTCs from the blood of patients with metastatic lung or breast cancer.

[0135] According to a recently published paper (Lin et al., Small (2015), Vol. 11: pp. 4394-4402), fluorescent nanodiamonds (FNDs) can be used to label and isolate low-proliferation / quiescent cancer stem cells. The authors of the study stated that fluorescent nanodiamonds were difficult to isolate and track over the long term using traditional fluorescent markers. They concluded that FNDs do not cause nanoparticle DNA damage or impair cell proliferation and offer superior long-term tracking capabilities compared to EdU and CFSE fluorescent labeling.

[0136] It should be understood that cell purification or isolation may also include combinations of the above methods. A typical combination may include in the first method a method effective in removing many unwanted cells and cellular material. The second step may include isolating cells expressing a marker common to one or more of the progenitor cell populations by immunoadsorption onto an antibody bound to a substrate. Additional steps that provide a variety of cell types with higher resolution, such as FACS sorting using antibodies against a set of specific cell markers, may be used to obtain a substantially pure population of the desired cells.

[0137] Modification and alteration of cancer cells The cancer cells contained in the aforementioned cancer vaccine are PTEN-deficient and p53-deficient. In some embodiments, the cancer cells are PTEN-deficient and p53-deficient due to genetic mutations acquired by the cancer cells during cancer transformation or progression. In some other embodiments, the cancer cells are PTEN-deficient and p53-deficient by being modified with agents that reduce the copy number, quantity, and / or activity of PTEN and / or p53.

[0138] The agents that reduce the copy number, quantity, and / or activity of PTEN and / or p53 may be small molecule inhibitors, CRISPR guide RNA (gRNA), RNA interference agents, antisense oligonucleotides, peptide inhibitors or peptide mimeograph inhibitors, aptamers, antibodies, or intracellular antibodies.

[0139] In one embodiment, peptides or peptide mimes can be used to weaken the activity of PTEN and / or p53. In one embodiment, variants of PTEN and / or p53 that function as regulators for their respective full-length proteins can be identified by screening a combinatorial library of variants, e.g., truncated variants, for antagonist activity. In one embodiment, a diversified variant library is created by combinatorial mutagenesis at the nucleic acid level, and the diversified variant library is encoded by a diversified gene library. A diversified variant library can be created, for example, by enzymatically linking a mixture of synthetic oligonucleotides to a gene sequence so that possible polypeptide sequences of a certain degenerate set can be expressed as individual polypeptides containing the polypeptide sequences of that set. Various methods exist that can be used to create a library of polypeptide variants from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed using an automated DNA synthesizer, after which the synthetic gene is ligated into a suitable expression vector. By using a degenerate gene set, all of those sequences encoding the possible polypeptide sequences of the desired set are provided as a single mixture. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang, SA, Tetrahedron, Vol. 39:3, 1983; Itakura et al., Annu. Rev. Biochem., Vol. 53:323, 1984; Itakura et al., Science, Vol. 198:1056, 1984; and Ike et al., Nucleic Acid Res., Vol. 11:477, 1983).

[0140] Furthermore, libraries of polypeptide coding sequence fragments can be used to create diversified polypeptide fragment populations for screening and subsequent selection of variants of a given polypeptide. In one embodiment, a library of coding sequence fragments can be prepared by treating double-strand PCR fragments of polypeptide coding sequences with a nuclease under conditions where nick formation occurs approximately once per polypeptide, thereby denaturing the double-strand DNA; regenerating the DNA to form double-strand DNA that may contain sense / antisense pairs from different nick-containing products; removing single-strand portions from the regenerated double-strands by treatment with an S1 nuclease; and ligating the resulting fragment library into an expression vector. This method allows for the derivation of expression libraries encoding N-terminal, C-terminal, and internal fragments of the polypeptide of various sizes.

[0141] Several techniques are known in the art for screening gene products of combinatorial libraries created by point mutation or truncation, and for screening cDNA libraries for gene products having selected characteristics. Such techniques are applicable to rapid screening of gene libraries created by combinatorial mutagenesis of polypeptides. The most widely used techniques suitable for screening large gene libraries and applicable to high-throughput analysis typically involve cloning the gene library into a replicable expression vector, transforming suitable cells with the resulting vector library, and expressing the combinatorial genes under conditions that facilitate the isolation of the vector encoding the gene whose product is detected by detecting the desired activity. Recurrent ensemble mutagenesis (REM), a technique for increasing the frequency of functional mutants in the library, can be used in conjunction with the screening assay to identify the target mutant (Arkin and Youvan, (1992) Proc. Natl. Acad. Sci. USA, Vol. 89: pp. 7811-7815; Delagrave et al., (1993) Protein Eng., Vol. 6 (No. 3): pp. 327-331). In one embodiment, a cell-based assay can be used for the analysis of a diversified polypeptide library. For example, an expression vector library can be transfused into a cell line that routinely synthesizes PTEN and / or p53. Subsequently, full-length polypeptides and specific mutant polypeptides are produced, and these transfused cells are cultured so that the effect of the expression of the mutant on the full-length polypeptide activity in the cell supernatant can be detected by, for example, one of a number of functional assays. Plasmid DNA can then be recovered from cells that scored for inhibition or enhancement of full-length polypeptide activity, and the characteristics of individual clones can be further analyzed.

[0142] It is possible to create more stable peptides by systematically substituting one or more amino acids in the polypeptide amino acid sequence with the same type of D-amino acid (e.g., D-lysine instead of L-lysine). Furthermore, restrictive peptides containing the target polypeptide amino acid sequence or substantially identical sequence mutations can be created by methods known in the art (Rizo and Gierasch, (1992) Annu. Rev. Biochem., Vol. 61: 387; incorporated herein by reference), for example, by adding internal cysteine ​​residues that can form intramolecular disulfide crosslinks that make the peptide cyclic.

[0143] From the amino acid sequences disclosed herein, those skilled in the art can construct peptide sequences corresponding to polypeptides and their sequence variants. Such polypeptides can be produced in prokaryotic or eukaryotic host cells by the expression of a polynucleotide encoding the peptide sequence, often as part of a larger polypeptide. Alternatively, such peptides can be synthesized by chemical methods. Methods for the expression of heterologous proteins in recombinant hosts, methods for the chemical synthesis of polypeptides, and methods for in vitro translation are well known in this art, and include Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd edition, Cold Spring Harbor, New York; Berger and Kimmel, Methods in Enzymology, Vol. 152; Guide to Molecular Cloning Techniques (1987), Academic Press, San Diego, California; Merrifield, J., (1969) J. Am. Chem. Soc., Vol. 91:501; Chaiken IM, (1981) CRC Crit. Rev. Biochem., Vol. 11:255; Kaiser et al., (1989) Science, Vol. 243:187; Merrifield, Further discussion is provided in B., (1986) Science, Vol. 232: p. 342; Kent, SBH, (1988) Annu. Rev. Biochem., Vol. 57: p. 957; and Offord, RE, (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference.

[0144] Peptides can typically be prepared by direct chemical synthesis. Peptides can be prepared as modified peptides having a non-peptide moiety covalently attached to the N-terminus and / or C-terminus. In certain preferred embodiments, either or both of the carboxyl or amino terminus are chemically modified. The most common modifications of terminal amino and carboxyl groups are acetylation and amidation, respectively. Other terminal modifications, including amino-terminus modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation), carboxyl-terminus modifications such as amidation, and cyclization, can be invoked in various embodiments of the present invention. Certain amino-terminus modifications and / or carboxyl-terminus modifications and / or peptide elongation to the core sequence may provide advantageous physical, chemical, biochemical, and pharmacological properties, such as increased properties, stability, potency and / or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others. The peptides disclosed herein can be used for therapeutic purposes, for example, to treat diseases by altering co-stimulation in patients.

[0145] Peptide mimes (Fauchere, (1986) Adv. Drug Res., Vol. 15: p. 29; Veber and Freidinger, (1985) TINS, p. 392; and Evans et al., (1987) J. Med. Chem., Vol. 30: p. 1229; incorporated herein by reference) are typically developed with the assistance of computer molecular modeling. Peptide mimes that are structurally similar to therapeutically useful peptides can be used to produce equivalent therapeutic or prophylactic effects. Generally, peptide mimes are structurally similar to typical polypeptides (i.e., polypeptides with biological or pharmacological activity), but are known in the art and are incorporated herein by reference as follows: "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," edited by Weinstein, B., Marcel Decker Ltd., New York, p. 267, (1983), in which Spatola, AF; Spatola, AF, (1983), Vega Data Vol. 1, No. 3, "Peptide Backbone Modifications" (review), by Morley, JS, (1980), Trends Pharm. Sci., pp. 463-468 (review); Hudson, D. et al., (1979), Int. J. Pept. Prot. Res., Vol. 14: pp. 177-185 (-CH2NH-, CH2CH2-), Spatola, AF et al. (1986) Life Sci., Vol. 38: pp. 1243-1249 (-CH2-S), Hann, MM, (1982) J. Chem. Soc. Perkin Trans. I., pp. 307-314 (-CH=CH-, cis and trans), Almquist, RG et al. (1980) J. Med. Chem., Vol. 23: 1392-1398 (-COCH2-), Jennings-White, C. et al. (1982) Tetrahedron Lett. Magazine, Volume 23: 2533 (-COCH2-), Szelke, M. et al. (1982) European Appln.It has one or more peptide bonds which are optionally substituted by bonds selected from the group consisting of -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, as further described in EP 45665 CA:, Vol. 97: pp. 39405 (-CH(OH)CH2-), Holladay, MW et al., (1983) Tetrahedron Lett., Vol. 24: pp. 4401-4404 (-C(OH)CH2-), and Hruby, VJ, (1982) Life Sci., Vol. 31: pp. 189-199 (-CH2-S-). A particularly preferred non-peptide bond is -CH2NH-. Such peptide mimes may have significant advantages over polypeptide embodiments, including, for example, higher productivity, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, etc.), altered specificity (e.g., broader biological activity), reduced antigenicity, and others. Labeling of peptide mimes typically involves the direct or spacer-mediated covalent bonding of one or more labels to non-interfering positions on the peptide mime, as predicted by quantitative structure-activity data and / or molecular modeling. Such non-interfering positions are generally those that do not directly contact the macropolypeptide to which the peptide mime binds in order to produce a therapeutic effect. Derivatization (e.g., labeling) of the peptide mime should not substantially interfere with the desired biological or pharmacological activity of the peptide mime.

[0146] Small molecules capable of modulating (e.g., inhibiting) the activity of PTEN and / or p53, or their interactions with their natural binding partners, are also included in the scope of the present invention. These small molecules of the present invention can be obtained using any of the numerous approaches to combinatorial library methods known in the art, including spatially addressable parallel solid-phase or liquid-phase libraries, synthetic library methods requiring deconvolution, "one bead, one compound" library methods, and synthetic library methods using affinity chromatography selection (Lam, KS, (1997) Anticancer Drug Des., Vol. 12: p. 145).

[0147] Examples of methods for molecular library synthesis in this art include, for example, DeWitt et al., (1993) Proc. Natl. Acad. Sci. USA, Vol. 90: 6909; Erb et al., (1994) Proc. Natl. Acad. Sci. USA, Vol. 91: 11422; Zuckermann et al., (1994) J. Med. Chem., Vol. 37: 2678; Cho et al., (1993) Science, Vol. 261: 1303; Carrell et al., (1994) Angew. Chem. Int. Ed. Engl., Vol. 33: 2059; and Carell et al., (1994) Angew. Chem. Int. Ed. This can be found in the English journal, Vol. 33, p. 2061, and in Gallop et al., (1994) J. Med. Chem., Vol. 37, p. 1233.

[0148] Compound libraries can be placed in solution (e.g., Houghten, Biotechniques (1992), Vol. 13: pp. 412-421), on beads (Lam, Nature (1991), Vol. 354: pp. 82-84), on chips (Fodor, Nature (1993), Vol. 364: pp. 555-556), on bacteria (Ladner U.S. Patent No. 5223409), on spores (Ladner U.S. Patent No. 5223409), or on plasmids (Cull et al., Proc. Natl. Acad. Sci. (1992)). The compounds can be presented as follows: (USA, Vol. 89: pp. 1865-1869), or on phages (Scott and Smith, (1990) Science, Vol. 249: pp. 386-390), (Devlin, (1990) Science, Vol. 249: pp. 404-406), (Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA, Vol. 87: pp. 6378-6382), (Felici, (1991) J. Mol. Biol., Vol. 222: pp. 301-310), (Ladner, above). The compounds can be screened using cell-based or non-cell-based assays. The compounds can be screened collectively (e.g., multiple compounds per sample) or individually.

