Cancer vaccine compositions and methods for using the same to prevent and / or treat cancer

Abstract

To provide cancer vaccine compositions, and to provide methods for using the same to prevent and / or treat cancer.SOLUTION: The present invention is based, in part, on cancer vaccine compositions that comprise PTEN- and p53-deficient cancer cells with activated TGFβ-Smad / p63 signaling pathway, and methods for using same to prevent and / or treat cancer. In one aspect, provided herein is a cancer vaccine comprising cancer cells, the cancer cells being: (1) PTEN-deficient; (2) p53-deficient; and (3) modified to activate the TGF b-Smad / p63 signaling pathway.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 876,416, filed on Jul. 19, 2019, the entire contents of which are hereby incorporated by reference.

[0002] Statement Regarding Rights This invention was made with government support under National Institutes of Health grants P50 CA168504, CA233810, CA187918, and R35 CA210057. The U.S. government has certain rights in the invention.

Background Art

[0003] Transforming growth factor β (TGFβ) is a pleiotropic cytokine that plays an important role in the regulation of embryonic development, cell metabolism, tumor growth, and immune system homeostasis (David and Massague, Nat. Rev. Mol. Cell. Biol. 19, 419–435 (2018)). TGFβ controls the expression of its downstream genes in a manner that can be either Smad-dependent or Smad-independent upon binding to its receptor located on the cell membrane. TGFβ controls cancer initiation and progression in a stage- and cell context-dependent manner (Morikawa et al., Cold Spring Harb. Perspect. Biol. 8, a021873 (2016); Prunier et al., Trends Cancer 5, 66–78 (2019); Seoane and Gomis, Cold Spring Harb. Perspect. Biol. 9, a022277 (2017)). TGFβ suppresses tumorigenesis by inducing cell growth arrest and apoptosis in premalignant cells. Inhibition of the TGFβ signaling pathway promotes tumorigenesis in various mouse models (Cammareri et al., Nat. Commun. 7, 12493 (2016); Yu et al., Oncogene 33, 1538–1547 (2014); Cohen et al., Cancer Res. 69, 3415–3424 (2009)). Loss-of-function mutations in the TGFβ signaling pathway are also commonly found in various human cancers (Levy and Hill, Cytokine Growth Factor Rev. 17, 41–58 (2006)). However, in late-stage cancers, TGFβ promotes tumor metastasis and drug resistance. On the one hand, cancer cells themselves overcome the growth arrest and apoptosis induced by TGFβ due to the accumulation of oncogenic mutations. TGFβ induces epithelial-mesenchymal transition (EMT) in cancer cells, enhances the cancer stem cell properties of cancer cells, increases angiogenesis, and promotes drug resistance (Ahmadi et al., J. Cell Physiol. 234, 12173–12187 (2018)).On the other hand, TGFβ promotes CD4+ regulatory T cell (Treg) differentiation, myeloid-derived suppressor cell (MDSC) differentiation, and M2 macrophage differentiation, thereby suppressing the host's anti-tumor immunity, which supports cancer growth and metastasis (Dahmani and Delisle, Cancers (Basel), Vol. 10:194, 2018). Since 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 has become an important issue. Therefore, there is a great need in the art to identify anti-cancer therapies based on a deeper understanding of the role of the TGFβ signaling pathway in cancer.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0005] The present invention is based at least in part on the discovery that PTEN- and p53-deficient tumor cells having TGFβ-Smad / p63 signaling activated (e.g., by treatment with at least one type of TGFβ superfamily protein) fail to form tumors in a T cell-dependent manner in hosts with normal immunity. Administration of these tumor cells also protects the host from recurrent and metastatic tumor lesions. This cancer vaccine generated using these tumor cells is advantageous in overcoming the stubborn obstacles in this field, such as lack of presentation of tumor-specific antigens, tumor heterogeneity, and low immune cell infiltration, by inducing a broad range of immune responses. These effects have been shown to be mediated at least in part by activation in tumor cells of the Smad / p63 transcriptional complex that controls the expression of multiple pathways that promote the immune response and ultimately promote the activation of cytotoxic T cells and immune memory.

[0006] 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.