[0149] At least one, two, three, four, five, ten, twenty, or more types of short-chain nucleic acids or antisense oligonucleotides or derivatives thereof, which, under cellular conditions, are cellular nucleic acids (e.g., miRNA, pre-miRNA, pri-miRNA, miRNA *Compositions comprising one or more nucleic acids in cells that contain or can express short-chain nucleic acids or antisense oligonucleotides or derivatives thereof that specifically hybridize (e.g., bind) to (short-chain non-coding RNAs such as anti-miRNA, miRNA-binding sites, their variants or functional variants, cellular mRNA, or fragments thereof). In one embodiment, the expression of the short-chain nucleic acids or antisense oligonucleotides or derivatives thereof in cells can inhibit the expression or biological activity of cellular nucleic acids and / or proteins by inhibiting transcription, translation and / or short-chain nucleic acid processing, such as PTEN and / or p53. In one embodiment, the short-chain nucleic acids or antisense oligonucleotides or derivatives thereof are short-chain RNA (e.g., microRNA) or complements of short-chain RNA. In another embodiment, the short-chain nucleic acid or antisense oligonucleotide or derivative thereof may be single-stranded or double-stranded, and be at least 6 nucleotides long, and less than about 1000 nucleotides long, less than about 900 nucleotides long, less than about 800 nucleotides long, less than about 700 nucleotides long, less than about 600 nucleotides long, less than about 500 nucleotides long, less than about 400 nucleotides long, less than about 300 nucleotides long, less than about 200 nucleotides long, less than about 100 nucleotides long, less than about 50 nucleotides long, less than about 40 nucleotides long, less than about 30 nucleotides long, less than about 25 nucleotides long, less than about 24 nucleotides long, less than about 23 nucleotides long, less than about 22 nucleotides long, less than about 21 nucleotides long, less than about 20 nucleotides long, less than about 19 nucleotides long, less than about 18 nucleotides long, less than about 17 nucleotides long, less than about 16 nucleotides long, less than about 15 nucleotides long, or less than about 10 nucleotides long. In another embodiment, the composition may include a short-chain nucleic acid or antisense oligonucleotide or derivative thereof, or a library of nucleic acids that include or can express a pool of such short-chain nucleic acids or antisense oligonucleotides or derivatives thereof. The nucleic acid pool may contain about 2 to 5, 5 to 10, 10 to 20, 10 to 30, or more nucleic acids that include or can express short-chain nucleic acids or antisense oligonucleotides or derivatives thereof.

[0150] In one embodiment, binding may be by conventional base-pair complementarity, or, for example, in the case of binding to a DNA double helix, it may be via specific interactions within the major groove of the double helix. Generally, "antisense" refers to a range of techniques commonly used in the art and includes any process that relies on specific binding to an oligonucleotide sequence.

[0151] It is well known in the art that miRNA or pre-miRNA sequences can be modified without impairing miRNA activity. As used herein, the term “functional variant” of a miRNA sequence refers to an oligonucleotide sequence that is altered from a native miRNA sequence but retains one or more of the miRNA’s functional characteristics (e.g., inhibition of cancer cell proliferation, induction of cancer cell apoptosis, enhancement of cancer cell sensitivity to chemotherapeutic agents, or inhibition of specific miRNA targets). In some embodiments, the functional variant of a miRNA sequence retains all of the miRNA’s functional characteristics. In a particular embodiment, the functional variant of the miRNA is approximately 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 30 bases, 35 bases, 40 bases, 45 bases, 50 bases, 55 bases, 60 bases, 65 bases, 70 bases, 75 bases, 80 bases, 85 bases, and 90 bases. The functional variant has a nucleic acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the miRNA or its precursor over a region of about 95, about 100, or more nucleic acid bases, or the functional variant hybridizes to the complement of the miRNA or its precursor under stringent hybridization conditions. Thus, in a particular embodiment, the nucleic acid sequence of the functional variant is hybridizable to one or more target sequences of the miRNA.

[0152] The microRNAs and their corresponding stem-loop sequences described herein may be found in miRBase, an online searchable database of miRNA sequences and annotations located on the World Wide Web at microrna.sanger.ac.uk. Entries in the miRBase sequence database indicate the predicted hairpin portion (stem-loop) of a miRNA transcript, along with information about the location and sequence of its mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs); in some cases, they may include pre-miRNAs derived from the predicted early transcript and some adjacent sequences. The miRNA nucleic acid sequences described herein encompass all types of that miRNA, including sequences listed in miRBase sequence database release 10.0 and any earlier releases of the miRBase sequence database. Certain miRNAs may be renamed depending on the sequence database release. Variations of mature miRNA sequences may occur depending on the sequence database release.

[0153] In some embodiments, the miRNA sequence of the present invention may be bound to a second RNA sequence that may be located on the same RNA molecule as the miRNA sequence or on another RNA molecule. In such cases, the miRNA sequence may be called the active strand, while the second RNA sequence, which is at least partially complementary to the miRNA sequence, may be called the complementary strand. The active and complementary strands hybridize to produce a double-stranded RNA similar to a natural miRNA precursor. The activity of the miRNA can be optimized by maximizing the uptake of the active strand and minimizing the uptake of the complementary strand by the miRNA protein complex that controls gene translation. This optimization can be achieved by modifying and / or designing the complementary strand.

[0154] In some embodiments, the complementary chain is modified to have a chemical group other than a phosphate or hydroxyl group at its 5' end. The presence of this 5' modification clearly excludes the uptake of the complementary chain and subsequently supports the uptake of the active chain by the miRNA protein complex. The 5' modification can be any of the various molecules known in the art, including NH2, NHCOCH3, and biotin.

[0155] In another embodiment, the uptake of the complementary strand by the miRNA pathway is reduced by incorporating sugar-modified nucleotides into the first 2-6 nucleotides of the complementary strand. Note that such sugar modifications can be combined with the 5'-end modifications described above to further enhance miRNA activity.

[0156] In some embodiments, the complementary strand is designed such that the nucleotides at its 3' end are not complementary to the active strand. As a result, a double-stranded hybrid RNA is produced that is stable at the 3' end of the active strand but relatively unstable at its 5' end. This difference in stability suppresses the uptake of the complementary strand by the miRNA pathway while increasing the uptake of the active strand, thereby enhancing miRNA activity.

[0157] The short-chain nucleic acids and / or antisense constructs of the methods and compositions presented herein may be delivered, for example, as expression plasmids that, when transcribed within a cell, produce RNA that is complementary to at least a specific portion of cellular nucleic acids (e.g., short-chain RNA, mRNA, and / or genomic DNA). Alternatively, these short-chain nucleic acid molecules may be mRNA, miRNA, pre-miRNA, pri-miRNA, miRNA *RNAs encoding anti-miRNAs, miRNA-binding sites, or variants thereof may be produced. For example, the selection of plasmids suitable for the expression of these miRNAs, methods for inserting nucleic acid sequences into the plasmids, and methods for delivering the recombinant plasmids to target cells are within the scope of this art. For example, the entire disclosures incorporated herein by reference are: Zeng et al., (2002) Mol. Cell, Vol. 9: pp. 1327-1333; Tuschl, (2002) Nat. Biotechnol., Vol. 20: pp. 446-448; Brummelkamp et al., (2002) Science, Vol. 296: pp. 550-553; Miyagishi et al., (2002) Nat. Biotechnol., Vol. 20: pp. 497-500; Paddison et al., (2002) Genes Dev., Vol. 16: pp. 948-958; Lee et al., (2002) Nat. Biotechnol., Vol. 20: pp. 500-505; and Paul et al., (2002) Nat. See Biotechnol., Vol. 20, pp. 505-508.

[0158] Alternatively, the short-chain nucleic acid and / or antisense construct is an oligonucleotide probe that is prepared ex vivo and hybridizes with cellular nucleic acids upon introduction into cells. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, such as exonucleases and / or endonucleases, and are therefore stable in vivo. Examples of nucleic acid molecules used as short-chain nucleic acids and / or antisense oligonucleotides include phosphoramidate analogs, phosphothioate analogs, and methylphosphonate analogs of DNA (see also U.S. Patents No. 5,176,996, 5,264,564, and 5,256,775). Furthermore, general approaches to constructing oligomers useful for antisense therapy are outlined, for example, by Van der Krol et al., BioTechniques (1988), Vol. 6: pp. 958-976, and by Stein et al., Cancer Res (1988), Vol. 48: pp. 2659-2668.

[0159] The antisense approach may involve designing oligonucleotides (either DNA or RNA) that are complementary to cellular nucleic acids (e.g., complementary to the PTEN gene and / or the p53 gene). Absolute complementarity is not required. Therefore, in the case of double-stranded antisense nucleic acids, one strand of the double-stranded DNA may be examined, and triple helix formation may be analyzed. Hybridization ability depends on both the degree of complementarity and the length of the antisense nucleic acid. The longer the nucleic acid to hybridize, the more mismatches it may contain with other nucleic acids (e.g., RNA), yet it may still form a stable double (or possibly triple) helix. Those skilled in the art can determine an acceptable degree of mismatch by using standard methods for determining the melting point of the hybridized complex.

[0160] Oligonucleotides complementary to the 5' untranslated sequence of the mRNA, including the 5' end, for example, up to the AUG start codon, should most efficiently inhibit translation. However, it has recently been shown that sequences complementary to the 3' untranslated sequence of the mRNA are equally effective in inhibiting mRNA translation (Wagner, Nature (1994), Vol. 372: p. 333). Therefore, oligonucleotides complementary to either the 5' untranslated non-coding region or the 3' untranslated non-coding region of a gene may be used in antisense approaches to inhibit the translation of endogenous mRNA. The oligonucleotide complementary to the 5' untranslated region of the mRNA may include a complement to the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region are not as efficient translation inhibitors, but they may also be used in accordance with the methods and compositions presented herein. Whether designed to hybridize to the 5' region, 3' region, or coding region of cellular mRNA, short nucleic acids, and / or antisense nucleic acids, it should be at least 6 nucleotides long and may be less than approximately 1000 nucleotides, less than approximately 900 nucleotides, less than approximately 800 nucleotides, less than approximately 700 nucleotides, less than approximately 600 nucleotides, less than approximately 500 nucleotides, less than approximately 400 nucleotides, less than approximately 300 nucleotides, less than approximately 200 nucleotides, less than approximately 100 nucleotides, less than approximately 50 nucleotides, less than approximately 40 nucleotides, less than approximately 30 nucleotides, less than approximately 25 nucleotides, less than approximately 24 nucleotides, less than approximately 23 nucleotides, less than approximately 22 nucleotides, less than approximately 21 nucleotides, less than approximately 20 nucleotides, less than approximately 19 nucleotides, less than approximately 18 nucleotides, less than approximately 17 nucleotides, less than approximately 16 nucleotides, less than approximately 15 nucleotides, or less than approximately 10 nucleotides.

[0161] To quantify the gene expression inhibitory ability of antisense oligonucleotides, it is preferable to first perform in vitro tests, regardless of the selection of the target sequence. In one embodiment, these tests utilize a control that distinguishes antisense gene inhibition from nonspecific biological effects of the oligonucleotide. In another embodiment, these tests compare the level of the target nucleic acid or protein with the level of an internal control nucleic acid or protein. It is also envisioned that the results obtained using the control oligonucleotide will be compared with those obtained using the antisense oligonucleotide. It is preferable that the control oligonucleotide is approximately the same length as the test oligonucleotide, and that the nucleotide sequence of the oligonucleotide is not more different from the antisense sequence than necessary to interfere with specific hybridization to the target sequence.

[0162] Short-chain nucleic acids and / or antisense oligonucleotides may be DNA or RNA or chimeric mixtures thereof or derivatives thereof or modified forms thereof, and may be single-stranded or double-stranded. Short-chain nucleic acids and / or antisense oligonucleotides may have modifications to their base moieties, sugar moieties, or phosphate backbone to improve the stability or hybridization of the molecule, for example, other accompanying groups such as peptides (for example, to target receptors on host cells), cell membranes (e.g., Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA, Vol. 86: pp. 6553-6556, Lemaitre et al., (1987) Proc. Natl. Acad. Sci. The formulation may include agents that facilitate transport across the blood-brain barrier (see, for example, the Journal of USA, Vol. 84: pp. 648-652, PCT International Publication No. 88 / 09810), hybridization-inducing cleavage agents (see, for example, Krol et al., BioTech. (1988), Vol. 6: pp. 958-976), or insertors (see, for example, Zon, Pharm. Res. (1988), Vol. 5: pp. 539-549). For this purpose, short-chain nucleic acids and / or antisense oligonucleotides may be complexed with other molecules, such as peptides, hybridization-inducing crosslinking agents, transport factors, hybridization-inducing cleavage agents, etc.

[0163] Short-chain nucleic acids and / or antisense oligonucleotides include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxytriethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl quosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil It may contain at least one modified base moiety selected from the group comprising: 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylquosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), weybutoxosin, pseudouracil, quosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Short-chain nucleic acids and / or antisense oligonucleotides may also include, but are not limited to, at least one modified sugar moiety selected from the group including arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0164] In certain embodiments, the compound comprises an oligonucleotide (e.g., miRNA or an oligonucleotide encoding miRNA) that is complexed with one or more moieties that improve the activity, intracellular distribution, or intracellular uptake of the oligonucleotide. In certain such embodiments, the moieties are cholesterol moieties (e.g., antagonistic acid), lipid moieties, or liposome complexes. Other moieties for complexation include carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In certain embodiments, the complex group is directly attached to the oligonucleotide. In certain embodiments, the oligonucleotide is compounded by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturated group (e.g., double or triple bond), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In certain such embodiments, substituents are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0165] In certain such embodiments, the compound comprises an oligonucleotide having one or more stabilizing groups attached to one or both ends of the oligonucleotide to improve properties such as nuclease stability. Examples of stabilizing groups include cap structures. These end modifications may protect the oligonucleotide from exonuclease degradation and aid in delivery and / or intracellular localization. The cap may be present at the 5' end (5' cap) or the 3' end (3' cap), or at both ends. Examples of cap structures include inverted deoxydecay caps.

[0166] Appropriate cap structures include 4',5'-methylene nucleotide, 1-(β-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, α-nucleotide, modified base nucleotide, phosphorodithioate bond, threopentofuranosyl nucleotide, acyclic 3',4'-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted debase moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted debase moiety, 1,4-butanediol phosphate, and 3'-phosphoramide. Examples include phosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, cross-linked methylphosphonate moiety and non-cross-linked methylphosphonate moiety, 5'-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 5'-5'-inverted nucleotide moiety, 5'-5'-inverted debase moiety, 5'-phosphoramidate, 5'-phosphorothioate, 5'-amino, cross-linked and / or non-cross-linked 5'-phosphoramidate, phosphorothioate, and 5'-mercapto moiety.

[0167] Short-chain nucleic acids and / or antisense oligonucleotides may also contain a neutral peptide-like backbone. Such molecules are called peptide nucleic acid (PNA) oligomers and are described, for example, in Perry-O'Keefe et al., (1996) Proc. Natl. Acad. Sci. USA, Vol. 93: pp. 14670, and Eglom et al., (1993) Nature, Vol. 365: pp. 566. One advantage of PNA oligomers is their ability to bind complementaryly to the medium, essentially independent of the ionic strength, because their DNA backbone is neutral. In yet another embodiment, the short-chain nucleic acids and / or antisense oligonucleotides contain at least one modified phosphate backbone selected from the group consisting of phosphorothioates, phosphorodithioates, phosphoramidothioates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkylphosphotryesters, and formacetals or analogs thereof.

[0168] In other embodiments, the short-chain nucleic acids and / or antisense oligonucleotides are α-anomeric oligonucleotides. α-anomeric oligonucleotides form a specific double-stranded hybrid with complementary RNA, in contrast to the usual β-unit, where the strands extend parallel to each other (Gautier et al., Nucl. Acids Res., Vol. 15: pp. 6625-6641, 1987). These oligonucleotides are either 2'-O-methylribonucleotides (Inoue et al., Nucl. Acids Res., Vol. 15: pp. 6131-6148, 1987) or chimeric RNA-DNA analogs (Inoue et al., FEBS Lett., Vol. 215: pp. 327-330, 1987).

[0169] Short-chain nucleic acids and / or antisense oligonucleotides of the methods and compositions presented herein can be synthesized by standard methods known in the art, such as using automated DNA synthesizers (commercially available from Biosearch, Applied Biosystems, etc.). Examples include the synthesis of phosphorothioate oligonucleotides by the method described by Stein et al., (1988) Nucl. Acids Res., Vol. 16: p. 3209, and the preparation of methylphosphonate oligonucleotides using controlled porous glass polymer supports (Sarin et al., (1988) Proc. Natl. Acad. Sci. USA, Vol. 85: pp. 7448-7451). For example, miRNA isolates can be chemically synthesized or recombinantly produced using methods known in the art. In some examples, miRNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. Examples of commercial suppliers of synthetic RNA molecules or synthetic reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colorado, USA), Pierce Chemical (part of Perbio Science, Rockford, Illinois, USA), Glen Research (Sterling, Virginia, USA), ChemGenes (Ashland, Massachusetts, USA), Cruachem (Glasgow, UK), and Exiqon (Vedbeck, Denmark).

[0170] Short-chain nucleic acids and / or antisense oligonucleotides can be delivered to cells in vivo. Numerous methods have been developed to deliver short-chain nucleic acids and / or antisense oligonucleotides (DNA or RNA) to cells. For example, antisense molecules can be injected directly into tissue sites, or modified antisense molecules designed to target desired cells (e.g., antisense linked to peptides or antibodies that specifically bind to receptors or antigens expressed on the surface of target cells) can be administered systemically.

[0171] In one embodiment, short nucleic acids and / or antisense oligonucleotides may comprise, or may arise from, double-stranded small interfering RNAs (siRNAs) that, when expressed in a cell, establish degradation by a sequence that is fully complementary to the cellular nucleic acid (e.g., mRNA) or establish translational repression by a sequence that is incompletely complementary to the cellular nucleic acid (e.g., mRNA). In another embodiment, double-stranded siRNAs may be processed into single-stranded antisense RNAs that bind to single-stranded cellular RNAs (e.g., microRNAs) and inhibit their expression. RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing process in animals and plants initiated by double-stranded RNA (dsRNA) whose sequence is homologous to the gene to be silenced. In vivo, long dsRNAs are cleaved by ribonuclease III to produce 21-nucleotide and 22-nucleotide siRNAs. 21-nucleotide siRNA double helix has been shown to specifically suppress the expression of endogenous heterogeneity genes in various mammalian cell lines, including human embryonic kidney (293) cells and HeLa cells (Elbashir et al., Nature, Vol. 411, pp. 494-498, 2001). Therefore, intracellular gene translation can be inhibited by contacting the cell with short double-stranded RNA having a length of approximately 15-30 nucleotides, 18-21 nucleotides, or 19-21 nucleotides. Alternatively, vectors encoding such siRNA or short hairpin RNA (shRNA) that are metabolized into siRNA can be introduced into target cells (see, for example, McManus et al., RNA, Vol. 8, p. 842, Xia et al., Nature Biotechnology, Vol. 20, p. 1006, and Brummelkamp et al., Science, Vol. 296, p. 550, 2002). Available vectors are commercially available, for example, under the name pSuperRNAi System™ by OligoEngine.

[0172] Ribozyme molecules designed to catalytically cleave cellular mRNA transcripts can also be used to interfere with the translation of cellular mRNA and / or the expression of cellular polypeptides (see, for example, PCT International Publication No. 90 / 11364, published October 4, 1990, by Sarver et al., Science (1990), Vol. 247: pp. 1222-1225, and U.S. Patent No. 5093246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used for the disruption of cellular mRNA, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNA at a position defined by an adjacent region that forms a complementary base pair with the target mRNA. The only requirement is that the target mRNA has the following two-base sequence, namely 5'-UG-3'. The construction and preparation of hammerhead ribozymes are well known in this art and are further described in detail in Haseloff and Gerlach, (1988) Nature, Vol. 334: pp. 585-591. Ribozymes can be modified so that the cleavage recognition site is located near the 5' end of cellular mRNA, that is, to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts.

[0173] The ribozymes of the methods presented herein include RNA endoribonucleases (hereinafter referred to as "Cech-type ribozymes") such as the ribozyme (known as IVS or L-19 IVS RNA) that occurs naturally in Tetrahymena thermophylla and has been extensively described by Thomas Cech and his collaborators (Zaug et al., (1984) Science, Vol. 224: pp. 574-578; Zaug et al., (1986) Science, Vol. 231: pp. 470-475; Zaug et al., (1986) Nature, Vol. 324: pp. 429-433, International Publication No. 88 / 04300; and Been et al., (1986) Cell, Vol. 47: pp. 207-216). Cech-type ribozymes have an 8-base pair active site that hybridizes to a target RNA sequence and subsequently causes cleavage of the target RNA. The methods and compositions presented herein include these Cech-type ribozymes that target 8-base pair active site sequences present in cellular genes.

[0174] Like antisense approaches, the ribozyme may be composed of modified oligonucleotides (for example, to improve stability, targeting, etc.). A preferred delivery method involves using a DNA construct "encoding" the ribozyme under the control of a potent constitutive promoter, such as the pol III promoter or the pol II promoter, so that the transfected cell produces a sufficient amount of the ribozyme to disrupt endogenous cellular messages and inhibit translation. Unlike antisense molecules, ribozymes are catalytic, and therefore require lower intracellular concentrations to be efficient.

[0175] Nucleic acid molecules used in the formation of a triple helix to inhibit the transcription of cellular genes are preferably single-stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides promotes triple helix formation via Hougsteen-type base pairing rules, which generally require a fairly large elongation of either purine or pyrimidine on one of the double helices. The nucleotide sequence may be pyrimidine-based, so that TAT triplets and CGC triplets are present across the three associated strands of the resulting triple helix. These high-pyrimidine molecules achieve base complementarity with the high-purine region on one of the double helices, oriented parallel to that strand. Alternatively, high-purine nucleic acid molecules, such as those containing G residue elongation regions, may be selected. These molecules form a triple helix with a GC-pair-rich DNA double helix, in which the majority of purine residues are located on one of the targeted double helices, so that CGC triplets are present across the three strands of the triple helix.

[0176] Alternatively, the number of sequences that could potentially be targets for triple helix formation can be increased by creating so-called "switchback" nucleic acid molecules. Switchback molecules are synthesized alternately from 5' to 3' and 3' to 5' so that they base-pair with the first strand of the double helix and then with the other strand, thereby eliminating the need for a significant extension of either purine or pyrimidine to be present on one of the strands of the double helix.

[0177] Short-chain nucleic acids (e.g., miRNA, pre-miRNA, pri-miRNA, miRNA) of the methods and compositions presented herein. *Antisense RNA molecules (anti-miRNA, or miRNA-binding sites, or variants thereof), antisense oligonucleotides, ribozymes, and triple helix molecules can be prepared by any method known in the art for synthesizing DNA and RNA molecules. These methods include, for example, well-known chemical synthesis techniques for oligodeoxyribonucleotides and oligoribonucleotides in the art, such as solid-phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecules. Such DNA sequences can be incorporated into a variety of vectors incorporating appropriate RNA polymerase promoters, such as the T7 polymerase promoter or the SP6 polymerase promoter. Alternatively, antisense cDNA constructs that constitutively or inductively synthesize antisense RNA depending on the promoter used can be stably introduced into cell lines.

[0178] Furthermore, various well-known modifications to nucleic acid molecules can be introduced as means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and / or 3' ends of the molecule, or the use of phosphorothioates or 2'O-methyl molecules rather than phosphodiesterase binding within the oligodeoxyribonucleotide backbone. Those skilled in the art will readily understand that polypeptides, short-chain nucleic acids, and antisense oligonucleotides can be further bound to other peptides or polypeptides (e.g., heterologous peptides) that serve, for example, as means of protein detection. Non-limiting examples of labeled peptide or labeled polypeptide moieties useful for detection in the present invention include, but are not limited to, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; epitope tags such as FLAG tags, MYC tags, HA tags, or HIS tags; fluorophores such as green fluorescent protein; dyes; radioisotopes; digoxigenin; biotin; antibodies; polymers; and other known in the art, such as those found in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (ed.), Plenum Press, 2nd edition (July 1999).