[0007] In another aspect, a method of preventing cancer development, delaying cancer onset, preventing cancer recurrence, and / or treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount 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 optionally the subject has cancer. In one embodiment, the cancer cells are derived from a cancer of the same type as the cancer to be treated with the cancer vaccine. In another embodiment, the cancer cells are derived from a cancer of a different type than the cancer to be treated with the cancer vaccine. In yet another embodiment, the cancer to be treated with the cancer vaccine is characterized by loss of PTEN, p53, and / or p110, and optionally the cancer further expresses Myc. In yet another embodiment, the cancer to be treated with the cancer vaccine has functional PTEN and / or p53, and optionally the cancer has a Kras activating mutation G12D. In another embodiment, the cancer vaccine is syngeneic or xenogeneic to the subject. In yet another embodiment, the cancer vaccine is autologous, syngeneic, allogeneic, or isogeneic to the subject. In yet another embodiment, the cancer to be treated with the cancer vaccine is selected from the group consisting of breast cancer, ovarian cancer, or brain cancer, such as a breast tumor, an ovarian tumor, or a brain tumor.

[0008] Numerous embodiments are further provided that are applicable to any aspect of the invention described herein. 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 selected from the group consisting of 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-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, decapentaplegic / DPP, artemin, GDNF, neurturin, persephin, 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 contacted with the TGFβ superfamily protein in vitro, in vivo, and / or ex vivo. For example, the cancer cells may be contacted 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 contact the cancer cells 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, amount, and / or activity of at least one biomarker described in Table 1 in the cancer cells and / or decreasing the copy number, amount, and / or activity of at least one biomarker described in Table 2. For example, the copy number, amount, and / or activity of at least one biomarker described in Table 1 can be increased by contacting the cancer cells with a nucleic acid molecule encoding at least one biomarker described in Table 1 or a fragment thereof, a polypeptide of at least one biomarker described in Table 1 or a fragment thereof, or a small molecule that binds to at least one biomarker described in Table 1. In 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 and Smad2 in the nucleus of the cancer cells. In yet another embodiment, the copy number, amount, and / or activity of at least one biomarker described in Table 2 is decreased by contacting the cancer cells with a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interference agent, antisense oligonucleotide, peptide inhibitor or peptidomimetic inhibitor, aptamer, antibody, and / or intracellular antibody.

[0009] In yet another embodiment, the cancer cells are derived from solid cancer or hematological cancer. In 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 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 said cancer cells. In yet another embodiment, activation of the TGFβ-Smad / p63 signaling pathway increases the expression levels of ICOS-L, PYCARD, SFN, PERP, RIPK3, CASP9, and / or SESN1 in said cancer cells. In 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 said cancer cells. In yet another embodiment, said cancer cells are capable of activating dendritic cells (DCs) co-cultured in vitro. In yet another embodiment, said cancer cells are capable of upregulating CD40, CD80, CD86, CD103, CD8, HLA-DR, MHC-II, and / or IL1-β in said co-cultured dendritic cells in vitro. In another embodiment, said cancer cells are capable of activating T cells co-cultured in the presence of DCs in vitro. In yet another embodiment, said cancer cells are capable of increasing the secretion of TNFα and / or IFNγ by said co-cultured T cells in the presence of DCs in vitro. In yet another embodiment, said cancer cells do not form tumors in immunocompetent subjects. In another embodiment, said cancer vaccine elicits cytotoxic T cell-mediated antitumor immunity. In yet another embodiment, said cancer vaccine increases CD4+ T cells and CD8+ T cells in the blood and / or in the tumor microenvironment. In yet another embodiment, said cancer vaccine increases TNFα- and INFγ-secreting CD4+ T cells and CD8+ T cells in the blood and / or in the tumor microenvironment. In another embodiment, said 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 tissues. In yet another embodiment, said 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 another embodiment, the cancer vaccine decreases the number of proliferating cells in the cancer and / or decreases the volume or size of the tumor containing cancer cells. In yet another embodiment, the cancer vaccine decreases the number of proliferating cells in the cancer and / or decreases the volume or size of the tumor containing cancer cells at the first immunization site. In yet another embodiment, the cancer vaccine decreases the number of proliferating cells in the cancer and / or decreases the volume or size of the tumor containing cancer cells in tissues distal to the immunization site. In another embodiment, the cancer vaccine induces a tumor-specific memory T cell response. In yet another embodiment, the cancer vaccine increases 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. In yet another embodiment, the cancer vaccine increases the percentage of splenic CD8+ T CM cells. In another embodiment, the cancer vaccine increases the percentage of CD8+ T EM cells in the spleen and / or lymph nodes. 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 cells. In another embodiment, the cancer vaccine increases the amount of tumor-infiltrating CD8+ T CM cells and / or CD8+ T EM cells. In yet another embodiment, the cancer cells do not replicate. In yet another embodiment, the cancer cells do not replicate for radiation treatment. In another embodiment, the radiation treatment is a sub-lethal dose.