[0179] The present invention also considers well-known methods for genetically modifying the genome of an organism or cell such that, once the genetic modification is complete, the expression and / or activity of PTEN and / or p53 are altered without contact with a drug. For example, cancer cells can be genetically modified using recombinant techniques to regulate the expression and / or activity of PTEN and / or p53 without the need to contact the cancer cells with a drug to regulate the expression and / or activity of PTEN and / or p53. For example, targeted gene knockout or non-targeted gene knockout methods can be used to genetically modify target cancer cells ex vivo before intravenous infusion. For example, target DNA in the genome can be manipulated by retroviral insertion, artificial chromosome techniques, gene insertion, deletion, insertion, and / or mutation using tissue-specific promoters, gene targeting, transposable elements, and / or any other method for introducing exogenous DNA or creating modified DNA / modified nuclear DNA. Other modification techniques include deletion of DNA sequences from the genome and / or modification of nuclear DNA sequences. For example, nuclear DNA sequences can be modified by site-directed mutagenesis. Such methods generally utilize host cells into which the recombinant expression vector of the present invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to specific target cells but also to the progeny cells or potentially progeny cells of such cells. Such progeny may not actually be identical to the parent cells because certain changes may occur in the offspring due to either mutation or environmental influences, but they are still included within the scope of the term as used herein. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or translocation techniques. As used herein, the terms “transformation” and “translocation” refer to various techniques understood in the art for introducing exogenous nucleic acids into host cells, including co-precipitation with calcium phosphate or calcium chloride, DEAE dextran-mediated translocation, lipofection, or electroporation.Appropriate methods for transforming or translocating host cells can be found in Sambrook et al. (cited above) and other experimental manuals. Stable translocation of mammalian cells is known to result in only a small percentage of cells incorporating the exogenous DNA into their genome, depending on the expression vector and translocation technique used. To identify and select these insertions, it is common practice to introduce a gene encoding a selection marker (e.g., a marker for antibiotic resistance) into the host cell along with the target gene. Preferred selection markers include drug resistance markers such as G418, hygromycin, and methotrexate. Cells that have stably translocated the introduced nucleic acid can be identified by drug selection (e.g., cells incorporating the selection marker gene survive, while other cells die).

[0180] Similarly, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g., for the creation of null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes may be expressed. For example, a vector containing only this guide RNA may be administered to animals or cells genetically modified with the Cas9 enzyme. Similar strategies (e.g., designer zinc fingers, activator-like effectors (TALEs), or homing meganucleases) may be used. Such systems are well known in the art (for example, U.S. Patent No. 8,697,359, by Sander and Joung, (2014) Nat. Biotech., Vol. 32: pp. 347-355; Hale et al., (2009) Cell, Vol. 139: pp. 945-956; Karginov and Hannon, (2010) Mol. Cell, Vol. 37: p. 7; U.S. Patent Application Publication No. 2014 / 0087426 and No. 2012 / 0178169, by Boch et al., (2011) Nat. Biotech., Vol. 29: pp. 135-136, Boch et al., (2009) Science, Vol. 326: pp. 1509-1512, Moscow and Bogdanove, (2009) Science, Vol. 326: p. 1501, Weber et al., (2011) PLoS One, Vol. 6: p. 19722, Li et al., (2011) Nucl. Acids Res., Vol. 39: pp. 6315-6325, Zhang et al., (2011) Nat. Biotech., Vol. 29: pp. 149-153, Miller et al., (2011) Nat. Biotech., Vol. 29: pp. 143-148, Lin et al., (2014) Nucl. Acids See Res., Vol. 42: e47). Such genetic strategies can be implemented using constitutive or inducible expression systems according to methods well known in the art.

[0181] In some embodiments, the cancer cells do not replicate. In certain embodiments, the cancer cells do not replicate due to irradiation (e.g., gamma and / or UV irradiation) and / or administration of agents that render cell replication ineffective (e.g., cell membrane disruptors, DNA replication inhibitors, inhibitors of spindle formation during cell division, etc.). Typically, a minimum dose of about 3,500 rad is sufficient, but doses up to about 30,000 rad are acceptable. In some embodiments, sublethal doses of irradiation may be used. For example, the cancer cells may be irradiated to suppress cell proliferation before administration of the cancer vaccine to reduce the risk of new neoplastic lesions developing. It should be understood that irradiation is only one method of making cells non-replicating, and that other methods that render cancer cells unable to divide but retain the ability to induce anti-tumor immunity when the TGFβ-Smad / p63 signaling pathway is activated are also included in the present invention.

[0182] Drugs that activate the TGFβ-Smad / p63 signaling pathway This specification demonstrates that activation of the TGFβ-Smad / p63 axis in cancer cells promotes the immune response and ultimately controls the expression of multiple pathways that promote cytotoxic T cell activation and immunological memory. Accordingly, the cancer cells included in the scope of the invention as described herein are modified to activate the TGFβ-Smad / p63 signaling pathway. In one embodiment, the cancer cells are contacted with TGFβ superfamily proteins to activate the TGFβ-Smad / p63 signaling pathway. In another embodiment, the cancer cells are contacted with modifiers of the copy number, expression, and / or activity of one or more biomarkers listed in Table 1 that can activate the TGFβ-Smad / p63 signaling pathway. The cancer cells (e.g., cancer cell lines or tumor tissue) can be cultured in vitro or ex vivo in 2D or 3D (e.g., as tumor spheres or tumor organoids).

[0183] In some embodiments, the cancer vaccine comprising the modified cancer cells described herein may be tested for certain desirable characteristics or functions before administration to a subject. In one embodiment, deficiencies in PTEN and p53 are confirmed in the modified cancer cells. In another embodiment, activation of the TGFβ-Smad / p63 signaling pathway is detected in the modified cancer cells. In yet another embodiment, the modified cancer cells have the following characteristics, namely: A decrease in the growth rate in either the 2D culture system or the 3D culture system. Activation of the TGFβ-Smad / p63 signature, e.g., upregulation of ICOSL, PYCARD, SFN, PERP, RIPK3, CASP9, and / or SESN1, and / or downregulation of KSR1, EIF4EBP1, ITGA5, EMILIN1, CD200, and / or CSF1. Upregulation of one or more dendritic cell (DC) activation markers, including but not limited to CD40, CD80, CD86, CD8, HLA-DR, IL1β, and / or T cell activation in the presence of dendritic cells, such as increased secretion of TNFα and / or IFNγ by T cells in the presence of dendritic cells. One or more of these will be tested.

[0184] i. TGFβ superfamily proteins In one embodiment, PTEN and p53-deficient cancer cells described herein are contacted with TGFβ superfamily proteins to activate the TGFβ-Smad / p63 signaling pathway. These TGFβ superfamily proteins may be any member of the TGFβ superfamily capable of activating the TGFβ-Smad / p63 signaling pathway. The TGFβ superfamily proteins may be derived from the TGFβ family, which includes, but is not limited to, LAP, TGFβ1, TGFβ2, TGFβ3, and TGFβ5. The TGFβ superfamily proteins may also be derived from the activin family, which includes, but is not limited to, activin A, activin AB, activin AC, activin B, activin C, C17ORF99, INHBA, INHBB, inhibin, inhibin A, and inhibin B. The TGFβ superfamily protein may be derived from the BMP (bone morphogenetic protein) family, and may be derived from BMP-1 / PCP, BMP-2, BMP-2 / BMP-6 heterodimer, BMP-2 / BMP-7 heterodimer, BMP-2a, BMP-3, BMP-3b / GDF-10, BMP-4, BMP-4 / BMP-7 heterodimer, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-15 / GDF-9B, and decapentaplesic / DPP. The TGFβ superfamily protein may be derived from the GDNF family, and may be derived from artemin, GDNF, neurturin, and parcefin. The TGFβ superfamily proteins may be derived from families other than those described above, including but not limited to Lefty A, Lefty B, MIS / AMH, Nordal, and SCUBE3. In certain embodiments, the TGFβ superfamily proteins are TGFβ1, TGFβ2, and / or TGFβ3. In one embodiment, the cancer cells are brought into contact with a single TGFβ superfamily protein (e.g., TGFβ1, TGFβ2, or TGFβ3).In another embodiment, the cancer cells are brought into contact with a combination of TGFβ superfamily proteins (for example, a combination of TGFβ1, TGFβ2, and TGFβ3).

[0185] The cancer cells may be contacted with TGFβ superfamily proteins in vitro, in vivo, and / or ex vivo. In one embodiment, the cancer cells are contacted with TGFβ superfamily proteins in vitro or ex vivo, and then the cancer cells are administered to the subject without the TGFβ superfamily proteins being administered in vivo. In another embodiment, the cancer cells are administered to the subject, and then the TGFβ superfamily proteins are administered to the subject to contact the cancer cells in vivo. In yet another embodiment, the cancer cells are contacted with TGFβ superfamily proteins in vitro or ex vivo, and then the TGFβ superfamily proteins are administered in vivo to the subject, and then the cancer cells are administered to the subject. The TGFβ superfamily proteins may be administered to the subject before, after, and / or simultaneously with the administration of the cancer cells. In some embodiments, the cancer cells are contacted with TGFβ superfamily proteins in vitro, in vivo, and / or ex vivo in combination with an immune checkpoint inhibitor. The subjects may be administered an immune checkpoint inhibitor before, after, and / or concurrently with the administration of the cancer vaccine.

[0186] The dosage of the TGFβ superfamily protein may be varied to obtain the degree of TGFβ-Smad / p63 signaling pathway activation that is effective in achieving the desired therapeutic response and remaining non-toxic to the patient for a particular patient, composition, and mode of administration.

[0187] The selected dosage level will depend on a variety of factors, including the activity of the specific TGFβ superfamily protein used, the specific type of cancer cells being contacted, the route of administration, the time of administration, the efflux or metabolic rate of the specific TGFβ superfamily protein used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the specific TGFβ superfamily protein used, the age, body weight, condition, general health status, and medical history of the patient being treated with the cancer vaccine, and similar factors well known in the medical field.

[0188] In some embodiments, the cancer cells are exposed to TGFβ superfamily proteins at doses greater than 0.1 ng / ml, for example, greater than 0.2 ng / ml, greater than 0.3 ng / ml, greater than 0.4 ng / ml, greater than 0.5 ng / ml, greater than 0.6 ng / ml, greater than 0.7 ng / ml, greater than 0.8 ng / ml, greater than 0.9 ng / ml, greater than 1 ng / ml, greater than 1.5 ng / ml, greater than 2 ng / ml, greater than 2.5 ng / ml, greater than 3 ng / ml, greater than 3.5 ng / ml, greater than 4 ng / ml, greater than 4.5 ng / ml, greater than 5 ng / ml, greater than 5.5 ng / ml, greater than 6 ng / ml, greater than 6.5 ng / ml, greater than 7 ng / ml, greater than 7.5 ng / ml, greater than 8 ng / ml, greater than 8.5 ng / ml, greater than 9 ng / ml, greater than 9.5 ng / ml, greater than 10 ng / ml, etc.

[0189] In some embodiments, the cancer cells are exposed to TGFβ superfamily proteins in doses ranging from approximately 0.1 ng / ml to approximately 100 ng / ml. In preferred embodiments, the cancer cells are exposed to TGFβ superfamily proteins in doses ranging from approximately 1 ng / ml to approximately 10 ng / ml, for example, approximately 1 ng / ml, 1.5 ng / ml, 2 ng / ml, 2.5 ng / ml, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.5 ng / ml, 6 ng / ml, 6.5 ng / ml, 7 ng / ml, 7.5 ng / ml, 8 ng / ml, 8.5 ng / ml, 9 ng / ml, 9.5 ng / ml, or 10 ng / ml, or any value in between.

[0190] In some embodiments, the cancer cells are exposed to TGFβ superfamily proteins for a certain period of time. This period can range from a few minutes to four weeks and may be, for example, 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 36 hours, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days or any value in between. The preferred range for that period is approximately 6 hours to 21 days, approximately 12 hours to 15 days, approximately 1 day to 10 days, or approximately 3 days to 7 days.

[0191] ii. Agents that increase the copy number, quantity, and / or activity of at least one biomarker listed in Table 1. In another embodiment, the PTNE and p53-deficient cancer cells described herein are exposed to modifiers of the copy number, expression, and / or activity of one or more biomarkers listed in Table 1, thereby activating the TGFβ-Smad / p63 signaling pathway. Agents that increase the copy number, expression, and / or activity of one or more biomarkers listed in Table 1 can do so directly or indirectly.

[0192] Useful agents in methods within the scope of the present invention include antibodies, small molecules, peptides, peptide mimes, natural ligands, derivatives of natural ligands, etc., that can bind to and / or modulate one or more biomarkers or fragments thereof listed in Table 1, and RNA interference, antisense, nucleic acid aptamers, nucleic acids, polypeptides, etc., that can increase the expression and / or activity of one or more biomarkers or fragments thereof listed in Table 1.

[0193] In one embodiment, an isolated nucleic acid molecule that specifically hybridizes with or encodes one or more biomarkers listed in Table 1 or their biologically active portions. As used herein, the term “nucleic acid molecule” encompasses DNA molecules (i.e., cDNA or genomic DNA) and RNA molecules (i.e., mRNA) and DNA or RNA analogs created using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. An “isolated” nucleic acid molecule is a nucleic acid molecule that has been separated from other nucleic acid molecules present in the natural origin of the nucleic acid. It is preferable that an “isolated” nucleic acid does not contain sequences that are naturally adjacent to the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (i.e., sequences located at the 5' end and 3' end of the nucleic acid). For example, isolated nucleic acid molecules corresponding to one or more biomarkers listed in Table 1 in various embodiments may contain nucleotide sequences of less than approximately 5 kb, less than approximately 4 kb, less than approximately 3 kb, less than approximately 2 kb, less than approximately 1 kb, less than approximately 0.5 kb, or less than approximately 0.1 kb that are naturally adjacent to the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates (i.e., lymphoma cell). Furthermore, “isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0194] Nucleic acid molecules that fall within the scope of the present invention, for example, nucleic acid molecules having a nucleotide sequence that is at least about 50%, preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% (e.g., about 98%) homologous to the nucleotide sequences of one or more biomarkers listed in Table 1 or a portion thereof (i.e., 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 450 nucleotides, 500 nucleotides, or more nucleotides) listed in Table 1, can be isolated using standard molecular biology techniques and sequence information provided herein. For example, human cDNA can be isolated from human cell lines (from Stratagene, La Jolla, California or Clontech, Palo Alto, California) using the whole or a portion of its nucleic acid molecule or fragment as a hybridization probe and standard hybridization techniques (i.e., hybridization techniques as described in Sambrook, J., Fritsh, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Furthermore, nucleic acid molecules comprising all or part of a nucleotide sequence that is at least about 50% homologous, preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% homologous, to one or more biomarkers listed in Table 1, or a nucleotide sequence or fragment thereof, can be isolated by polymerase chain reaction using one or more biomarkers listed in Table 1 or fragment thereof, or oligonucleotide primers designed based on the homologous nucleotide sequence.For example, mRNA can be isolated from muscle cells (i.e., by the guanidine thiocyanate extraction method described in Chirgwin et al., Biochemistry (1979), Vol. 18: pp. 5294-5299), and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase available from Gibco / BRL, Bethesda, Maryland, or AMV reverse transcriptase available from Seikagaku America, St. Petersburg, Florida). Synthetic oligonucleotide primers for PCR amplification can be designed according to methods well known in the art. Nucleic acids included in the scope of this invention can be amplified using cDNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The thus amplified nucleic acids can be cloned into appropriate vectors and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to the nucleotide sequences of one or more biomarkers listed in Table 1 can be prepared by standard synthetic techniques, i.e., using an automated DNA synthesizer.

[0195] Probes based on the nucleotide sequences of one or more biomarkers listed in Table 1 may be used to detect or confirm a desired transcript or genomic sequence encoding the same or homologous protein. In a preferred embodiment, the probe further comprises a labeling group attached thereto, i.e., the labeling group may be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used as part of a diagnostic test kit to identify cells or tissues expressing one or more biomarkers listed in Table 1 by measuring the nucleic acid levels of one or more biomarkers listed in Table 1 in a cell sample from a target, i.e., by detecting the mRNA levels of one or more biomarkers listed in Table 1.

[0196] Nucleic acid molecules encoding proteins corresponding to one or more biomarkers listed in Table 1, derived from various species, have also been considered. For example, rat or monkey cDNA can be identified based on human nucleotide sequences and / or mouse sequences, and such sequences are well known in the art. In one embodiment, the nucleic acid molecule included in the scope of the present invention is a protein or moiety containing an amino acid sequence that is sufficiently homologous to regulate (e.g., enhance) one or more of the following biological activities: (a) binding to the biomarker, (b) regulating the copy number of the biomarker, (c) regulating the expression level of the biomarker, and (d) regulating the activity level of the biomarker, wherein the protein or moiety contains an amino acid sequence that is sufficiently homologous to the amino acid sequences of one or more biomarkers listed in Table 1.

[0197] As used herein, the term “sufficiently homologous” means a protein or portion thereof having an amino acid sequence containing identical or equivalent amino acid residues to such an extent that one or more of the following biological activities are regulated (e.g., enhanced): (a) binding to the biomarker, (b) regulating the copy number of the biomarker, (c) regulating the expression level of the biomarker, and (d) regulating the activity level of the biomarker, wherein the protein or portion thereof has an amino acid sequence containing the minimum number of amino acid residues (e.g., amino acid residues having the same side chain as the amino acid residues in the biomarker or portion thereof) that are identical or equivalent to the amino acid sequence of one or more biomarkers or fragments of the biomarker listed in Table 1.

[0198] In another embodiment, the protein is homologous to the entire amino acid sequence of the biomarker or fragment thereof by at least about 30%, preferably at least about 60%, more preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a higher percentage.

[0199] The protein portion encoded by the nucleic acid molecules of one or more biomarkers listed in Table 1 is preferably the biologically active portion of that protein. As used herein, the term "biologically active portion" of one or more biomarkers listed in Table 1 includes one or more portions of the full-length protein having one or more biological activities, such as domains / motifs.

[0200] Standard binding assays, such as immunoprecipitation and yeast two-hybrid assays, or functional assays, such as RNAi or overexpression experiments, as described herein may be performed to determine the ability of the protein or its biologically active fragments to maintain the biological activity of the full-length protein.

[0201] The present invention further encompasses nucleic acid molecules that, unlike the nucleotide sequences or fragments thereof of one or more biomarkers listed in Table 1 due to the degeneracy of the genetic code, encode the same protein as the protein encoded by said nucleotide sequence or fragment. In another embodiment, an isolated nucleic acid molecule included in the scope of the present invention has a nucleotide sequence encoding a protein having the amino acid sequence of one or more biomarkers or fragments thereof listed in Table 1, or a nucleotide sequence encoding a protein having an amino acid sequence homologous to the amino acid sequence of one or more biomarkers or fragments thereof listed in Table 1 by at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In another embodiment, the nucleic acid encoding the polypeptide consists of a nucleic acid sequence encoding a portion of a target full-length fragment having an amino acid length of less than 195, less than 190, less than 185, less than 180, less than 175, less than 170, less than 165, less than 160, less than 155, less than 150, less than 145, less than 140, less than 135, less than 130, less than 125, less than 120, less than 115, less than 110, less than 105, less than 100, less than 95, less than 90, less than 85, less than 80, less than 75, or less than 70.

[0202] Those skilled in the art will understand that DNA sequence polymorphisms leading to changes in the amino acid sequence of one or more biomarkers listed in Table 1 may exist within a population (e.g., a mammalian population and / or a human population). Such genetic polymorphisms may exist among individuals within a population due to natural allele variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules containing an open reading frame that encode one or more biomarkers listed in Table 1, preferably mammalian proteins, e.g., human proteins. Such natural allele variation typically results in a 1-5% mismatch in the nucleotide sequence of one or more biomarkers listed in Table 1. All such nucleotide variation and the resulting amino acid polymorphisms within one or more biomarkers listed in Table 1 that are the result of natural allele variation and do not alter the functional activity of one or more biomarkers listed in Table 1 are considered to be within the scope of the present invention. Furthermore, nucleic acid molecules encoding proteins of one or more biomarkers listed in Table 1 from other species.

[0203] In addition to the natural allele variants of one or more biomarkers listed in Table 1 that may exist in the aforementioned population, those skilled in the art will further understand that mutations can introduce changes within the nucleotide sequence or fragment thereof, thereby causing changes in the amino acid sequence of one or more encoded biomarkers listed in Table 1 without altering the functional capacity of the one or more biomarkers listed in Table 1. For example, nucleotide substitutions causing amino acid substitutions at "non-essential" amino acid residues can be made within the sequence or fragment thereof. "Non-essential" amino acid residues are those that can be altered from the wild-type sequence of one or more biomarkers listed in Table 1 without altering the activity of the one or more biomarkers listed in Table 1, while "essential" amino acid residues are necessary for the activity of one or more biomarkers listed in Table 1. However, other amino acid residues (e.g., amino acid residues that are not conserved or are only semi-conserved between mouse and human) may not be essential for activity and therefore may be altered without altering the activity of one or more biomarkers listed in Table 1.

[0204] The term "sequence identity or sequence homology" refers to the similarity of sequences between two polypeptide molecules or two nucleic acid molecules. When certain positions in both of the two sequences being compared are occupied by the same base monomer subunit or amino acid monomer subunit, for example, when certain positions in each of two DNA molecules are occupied by adenine, those molecules are homologous or sequence-identical at that position. The percentage of homology or sequence identity between two sequences is a function of dividing the number of matching or homologous identical positions shared by the two sequences by the number of positions being compared and multiplying by 100. For example, if 6 out of 10 positions in two sequences are the same, those two sequences have 60% homology or 60% sequence identity. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. It is common practice to compare two sequences when they are aligned to give maximum homology. Unless otherwise specified, "loop-out regions," such as those resulting from the deletion or insertion of one of the sequences, are counted as mismatches.

[0205] Sequence comparison and determination of percentage homology between two sequences can be achieved using mathematical algorithms. It is preferable to perform alignment using the Clustal method. Multiple alignment parameters include a gap penalty of 10 and a gap length penalty of 10. For DNA alignment, pairwise alignment parameters may be H-tuple = 2, gap penalty = 5, window = 4, and diagonal-saved = 4. For protein alignment, pairwise alignment parameters may be K-tuple = 1, gap penalty = 3, window = 5, and diagonal-saved = 5.

[0206] In a preferred embodiment, the percentage identity between two amino acid sequences is determined using either the Blossom62 matrix or the PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6, using the Needleman and Wunsch algorithm (J. Mol. Biol. (Vol. 48): pp. 444-453 (1970)) incorporated into the GAP program in the GCG software package (available online). In yet another preferred embodiment, the percentage identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix, with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6, using the GAP program in the GCG software package (available online). In another embodiment, the percentage identity between two amino acid sequences or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, Vol. 4: pp. 11-17 (1989)), which is incorporated into the ALIGN program (version 2.0) (available online).

[0207] Isolated nucleic acid molecules encoding proteins homologous to one or more biomarkers or fragments thereof listed in Table 1 can be produced by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence, fragment thereof, or homologous nucleotide sequence such that one or more amino acid substitutions, additions, or deletions are introduced into the encoding protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. It is preferable that conservative amino acid substitutions be performed at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been clearly defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable that the predicted non-essential amino acid residues in one or more biomarkers listed in Table 1 be replaced with other amino acid residues from the same side chain family. Alternatively, in another embodiment, mutations can be randomly introduced into all or part of the coding sequences of one or more biomarkers listed in Table 1 by saturation mutagenesis or the like, and the resulting mutants can be screened for the activity described herein to identify mutants that retain the desired activity. After mutagenesis, the encoded protein can be expressed by recombinant technology according to methods well known in the art, and the activity of the protein can be determined, for example, using assays described herein.

[0208] The levels of one or more biomarkers listed in Table 1 can be assessed by any of the various well-known methods for detecting the expression of the transcribed molecules or proteins. Non-exclusive examples of such methods include immunological methods for protein detection, protein purification methods, protein function assays or protein activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0209] In preferred embodiments, the levels of one or more biomarkers listed in Table 1 are confirmed by measuring gene transcripts (e.g., mRNA), the amount of translated protein, or the activity of gene products. Expression levels can be monitored by various methods, including mRNA detection, protein detection, or protein activity detection, all of which are measurable using standard techniques. Detection may involve quantification of gene expression levels (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or it may be a qualitative assessment of gene expression levels, particularly a qualitative assessment of gene expression levels compared to control levels. The type of level detected will be evident from the background.

[0210] In certain embodiments, the mRNA expression level can be determined in biological samples both in situ and in vitro using methods known in the art. The term “biological sample” includes tissues, cells, bodily fluids, and their isolates isolated from a subject, as well as tissues, cells, and bodily fluids present within the subject. Numerous expression detection methods use isolated RNA. Any RNA isolation technique not selected for mRNA isolation for in vitro methods can be used for the purification of RNA from cells (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987–1999). Furthermore, numerous tissue samples can be easily processed using techniques well known to those skilled in the art, such as the single-step RNA isolation method of Chomczynski (1989, U.S. Patent No. 4843155).

[0211] The isolated mRNA may be used in hybridization assays or amplification assays, including but not limited to Southern or Northern analysis, polymerase chain reaction analysis, and probe assays. One preferred diagnostic method for detecting mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene to be detected. The nucleic acid probe may be full-length cDNA or a portion thereof, such as an oligonucleotide sufficient to specifically hybridize under stringent conditions to mRNA or genomic DNA encoding one or more biomarkers listed in Table 1, and having a length of at least 7 nucleotides, 15 nucleotides, 30 nucleotides, 50 nucleotides, 100 nucleotides, 250 nucleotides, or 500 nucleotides. Other suitable probes for use in diagnostic assays included in the scope of the present invention are described herein. Hybridization of mRNA with the probe indicates the expression of one or more biomarkers listed in Table 1.

[0212] In one form, the mRNA is immobilized on a solid surface by, for example, electrophoresis of the isolated mRNA on an agarose gel and transfer of the mRNA from the gel to a membrane such as nitrocellulose, and then brought into contact with the probe. In another form, in a gene chip array, such as the Affymetrix® gene chip array, the probe is immobilized on a solid surface and the mRNA is brought into contact with the probe. Those skilled in the art can readily apply known mRNA detection methods for use in detecting the expression levels of one or more biomarker mRNAs listed in Table 1.