[0011] In yet another embodiment, the cancer vaccine is administered to a subject in combination with immunotherapy and / or cancer therapy, and optionally, the immunotherapy and / or cancer therapy is administered before, after, or simultaneously with the cancer vaccine. In yet another embodiment, the immunotherapy is cell-based. In another embodiment, the immunotherapy comprises a cancer vaccine and / or a virus. In yet another embodiment, the immunotherapy inhibits an immune checkpoint. 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, butyrophilin, and A2aR. In 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 radiation, radiosensitizers, and chemotherapeutic agents.

[0012] In yet another aspect, a method for evaluating the efficacy of the cancer vaccine for treating a subject suffering from cancer, comprising: (a) detecting the number of proliferating cells in the cancer and / or the volume or size of a 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 a tumor containing the cancer cells detected in steps (a) and (b), wherein the absence or significant decrease in the number of proliferating cells in the cancer and / or the volume or size of a tumor containing the cancer cells in the subsequent sample as compared to the number and / or volume or size in the sample at the first time point indicates that the cancer of the subject is being treated by the cancer vaccine. A method is provided herein. In one embodiment, between the first time point and the subsequent time point, the subject has received treatment, completed treatment, and / or is in a remission state with respect to the cancer. In another embodiment, the first sample and / or at least one subsequent sample is 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 part of a single sample or a part of a pooled sample obtained from the subject. In yet another embodiment, the sample comprises cells, serum, peripheral lymphoid organs, and / or intratumoral tissue obtained from the subject. In another embodiment, the method described herein further comprises determining the responsiveness to the agent by evaluating at least one criterion selected from the group consisting of clinical utility rate, survival period until death, pathological complete response, semi-quantitative pathological response scale, clinical complete remission, clinical partial remission, clinical disease stabilization, recurrence-free survival period, metastasis-free survival period, disease-free survival period, decrease in 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, a particular embodiment is applicable to any aspect of the invention described herein. For example, in one embodiment, the cancer vaccine prevents recurrent tumor lesions 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 remission state with respect to the cancer. In another embodiment, the mammal is a mouse or a human. For example, the mammal is a human.

Brief Description of the Drawings

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

[0016] In this specification, PTEN and p53-deficient tumor cells having activated TGFβ-Smad / p63 signaling (e.g., treated with at least one type of TGFβ superfamily protein) have been shown to fail to form tumors in a T cell-dependent manner in hosts with normal immunity. For example, by treating tumor cells derived from a syngeneic mouse breast tumor model generated by simultaneous deficiency of p53 and Pten with TGFβ in vitro, the tumor-forming ability of these cells was completely suppressed in a T cell-dependent manner in mice with normal immunity. These cells have also been shown to induce strong antitumor immunity through the binding and activation of dendritic cells (DCs) and subsequent activation of T cells against target tumor cells. Further, p63 was found to be an important cofactor for TGFβ / Smad-mediated transcription in response to TGFβ stimulation. For example, activation of the TGFβ-Smad / p63 axis upregulates transcriptional production that induces activation of multiple immune pathways, and these effects disappeared when either p63 or Smad2 was lacking. Additionally, administration of tumor cells having activated TGFβ-Smad / p63 signaling protects the host from recurrent and metastatic tumor lesions through induction of a long-term memory T cell response. It was also found that the survival rate of breast cancer patients highly correlates with the TGFβ-Smad / p63 signature. From these results, a new molecular switch underlying the opposing actions of TGFβ in tumorigenesis has been discovered, providing a strategy 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) deficient in Pten, (2) deficient in p53, and (3) modified to activate the TGFβ-Smad / p63 signaling pathway. Also provided is a method for evaluating the efficacy of the cancer vaccine for preventing and / or treating cancer.