[0213] Other methods for determining mRNA expression levels in a sample include nucleic acid amplification, e.g., RT-PCR (Mullis, experimental embodiment shown in U.S. Patent No. 4683202, 1987), Liges chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, Vol. 88: pp. 189-193), autologous persistent sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA, Vol. 87: pp. 1874-1878), and transcription amplification systems (Kwoh et al., 1989, Proc. Natl. Acad. Sci. This includes the process of nucleic acid amplification by any nucleic acid amplification method, such as the USA journal, Vol. 86: pp. 1173-1177, Qβ replicase (Lizardi et al., 1988, Bio / Technology, Vol. 6: p. 1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5854033), or any other nucleic acid amplification method, and the subsequent detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when they are present in very small numbers. As used herein, amplification primers are defined as a pair of nucleic acid molecules that can anneal to the 5' or 3' region of a gene (the positive and negative strands, or vice versa, respectively) and may contain a short region between them. Generally, amplification primers are about 10-30 nucleotides long and adjacency to a region about 50-200 nucleotides long. Nucleic acid molecules containing the nucleotide sequences adjacent to these primers are amplified by those primers using appropriate reagents under appropriate conditions.

[0214] For in-situ detection, it is not necessary to isolate mRNA from cells before detection. In such methods, cell or tissue samples are prepared / processed using known histological methods. The samples are then immobilized on a support, typically a glass slide, and subsequently contacted with probes capable of hybridizing to the mRNA of one or more biomarkers listed in Table 1.

[0215] As an alternative to making decisions based on absolute expression levels, decisions can be made based on the normalized expression levels of one or more biomarkers listed in Table 1. Expression levels are normalized by correcting the absolute expression level by comparing their expression to that of non-biomarker genes, such as constitutively expressed housekeeping genes. Suitable genes for normalization include housekeeping genes such as actin genes or epithelial cell-specific genes. This normalization allows comparison of expression levels in one sample, such as a control sample, to those in another sample, such as a normal sample, or between samples from different origins.

[0216] The levels or activity of proteins corresponding to one or more biomarkers listed in Table 1 can also be detected and / or quantified by detection or quantification of expressed polypeptides. These polypeptides can be detected and quantified by any of the many methods well known to those skilled in the art. These methods may include analytical biochemical methods such as electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and high-diffusion chromatography, or various immunological methods such as liquid or gel precipitation reactions, immunodiffusion (one- or two-way), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and Western blotting. Those skilled in the art can readily apply known protein / antibody detection methods to determine whether cells express the biomarker of interest.

[0217] The present invention further provides one or more biomarkers or fragments thereof listed in Table 1 in the form of soluble purified and / or isolated polypeptides. It should also be understood that all properties of the polypeptides described herein, such as percentage identity, polypeptide length, polypeptide fragments, biological activity, and antibodies, can be combined in any order or combination with respect to one or more biomarkers listed in Table 1.

[0218] In one embodiment, the polypeptide may comprise a full-length amino acid sequence corresponding to one or more biomarkers listed in Table 1, or a full-length amino acid sequence having 1 to about 20 conserved amino acid substitutions. Any amino acid sequence described herein may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the full-length sequences described herein, full-length sequences well known in the art, or fragments thereof of one or more biomarkers listed in Table 1. In another embodiment, the present invention considers a composition comprising an isolated polypeptide corresponding to one or more biomarkers listed in Table 1, and less than about 25%, or less than 15%, or less than 5% of contaminating biological macromolecules or contaminating polypeptides.

[0219] The present invention further provides compositions, such as nucleic acids, vectors, and host cells, related to the preparation, detection, or characterization of such polypeptides or fragments thereof. Such compositions can function as compounds that regulate (e.g., enhance) the expression and / or activity of one or more biomarkers listed in Table 1.

[0220] Isolated polypeptides or fragments thereof (or nucleic acids encoding such polypeptides) corresponding to one or more biomarkers listed in Table 1 may be used to generate antibodies that bind to an immunogen using standard techniques for the preparation of polyclonal and monoclonal antibodies according to methods well known in the art. The antigen peptide comprises at least eight amino acid residues and includes epitopes present in each full-length molecule such that the antibody produced against the peptide forms a specific immune complex with each full-length molecule. Preferably, the antigen peptide comprises at least ten amino acid residues. In one embodiment, such epitopes may be specific to a given polypeptide molecule derived from one species, e.g., mouse or human (i.e., an antigen peptide extending to a region of a polypeptide molecule that is not conserved across multiple species is used as an immunogen. Such non-conserved residues may be determined using an alignment, e.g., the alignment provided herein).

[0221] In one embodiment, an antibody, particularly an intracellular antibody, binds substantially specifically to one or more biomarkers listed in Table 1, enhancing their biological function. In another embodiment, an antibody, particularly an intracellular antibody, binds substantially specifically to a binding partner of one or more biomarkers listed in Table 1, enhancing their biological function.

[0222] Antibodies used in accordance with the present invention can be produced according to methods well known in the art. For example, polypeptide immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., rabbits, goats, mice, or other mammals) with an immunogen. A suitable immunogenic preparation may include recombinant or chemically synthesized molecules or fragments thereof that induce an immune response. The preparation may further include adjuvants such as Freund's complete adjuvant or Freund's incomplete adjuvant, or similar immunostimulants. Immunization of a suitable subject with the immunogenic preparation induces a polyclonal antibody response to the antigen peptide contained therein.

[0223] Polyclonal antibodies can be prepared as described above by immunizing a suitable target with a polypeptide immunogen. The polypeptide antibody titer in the immunized target can be monitored over time by standard techniques, such as the use of enzyme-linked immunosorbent assay (ELISA) using immobilized polypeptides. If desired, antibodies against the antigen can be isolated from mammals (e.g., from blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, at the time when the antibody titer is highest, antibody-producing cells are obtained from the subject using hybridoma technology (originally described by Kohler and Milstein, (1975) Nature, Vol. 256: pp. 495-497) (Brown et al., (1981) J. Immunol., Vol. 127: pp. 539-546; Brown et al., (1980) J. Biol. Chem., Vol. 255: pp. 4980-4983; Yeh et al., (1976) Proc. Natl. Acad. Sci., Vol. 76: pp. 2927-2931; Yeh et al., (1982) Int. J. It can also be used to prepare monoclonal antibodies by standard techniques such as Cancer, Vol. 29: pp. 269-275, as well as more recent human B-cell hybridoma technology (Kozbor et al., (1983) Immunol. Today, Vol. 4: p. 72), EBV hybridoma technology (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96), or trioma technology. The techniques for producing monoclonal antibody hybridomas are well known (see, in general, *Monoclonal Antibodies: A New Dimension In Biological Analyses* by Kenneth, RH, published by Plenum Press, New York, New York (1980); *Lerner, EA*, *Yale J. Biol. Med.*, Vol. 54: pp. 387-402 (1981); and *Gefter, ML et al.*, *Somatic Cell Genet.*, Vol. 3: pp. 231-236 (1977)).In short, an immortal cell line (typically myeloma) is fused with lymphocytes (typically spleen cells) derived from mammals immunized with an immunogen as described above, and the culture supernatant of the resulting hybridoma cells is screened to identify hybridomas that produce monoclonal antibodies that preferably specifically bind to the polypeptide antigen.

[0224] Any of the many well-known protocols used to fuse lymphocytes with immortalized cell lines can be applied to the purpose of generating monoclonal antibodies against one or more biomarkers or fragments thereof listed in Table 1 (see, for example, Galfre, G. et al., (1977) Nature, Vol. 266: pp. 550-52; Gefter et al., (1977) op. cit.; Lerner, (1981) op. cit.; Kenneth, (1980) op. cit.). Furthermore, those skilled in the art will understand that there are numerous equally useful variations of such methods. The immortalized cell line (e.g., myeloma cell line) is typically derived from the same mammalian species as the lymphocytes. For example, a mouse hybridoma can be produced by fusing lymphocytes derived from a mouse immunized with an immunogenic preparation included in the scope of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to a culture medium containing hypoxanthine, aminopterin, and thymidine ("HAT medium"). Any of the many myeloma cell lines can be used as fusion partners according to standard techniques, for example, the P3-NS1 / 1-Ag4-1 myeloma line, the P3-x63-Ag8.653 myeloma line, or the Sp2 / O-Ag14 myeloma line. These myeloma lines are available from the American Cell Culture and Cell Line Preservation Center (ATCC) in Rockville, Maryland. HAT-sensitive mouse myeloma cells are typically fused to mouse splenocytes using polyethylene glycol ("PEG"). The resulting hybridoma cells are then sorted using HAT medium, which kills myeloma cells that did not fuse and myeloma cells that fused but did not produce any cells (unfusing splenocytes are not transformed and will die after a few days). Hybridoma cells that produce monoclonal antibodies within the scope of the present invention can be detected by screening the hybridoma culture supernatant for antibodies that bind to a given polypeptide using a standard ELISA assay or the like.

[0225] As an alternative to the preparation of monoclonal antibody-secreting hybridomas, a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) can be screened with a suitable polypeptide, thereby isolating immunoglobulin library members that bind to the polypeptide, and a monoclonal specific to one of the polypeptides can be identified and isolated. Kits for preparing and screening phage display libraries are commercially available (e.g., Pharmacia's Recombinant Phage Antibody System, catalog no. 27-9400-01, and Stratagene's SurfZAP® Phage Display Kit, catalog no. 240612).Furthermore, examples of methods and reagents usable for the preparation and screening of antibody display libraries include, for example, U.S. Patent No. 5223409 by Ladner et al., International Publication No. 92 / 18619 by Kang et al., International Publication No. 91 / 17271 by Dower et al., International Publication No. 92 / 20791 by Winter et al., International Publication No. 92 / 15679 by Markland et al., International Publication No. 93 / 01288 by Breitling et al., International Publication No. 92 / 01047 by McCafferty et al., International Publication No. 92 / 01047 by Garrard et al., International Publication No. 92 / 09690 by Ladner et al., International Publication No. 90 / 02809 by Fuchs et al., (1991) Biotechnology (New York), Vol. 9: pp. 1369-1372, and Hay et al., (1992) Hum. Antibod. Hybridomas, Vol. 3: pp. 81-85, by Huse et al., (1989) Science, Vol. 246: pp. 1275-1281, by Griffiths et al., (1993) EMBO J., Vol. 12: pp. 725-734, by Hawkins et al., (1992) J. Mol. Biol., Vol. 226: pp. 889-896, by Clarkson et al., (1991) Nature, Vol. 352: pp. 624-628, by Gram et al., (1992) Proc. Natl. Acad. Sci. It can be found in the following publications: USA, Vol. 89: pp. 3576-3580; Garrard et al., (1991); Biotechnology (New York), Vol. 9: pp. 1373-1377; Hoogenboom et al., (1991); Nucleic Acids Res., Vol. 19: pp. 4133-4137; Barbas et al., (1991); Proc. Natl. Acad. Sci. USA, Vol. 88: pp. 7978-7982; and McCafferty et al., (1990); Nature, Vol. 348: pp. 552-554.

[0226] Since the CDR3 domains of the heavy and light chains of an antibody are well known in the art to play a particularly important role in the binding specificity / affinity of the antibody to an antigen, it is preferable that recombinant monoclonal antibodies within the scope of the present invention, prepared as shown above, contain the CDR3 of the variable regions of the heavy and light chains of the antibody of interest. The antibody may further contain the CDR2 of the variable region within the scope of the present invention. The antibody may further contain the CDR1 of the variable region within the scope of the present invention. In other embodiments, the antibody may contain any combination of the CDRs.

[0227] The CDR1, CDR2, and / or CDR3 regions of the modified antibody described above may contain precise amino acid sequences, such as those regions of the variable regions included in the scope of the present invention. However, those skilled in the art will understand that there may be some deviation from their precise CDR sequences (e.g., conservative sequence modifications) while still retaining the antibody's ability to efficiently bind to a target of interest, such as one or more biomarkers and / or one or more natural binding partners listed in Table 1. Thus, in another embodiment, the modified antibody may consist of one or more CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or more CDRs included in the scope of the present invention.

[0228] For example, the structural features of a non-human antibody or a human antibody (e.g., a rat anti-mouse / anti-human antibody) can be used to create a structurally related human antibody, particularly an introbody, that retains at least one functional property of the antibody, such as one or more biomarkers listed in Table 1, a binding partner / substrate for one or more biomarkers listed in Table 1, and / or binding to an immune checkpoint. Another functional property is the inhibition of binding of the original known non-human antibody or human antibody in a competitive ELISA assay.

[0229] Those skilled in the art will note that such percentage homology is equivalent to introducing one, two, three, four, five, six, seven, eight, nine, ten, or more conserved amino acid substitutions into a given CDR, and can be achieved by such introduction.

[0230] A monoclonal antibody within the scope of the present invention may include a heavy chain, the variable domain of which comprises at least one CDR having a sequence selected from the group consisting of heavy chain variable domains CDR described herein, and the monoclonal antibody may include a light chain, the variable domain of which comprises at least one CDR having a sequence selected from the group consisting of light chain variable domains CDR described herein.

[0231] Such monoclonal antibodies may include a light chain, the variable domain comprising at least one CDR having a sequence selected from the group consisting of CDR-L1, CDR-L2, and CDR-L3 as described herein, and / or the monoclonal antibody may include a heavy chain, the variable domain comprising at least one CDR having a sequence selected from the group consisting of CDR-H1, CDR-H2, and CDR-H3 as described herein. In some embodiments, the monoclonal antibody capable of binding to one or more biomarkers listed in Table 1 comprises or consists of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as described herein.

[0232] The heavy chain variable domain of the monoclonal antibody within the scope of the present invention may include or consist of the vH amino acid sequence shown herein, and / or the light chain variable domain of the monoclonal antibody within the scope of the present invention may include or consist of the vκ amino acid sequence shown herein.

[0233] The present invention further provides fragments of monoclonal antibodies, including but not limited to Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody, as well as multispecific antibodies formed from these antibody fragments. For example, a number of immunosuppressive molecules such as PD-L1, PD-1, and CTLA-4 can be bound bispecifically or multispecifically.

[0234] Other fragments of the monoclonal antibody that fall within the scope of the present invention are also considered. For example, individual immunoglobulin heavy and / or light chains are provided in which the variable domain comprises at least one CDR described herein. In one embodiment, the immunoglobulin heavy chain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to that of the heavy chain variable domain CDR or light chain variable domain CDR described herein. In another embodiment, the immunoglobulin light chain comprises at least one CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to that of the light chain variable domain CDR or heavy chain variable domain CDR described herein.

[0235] In some embodiments, the immunoglobulin heavy chain and / or light chain comprises a variable domain containing at least one of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 as described herein. Such an immunoglobulin heavy chain may contain or consist of at least one of CDR-H1, CDR-H2, and CDR-H3. Such an immunoglobulin light chain may contain or consist of at least one of CDR-L1, CDR-L2, and CDR-L3.

[0236] In other embodiments, the immunoglobulin heavy and / or light chains according to the present invention include or consist of the .vH variable domain sequence or the vκ variable domain sequence described herein, respectively.

[0237] The present invention further provides polypeptides having a sequence selected from the group consisting of the vH variable domain sequence, vκ variable domain sequence, CDR-L1 sequence, CDR-L2 sequence, CDR-L3 sequence, CDR-H1 sequence, CDR-H2 sequence, and CDR-H3 sequence described herein.

[0238] Antibodies, immunoglobulins, and polypeptides included in the scope of the present invention may be used in isolated (e.g., purified) forms or contained within vectors such as membranes or lipid vesicles (e.g., liposomes).

[0239] The amino acid sequence modifications of the antibodies described herein are being considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibodies. It is known that when humanized antibodies are produced by simply transplanting only the CDRs in the VH and VL of antibodies derived from non-human animals into the FRs of the VH and VL of human antibodies, their antigen-binding activity is reduced compared to that of the original antibodies derived from non-human animals. It is thought that several amino acid residues of the VH and VL of the non-human antibody, not only in the CDRs but also in the FRs, are directly or indirectly related to antigen-binding activity. Therefore, substitution of these amino acid residues with different amino acid residues derived from the FRs of the VH and VL of the human antibody reduces binding activity, and this substitution can be corrected by replacing these amino acids with amino acid residues from the original antibodies derived from non-human animals.

[0240] Modifications and alterations may be made to the structure of the antibodies and the DNA sequences encoding them that are within the scope of the present invention, and yet functional molecules encoding antibodies and polypeptides having the desired characteristics can be obtained. For example, certain amino acids within the protein structure may be replaced by other amino acids without clearly losing activity. Since the ability and nature of protein interactions define the biological functional activity of that protein, certain amino acid substitutions can be made within the protein sequence, and of course also to its DNA coding sequence, and yet a protein having similar properties is obtained. Thus, it is contemplated that various changes can be made within the antibody sequences or the corresponding DNA sequences encoding the polypeptides that are within the scope of the present invention without clearly losing biological activity.

[0241] In changing the amino acid sequence of a polypeptide, the hydrophobic-hydrophilic index of the amino acids may be taken into consideration. The importance of the hydrophobic-hydrophilic index of amino acids in conferring biological interaction functions on proteins is generally understood in the art. It is accepted that the relative hydrophobic-hydrophilic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of that protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophobic-hydrophilic index based on its hydrophobic and charge characteristics, which are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartic acid (<RTI3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0242] It is known in the art that certain amino acids may be replaced by other amino acids having similar hydrophobicity or hydrophilicity indices or scores, and that proteins with similar biological activity are still produced, i.e., proteins equivalent in terms of biological function are still obtained.

[0243] As broadly explained above, amino acid substitutions are therefore generally based on the relative similarity of substituents on the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Examples of substitutions that take into account the various properties mentioned above are well known to those skilled in the art, and examples of substitutions include the substitution of arginine with lysine, glutamic acid with aspartic acid, serine with threonine, glutamine with asparagine, and valine, leucine, and isoleucine.

[0244] Another type of amino acid m...

Claims

1. A cancer vaccine containing cancer cells, wherein the cancer cells (1) PTEN deficiency, (2) p53 is missing, and (3) A cancer vaccine comprising the TGFβ-Smad / p63 signaling pathway activated by contact with the TGFβ protein.

2. The cancer vaccine according to claim 1, wherein the TGFβ protein is selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3.

3. The cancer vaccine according to claim 2, wherein the TGFβ protein is TGFβ1.

4. The cancer vaccine according to any one of claims 1 to 3, comprising contacting the cancer cells with the TGFβ protein in vitro, in vivo, and / or ex vivo.

5. The cancer vaccine according to claim 4, wherein the cancer cells are brought into contact with the TGFβ protein in vitro or ex vivo.

6. The cancer vaccine according to claim 4, wherein the cancer cells are administerable to the target, and the TGFβ protein is administerable to the target in order to bring the TGFβ protein into contact with the cancer cells in vivo.

7. The cancer vaccine according to claim 6, wherein the TGFβ protein can be administered before, after, or simultaneously with the administration of the cancer cells.

8. The cancer vaccine according to any one of claims 1 to 7, wherein the cancer cells have increased nuclear localization of Smad2 and / or binding of p63 to Smad2 in the nucleus of the cancer cells compared to cancer cells that have not been in contact with the TGFβ protein.

9. The cancer vaccine according to any one of claims 1 to 8, wherein the cancer cells are derived from solid tumors or hematological cancers.

10. The cancer vaccine according to any one of claims 1 to 9, wherein the cancer cells are derived from a cancer cell line.

11. The cancer vaccine according to any one of claims 1 to 9, wherein the cancer cells are derived from primary cancer cells.

12. The cancer vaccine according to any one of claims 1 to 11, wherein the cancer cells are breast cancer cells.

13. The cancer vaccine according to any one of claims 1 to 12, wherein the cancer cells are derived from triple-negative breast cancer (TNBC).

14. A cancer vaccine according to any one of claims 1 to 13, which induces epithelial-mesenchymal transition (EMT) in cancer cells by contact with the TGFβ protein.

15. A cancer vaccine according to any one of claims 1 to 14, wherein contact with the TGFβ protein increases the expression levels of ICOSL, PYCARD, SFN, PERP, RIPK3, CASP9, and / or SESN1 in the cancer cells.

16. A cancer vaccine according to any one of claims 1 to 15, which reduces the expression levels of KSR1, KSR1, EIF4EBP1, ITGA5, EMILIN1, CD200, and / or CSF1 in the cancer cells by contact with the TGFβ protein.

17. The cancer vaccine according to any one of claims 1 to 16, wherein the cancer cells are capable of activating dendritic cells (DCs) co-cultured in vitro.

18. The cancer vaccine according to any one of claims 1 to 17, wherein the cancer cells are capable of increasing CD40, CD80, CD86, CD103, CD8, HLA-DR, MHC-II, and / or IL1-β in the co-cultured dendritic cells in vitro.

19. The cancer vaccine according to any one of claims 1 to 18, wherein the cancer cells are capable of activating T cells co-cultured in vitro in the presence of DCs.

20. The cancer vaccine according to any one of claims 1 to 19, wherein the cancer cells can increase the secretion of TNFα and / or IFNγ by T cells co-cultured in vitro in the presence of DCs.

21. The cancer vaccine according to any one of claims 1 to 20, wherein the cancer cells do not form tumors in immune-normal subjects.

22. The cancer vaccine according to any one of claims 1 to 21, wherein the cancer vaccine induces cytotoxic T cell-mediated antitumor immunity.

23. The cancer vaccine according to any one of claims 1 to 22, wherein the cancer vaccine increases CD4+ T cells and CD8+ T cells in the blood and / or tumor microenvironment.

24. The cancer vaccine according to any one of claims 1 to 23, wherein the cancer vaccine increases TNFα and INFγ-secreting CD4+ T cells and CD8+ T cells in the blood and / or tumor microenvironment.

25. The cancer vaccine according to any one of claims 1 to 24, wherein the cancer vaccine increases the expression of Icos, Klrc1, Il2rb, Pik3cd, H2-D1, Ccl8, Ifng, Icosl, Il2ra, Cxcr3, Ccr7, Cxcl10, Cd74, H2-Ab1, Hspa1b, Cd45, Lifr, and / or Tnf in tumor tissue.

26. The cancer vaccine according to any one of claims 1 to 25, wherein the amount of tumor-infiltrating dendritic cells increases by the cancer vaccine.

27. The cancer vaccine according to any one of claims 1 to 26, wherein the cancer vaccine increases CD80, CD103, and / or MHC-II in tumor-associated DCs.

28. The cancer vaccine according to any one of claims 1 to 27, wherein the cancer vaccine reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing cancer cells.

29. The cancer vaccine according to claim 28, wherein the cancer vaccine reduces the number of proliferating cells in the cancer at the initial immunization site and / or reduces the volume or size of the tumor containing cancer cells.

30. The cancer vaccine according to claim 28, wherein the cancer vaccine reduces the number of proliferating cells in the cancer in tissue distal to the immune site, and / or reduces the volume or size of the tumor containing cancer cells.

31. The cancer vaccine according to any one of claims 1 to 30, wherein the cancer vaccine induces a tumor-specific memory T cell response.

32. The aforementioned cancer vaccine stimulates CD4+ central memory T cells (T) in the spleen and / or lymph nodes. ＣＭ ) and / or CD4+ effector memory T cells (T ＥＭ The cancer vaccine according to any one of claims 1 to 31, wherein the percentage of ) increases.

33. The aforementioned cancer vaccine causes spleen CD8+ T ＣＭ A cancer vaccine according to any one of claims 1 to 32, wherein the percentage of cells increases.

34. The aforementioned cancer vaccine causes CD8+ T in the spleen and / or lymph nodes. ＥＭ A cancer vaccine according to any one of claims 1 to 33, wherein the percentage of cells increases.

35. The cancer vaccine according to any one of claims 1 to 34, wherein the cancer vaccine increases the amount of tumor-infiltrating CD4+ T cells and / or CD8+ T cells.

36. The aforementioned cancer vaccine causes tumor infiltration CD4+ T ＣＭ Cells and / or CD4+T ＥＭ A cancer vaccine according to any one of claims 1 to 35, which increases the amount of cells.

37. The aforementioned cancer vaccine causes tumor infiltration CD8+ T ＣＭ Cells and / or CD8+T ＥＭ A cancer vaccine according to any one of claims 1 to 36, which increases the amount of cells.

38. The cancer vaccine according to any one of claims 1 to 37, wherein the cancer cells do not replicate.

39. The cancer vaccine according to claim 38, wherein the cancer cells do not replicate due to radiation irradiation.

40. The cancer vaccine according to claim 39, wherein the radiation irradiation is a sublethal dose.

41. The cancer vaccine according to any one of claims 1 to 40, wherein the cancer vaccine can be administered to a target in combination with immunotherapy and / or cancer therapy.

42. The cancer vaccine according to claim 41, wherein the immunotherapy and / or cancer therapy is administered before, after, or simultaneously with the cancer vaccine.