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

[0018] The term "administering" is intended to encompass the route of administration by which a drug is caused to perform its intended function. Examples of routes of administration for the treatment of the body that can be used include injection (subcutaneous injection, intravenous injection, parenteral injection, intraperitoneal injection, intrathecal injection, etc.), oral route, inhalation route, and transdermal route. The injection can be a bolus injection or a continuous infusion. The drug may be coated or placed in a selected material depending on the route of administration to protect the drug from its natural state that may have an adverse effect on its ability to perform its intended function. 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 term "change in amount" or "change in level" refers to the copy number of the biomarker nucleic acid (e.g., germline and / or somatic) that has increased or decreased compared to the expression level or copy number of the biomarker nucleic acid in a control sample, e.g., the increased or decreased expression level in a cancer sample. The term "change in amount" of a biomarker also includes the increased or decreased protein level of the biomarker protein in a sample, e.g., a cancer sample, when compared to the corresponding protein level in a normal control sample. Furthermore, the change in the amount of a biomarker protein can be determined by detecting post-translational modifications such as the methylation state of the marker that may affect the expression or activity of the biomarker protein.

[0020] If the amount of the biomarker in question is greater than the standard error of the assay used for the assessment of the amount by an amount that is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or greater than that amount, or less than the normal level by such an amount, then the amount of that biomarker is "significantly" greater or less, respectively, than the normal amount of that biomarker. Alternatively, if the amount of that biomarker in the subject is at least about 2 times, preferably at least about 3 times, 4 times, or 5 times greater or less than the normal amount of that biomarker, then the amount of that biomarker may be considered to be "significantly" greater or less. Such "significance" may also apply to any other measurement parameters described herein, such as measurement parameters for expression, inhibition, cytotoxicity, cell proliferation, etc.

[0021] The term "change in expression level" of a biomarker refers to the expression level or copy number of the biomarker in a test sample, such as a sample derived from a patient with cancer, that is more or less than the standard error of the assay used to evaluate the expression or copy number, and is preferably at least 2 times, more preferably 3 times, 4 times, 5 times, or 10 times or more the expression level or copy number of the biomarker in a control sample (e.g., a sample derived from a healthy subject without a disease), preferably the average expression level or copy number of the biomarker in several control samples. This change in expression level is greater or smaller than the standard error of the assay used to evaluate the expression or copy number, and is preferably 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 the expression level or copy number of the biomarker in a control sample (e.g., a sample derived from a healthy subject without a disease), preferably 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 an unphosphorylated biomarker).

[0022] The term "change in activity" of a biomarker refers to the activity of the biomarker that is increased or decreased in a disease state, such as in a cancer sample, when compared to the activity of the biomarker in a normal control sample. The change in activity of the biomarker can be the result of, for example, a change in the expression of the biomarker, a change in the protein level of the biomarker, a change in the structure of the biomarker, or a change in the interaction of the biomarker with other proteins involved in the same or different pathways, or a change in the interaction of the biomarker with a transcriptional activator or transcriptional repressor.

[0023] The term "structural change" of a biomarker refers to the presence of a mutation or allelic variation within a biomarker nucleic acid or biomarker protein when compared to a normal or wild-type gene or protein, for example, the presence of a mutation that affects the expression or activity of that biomarker nucleic acid or biomarker protein. For example, mutations include, but are not limited to, substitution, deletion, or addition mutations. The mutation may be present in the coding region or non-coding region of the biomarker nucleic acid.

[0024] Unless otherwise defined herein, the terms "antibody" and "antibodies" 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 foregoing antibodies that have at least an antigen-binding site. Antibody derivatives may include a protein or chemical moiety conjugated to the antibody.