43. The cancer vaccine according to claim 41 or 42, wherein the immunotherapy is cell-based.

44. The cancer vaccine according to claim 43, wherein the immunotherapy comprises a cancer vaccine and / or a virus.

45. The cancer vaccine according to claim 44, wherein the immunotherapy inhibits immune checkpoints.

46. The cancer vaccine according to claim 45, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, and A2aR.

47. The cancer vaccine according to claim 46, wherein the immune checkpoint is PD1, PD-L1, or CD47.

48. The cancer vaccine according to claim 41, wherein the cancer therapy is selected from the group consisting of radiation, a radiosensitizer, and a chemotherapy agent.

49. A composition comprising a cancer vaccine for use in preventing the development of cancer, delaying the onset of cancer, preventing the recurrence of cancer, and / or treating cancer in a subject, characterized in that the composition is administered to the subject, wherein the cancer vaccine comprises cancer cells, and the cancer cells are (1) PTEN deficiency, (2) p53 is missing, and (3) A composition comprising the TGFβ-Smad / p63 signaling pathway activated by contact with the TGFβ protein.

50. The composition according to claim 49, wherein the subject is suffering from cancer.

51. The composition according to claim 49 or 50, wherein the TGFβ protein is selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3.

52. The composition according to claim 51, wherein the TGFβ protein is TGFβ1.

53. The composition according to any one of claims 49 to 52, wherein the cancer cells are brought into contact with the TGFβ protein in vitro, in vivo, and / or ex vivo.

54. The composition according to claim 53, wherein the cancer cells are brought into contact with the TGFβ protein in vitro or ex vivo.

55. The composition according to claim 53, characterized in that the composition is administered to the subject in combination with the TGFβ protein in order to bring the cancer cells into contact in vivo.

56. The composition according to claim 55, wherein the TGFβ protein is administered before, after, or simultaneously with the administration of the composition.

57. The composition according to any one of claims 49 to 56, wherein the cancer cells have increased nuclear localization of Smad2 and / or binding of p63 to Smad2 in the nucleus of the cancer cells compared to cancer cells that have not been in contact with the TGFβ protein.

58. The composition according to any one of claims 49 to 57, wherein the cancer cells are derived from a solid tumor or a hematological cancer.

59. The composition according to any one of claims 49 to 58, wherein the cancer cells are derived from a cancer cell line.

60. The composition according to any one of claims 49 to 58, wherein the cancer cells are derived from primary cancer cells.

61. The composition according to any one of claims 49 to 60, wherein the cancer cells are breast cancer cells.

62. The composition according to any one of claims 49 to 61, wherein the cancer cells are derived from triple-negative breast cancer (TNBC).

63. The composition according to any one of claims 49 to 62, wherein the cancer cells are derived from cancer of the same type as the cancer treated with the cancer vaccine.

64. The composition according to any one of claims 49 to 62, wherein the cancer cells are derived from a cancer of a different type than the cancer treated with the cancer vaccine.

65. The composition according to any one of claims 49 to 64, wherein the cancer treated with the cancer vaccine is characterized by the loss of PTEN, p53, and / or p110.

66. The composition according to claim 65, wherein the cancer further expresses Myc.

67. The composition according to any one of claims 49 to 64, wherein the cancer treated with the cancer vaccine has functional PTEN and / or p53.

68. The composition according to claim 67, wherein the cancer has the Kras activating mutation G12D.

69. The composition according to any one of claims 49 to 68, wherein the cancer vaccine is homogeneous or heterogeneous with respect to the subject.

70. The composition according to any one of claims 49 to 69, wherein the cancer vaccine is autologous, compatible allogeneic, incompatible allogeneic, or genetically related to the subject.

71. The composition according to any one of claims 49 to 70, wherein the cancer treated with the cancer vaccine is selected from the group consisting of breast tumors, ovarian tumors, or brain tumors.

72. The composition according to any one of claims 49 to 71, wherein contact with the TGFβ protein induces epithelial-mesenchymal transition (EMT) in the cancer cells.

73. The composition according to any one of claims 49 to 72, which increases the expression levels of ICOSL, PYCARD, SFN, PERP, RIPK3, CASP9, and / or SESN1 in the cancer cells by contact with the TGFβ protein.

74. The composition according to any one of claims 49 to 73, wherein contact with the TGFβ protein reduces the expression levels of KSR1, KSR1, EIF4EBP1, ITGA5, EMILIN1, CD200, and / or CSF1 in the cancer cells.

75. The composition according to any one of claims 49 to 74, wherein the cancer cells are capable of activating dendritic cells (DCs) co-cultured in vitro.

76. The composition according to any one of claims 49 to 75, wherein the cancer cells are capable of increasing CD40, CD80, CD86, CD103, CD8, HLA-DR, MHC-II, and / or IL1-β in dendritic cells co-cultured in vitro.

77. The composition according to any one of claims 49 to 76, wherein the cancer cells are capable of activating T cells co-cultured in vitro in the presence of DCs.

78. The composition according to any one of claims 49 to 77, wherein the cancer cells can increase the secretion of TNFα and / or IFNγ by T cells co-cultured in vitro in the presence of DCs.

79. The composition according to any one of claims 49 to 78, wherein the cancer cells do not form tumors in immunocompetent subjects.

80. The composition according to any one of claims 49 to 79, wherein the cancer vaccine induces cytotoxic T cell-mediated antitumor immunity.

81. The composition according to any one of claims 49 to 80, wherein the cancer vaccine increases CD4+ T cells and CD8+ T cells in the blood and / or tumor microenvironment.

82. The composition according to any one of claims 49 to 81, wherein the cancer vaccine increases TNFα and INFγ-secreting CD4+ T cells and CD8+ T cells in the blood and / or tumor microenvironment.

83. The composition according to any one of claims 49 to 82, wherein the cancer vaccine increases the expression of Icos, Klrc1, Il2rb, Pik3cd, H2-D1, Ccl8, Ifng, Icosl, Il2ra, Cxcr3, Ccr7, Cxcl10, Cd74, H2-Ab1, Hspa1b, Cd45, Lifr, and / or Tnf in tumor tissue.

84. The composition according to any one of claims 49 to 83, wherein the amount of tumor-infiltrating dendritic cells increases by the cancer vaccine.

85. The composition according to any one of claims 49 to 84, wherein the cancer vaccine increases CD80, CD103, and / or MHC-II in tumor-associated DCs.

86. The composition according to any one of claims 49 to 85, wherein the cancer vaccine reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing cancer cells.

87. The composition according to claim 86, wherein the cancer vaccine reduces the number of proliferating cells in the cancer at the initial immunization site and / or reduces the volume or size of the tumor containing cancer cells.

88. The composition according to claim 86, wherein the cancer vaccine reduces the number of proliferating cells in the cancer in tissue distal to the immune site, and / or reduces the volume or size of the tumor containing cancer cells.

89. The composition according to any one of claims 49 to 88, wherein the cancer vaccine induces a tumor-specific memory T cell response.

90. The percentage of CD4+ central memory T cells (T ＣＭ ), and / or CD4+ effector memory T cells (T ＥＭ ) in the spleen and / or lymph nodes is increased by the cancer vaccine, the composition according to any one of claims 49 to 89.

91. The aforementioned cancer vaccine causes spleen CD8+ T ＣＭ The composition according to any one of claims 49 to 90, wherein the percentage of cells increases.

92. The aforementioned cancer vaccine causes CD8+ T in the spleen and / or lymph nodes. ＥＭ The composition according to any one of claims 49 to 91, wherein the percentage of cells increases.

93. The composition according to any one of claims 49 to 92, wherein the cancer vaccine increases the amount of tumor-infiltrating CD4+ T cells and / or CD8+ T cells.

94. The aforementioned cancer vaccine causes tumor infiltration CD4+ T ＣＭ Cells and / or CD4+T ＥＭ The composition according to any one of claims 49 to 93, wherein the amount of cells increases.

95. The aforementioned cancer vaccine causes tumor infiltration CD8+ T ＣＭ Cells and / or CD8+T ＥＭ The composition according to any one of claims 49 to 94, wherein the amount of cells increases.

96. The composition according to any one of claims 49 to 95, wherein the cancer cells do not replicate.

97. The composition according to claim 96, wherein the cancer cells do not replicate due to radiation irradiation.

98. The composition according to claim 97, wherein the radiation irradiation is a sublethal dose.

99. The composition according to any one of claims 49 to 98, characterized in that the composition is administered to the subject in combination with immunotherapy and / or cancer therapy.

100. The composition according to claim 99, wherein the immunotherapy and / or cancer therapy is administered before, after, or simultaneously with the composition.

101. The composition according to claim 99 or 100, wherein the immunotherapy is cell-based.

102. The composition according to claim 99 or 100, wherein the immunotherapy comprises a cancer vaccine and / or a virus.

103. The composition according to claim 99 or 100, wherein the immunotherapy inhibits an immune checkpoint.

104. The composition according to claim 103, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, and A2aR.

105. The composition according to claim 104, wherein the immune checkpoint is PD1, PD-L1, or CD47.

106. The composition according to claim 99 or 100, wherein the cancer therapy is selected from the group consisting of radiotherapy, radiosensitizers, and chemotherapeutic agents.

107. A method for analyzing proliferating cells in a sample containing cells from the cancer of a subject as an indicator for evaluating the efficacy of a cancer vaccine according to any one of claims 1 to 48 for treating a subject suffering from cancer, the method comprising determining and comparing the number of proliferating cells in the cancer in a sample from the subject at a time prior to the administration of the cancer vaccine with the number of proliferating cells in a sample from the subject after the administration of the cancer vaccine, wherein the absence or significant decrease in the number of proliferating cells in the cancer in the sample from the subject after the administration of the cancer vaccine compared to the number in the sample from the subject at the earlier time indicates that the cancer of the subject has been treated by the cancer vaccine.

108. The method according to claim 107, wherein between the earlier time point and the subsequent time point, the subject has received treatment, completed treatment, and / or is in a palliative state for the cancer.

109. The method according to claim 107 or 108, wherein the preceding sample and / or subsequent sample are selected from the group consisting of ex vivo samples and in vivo samples.

110. The method according to any one of claims 107 to 109, wherein the preceding sample and / or subsequent sample is part of a single sample or part of a pooled sample obtained from the subject.

111. The method according to any one of claims 107 to 110, wherein the sample comprises cells, serum, peripheral lymphoid organs, and / or tumor tissue obtained from the subject.

112. The method according to any one of claims 107 to 111, further comprising determining the responsiveness to the drug by evaluating at least one criterion selected from the group consisting of clinical efficacy rate, survival to death, complete pathological response, semi-quantitative pathological response scale, complete clinical remission, partial clinical remission, clinical disease stability, recurrence-free survival, metastasis-free survival, disease-free survival, reduction of circulating tumor cells, circulating marker response, and RECIST criteria.

113. The composition according to any one of claims 49 to 106, wherein the cancer vaccine can be administered in a pharmaceutically acceptable formulation.

114. The cancer vaccine or composition according to any one of claims 1 to 106, wherein the cancer vaccine prevents recurrent tumor lesions and metastatic tumor lesions.

115. The cancer vaccine or composition according to any one of claims 1 to 106 and 113 to 114, wherein the cancer vaccine can be administered intratumorally or subcutaneously to the target.

116. The cancer vaccine or composition according to any one of claims 1 to 106 and 113 to 115, wherein the cancer vaccine is administerable to a subject, and the subject is an animal model of the cancer.

117. The cancer vaccine or composition according to claim 116, wherein the animal model is a mouse model.

118. The cancer vaccine or composition according to any one of claims 1 to 106 and 113 to 115, wherein the cancer vaccine is administerable to a target, and the target is a mammal.

119. The cancer vaccine or composition according to claim 118, wherein the mammal is in a mitigated state for cancer.

120. The cancer vaccine or composition according to claim 118 or 119, wherein the mammal is a mouse or a human.

121. The cancer vaccine or composition according to claim 120, wherein the mammal is a human.

122. The method according to any one of claims 107 to 112, wherein the cancer vaccine can be administered in a pharmaceutically acceptable formulation.

123. The method according to any one of claims 107 to 112, wherein the cancer vaccine prevents recurrent tumor lesions and metastatic tumor lesions.

124. The method according to any one of claims 107 to 112 and 123, wherein the cancer vaccine can be administered intratumorally or subcutaneously to the target.

125. The method according to any one of claims 107 to 112 and 124, wherein the subject is an animal model of the cancer.

126. The method according to claim 125, wherein the animal model is a mouse model.

127. The method according to any one of claims 107 to 112 and 122 to 124, wherein the subject is a mammal.

128. The method according to claim 127, wherein the mammal is in a palliative state for cancer.

129. The method according to claim 127 or 128, wherein the mammal is a mouse or a human.

130. The method according to claim 129, wherein the mammal is a human.

Images