[0025] Also, intracellular antibodies are well-known antigen-binding molecules that have the characteristics of antibodies but can be expressed intracellularly to bind and / or inhibit a target intracellular target (Chen et al., (1994) Human Gene Ther., Vol. 5: 595-601). Methods for applying antibodies to target (e.g., inhibit) intracellular moieties, such as the use of single-chain antibodies (scFv), modification of immunoglobulin VL domains for enhanced stability, modification of antibodies to resist the reducing intracellular environment, generation of fusion proteins to increase intracellular stability, and / or regulation of intracellular localization, are well-known in the art. It is also possible to introduce and express intracellular antibodies into one or more cells, tissues, or organs of a multicellular organism, for example, for prophylactic and / or therapeutic purposes (e.g., as gene therapy) (at least WO 08 / 020079, WO 94 / 02610, WO 95 / 22618, and WO 03 / 014960, US Patent No. 7004940, Cattaneo and Biocca, (1997) Intracellular Antibodies: Development and See Applications (Landes and Springer-Verlag), Kontermann, Methods, Vol. 34: 163-170 (2004), Cohen et al., Oncogene, Vol. 17: 2445-2456 (1998), Auf der Maur et al., FEBS Lett., Vol. 508: 407-412 (2001), Shaki-Loewenstein et al., J. Immunol. Meth., Vol. 303: 19-39 (2005).

[0026] As used herein, the term "antibody" includes the "antigen-binding portion" of the antibody (or simply the "antibody portion"). As used herein, the term "antigen-binding portion" refers to one or more fragments of an antibody that retain 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 within the term "antigen-binding portion" of an antibody include (i) the Fab fragment, which is a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain; (ii) the F(ab’)2 fragment, which is a divalent fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment, which consists of the VH domain and the CH1 domain; (iv) the Fv fragment, which consists of the single-chain VL domain and VH domain of one arm of the antibody; (v) the dAb fragment, which consists of the VH domain (Ward et al., (1989) Nature, Vol. 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide using recombinant DNA methods (known as single-chain Fv (scFv). See, for example, Bird et al., (1988) Science, Vol. 242:423-426, and Huston et al., (1988) Proc. Natl. Proceedings of the National Academy of Sciences of the United States of America, Vol. 85: pp. 5879 - 5883, and Osbourn et al., Nature Biotechnology, Vol. 16: p. 778, 1998). Such single-chain antibodies are also considered to be within the scope of the term "antigen-binding portion" of an antibody. Any VH and VL sequences of a particular scFv can be ligated to the cDNA or genomic sequence of a human immunoglobulin constant region to generate an expression vector encoding a complete IgG polypeptide or other isotype. VH and VL can also be used for the production of 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 the VH and VL domains are expressed on a single polypeptide chain but are paired with complementary domains on another chain by using a linker that is too short to allow pairing between those two domains on the same chain, thereby forming two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 90: pp. 6444 - 6448, 1993; Poljak et al., Structure, Vol. 2: pp. 1121 - 1123, 1994).

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

[0028] The antibody can be polyclonal or monoclonal, and can be heterologous, allogeneic, or syngeneic, or modified forms thereof (e.g., humanized, chimeric, etc.). The antibody can be a fully human antibody. The antibodies of the present invention preferably bind specifically or substantially specifically to a biomarker polypeptide or fragment thereof. As used herein, the terms “monoclonal antibody” and “monoclonal antibody composition” refer to a population of antibody polypeptides that contain only one type of 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 population of antibody polypeptides that contain multiple types of antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the specific antigen with which they immunoreact.

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

[0030] The term "biomarker" refers to a measurable substance of the invention that is determined to predict the effect of a cancer therapy. Biomarkers can include, but are not limited to, nucleic acids (e.g., genomic nucleic acids and / or transcribed nucleic acid products) and proteins. A number of 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 antibodies or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological activity of the antigen.

[0032] The term "body fluid" refers to fluids excreted or secreted from the body as well as fluids that are not normally excreted or secreted (e.g., amniotic fluid, aqueous humor, cerebrospinal fluid, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's gland fluid or pre-ejaculatory fluid, breast milk, bile, feces, smegma, interstitial fluid, intracellular fluid, lymph, menstrual blood, milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubricant, vitreous humor, vomit).

[0033] The terms "cancer", "tumor", or "hyperplasia" refer to the presence of cells having characteristics typical of cancer-causing cells, such as unrestricted growth, immortality, metastatic ability, rapid growth rate, and certain unique morphological features.

[0034] Cancer cells often form tumors, but such cells can exist alone within an animal or may be non-tumorigenic cancer cells such as leukemia cells. As used herein, the term "cancer" includes pre-malignant and malignant cancers. Cancers include B-cell cancers such as multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases such as alpha-chain disease, gamma-chain disease, and mu-chain disease, benign monoclonal gammopathy and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, urothelial bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine body cancer or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, cholangiocarcinoma, small intestine cancer or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancers of hematological tissues, etc., but are not limited thereto. Other non-limiting examples of types of cancer applicable to the methods within the scope of the present invention include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, angioendotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing 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, bronchiogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia such as acute lymphocytic leukemia and acute myeloblastic leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström type macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colorectal 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 still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell cancer, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. The characteristics of the epithelial cancer can be described in various other respects including, but 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 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., 5' untranslated region and 3' untranslated region).

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

[0037] As used herein, the terms "co-therapy" and "combination therapy" refer to the administration of two or more therapeutic substances. Those different agents that make up the combination therapy can be administered simultaneously with, before, or after the administration of one or more therapeutic agents.

[0038] The term "control" refers to any reference standard suitable 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 the 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 control samples may include any suitable sample, such as samples from control cancer patients with known outcomes (which may be archived samples or previous sample measurement results), normal tissues or cells isolated from subjects such as healthy subjects or cancer patients, primary cultured cells / tissues isolated from subjects such as healthy subjects or cancer patients, adjacent normal cells / tissues obtained from the same organ or body part of a cancer patient, tissue samples or cell samples isolated from healthy subjects, or primary cells / primary tissues obtained from a depository institution, but are not limited thereto. In another preferred embodiment, the control may include the levels of expression products as reference standards derived from any suitable source, such as housekeeping genes, the range of expression product levels in normal tissues (or other previously analyzed control samples), the range of expression product levels previously determined in the test samples of a group of patients, or the levels of expression products of a series of patients having a particular outcome (e.g., survival for 1 year, 2 years, 3 years, or 4 years, etc.) or having received a particular treatment (e.g., standard cancer treatment), but are not limited thereto. Those skilled in the art will understand that in the method of the present invention, such control samples and the levels of expression products as reference standards can be used in combination as controls. In one embodiment, the control may include normal cell / tissue samples or non-cancerous cell / tissue samples. In another preferred embodiment, the control may include the expression levels of a series of patients, such as a series of cancer patients, or the expression levels of a series of cancer patients receiving a particular treatment, or the expression levels of a series of patients having one outcome compared to another outcome. In the former example, the level of the expression product of each patient can be specified as a certain percentile of the expression level or expressed as higher or lower than the average or mean of the reference standard expression level. In another preferred embodiment, the control may include normal cells, cells of patients treated with combination chemotherapy, and cells of patients with benign cancers.In another embodiment, the control may also include the average expression level of a specific gene in the same population compared to a measured value, such as the expression level of a housekeeping gene in a population. Such a population may include healthy subjects, cancer patients who have not received any treatment (i.e., treatment-naive), cancer patients receiving standard treatment, or patients with benign cancer. In another preferred embodiment, the control includes a conversion of the ratio of the levels of the expression products, which includes determining the ratio of the levels of the expression products 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 levels of the expression products of two or more genes in the test sample, determining the difference in the levels of the expression products in any appropriate control, and determining the levels of the expression products of two or more genes in the test sample, normalizing the expression of the genes against the expression of a housekeeping gene in the test sample, and comparing this to any appropriate control, but is not limited thereto. In a particularly preferred embodiment, the control includes a control sample that is of the same lineage and / or type as the test sample. In another embodiment, the control may include the levels of the expression products grouped as percentiles or percentiles based on those samples in a series of patient samples such as all cancer patients. In one embodiment, for example, the level of the expression product is set as a control such that whether the level of the expression product is higher or lower than a specific percentile is used as a criterion for outcome prediction. In another preferred embodiment, the level of the expression product as a control is set using the level of the expression product of a cancer control patient with a known outcome, and the level of the expression product derived from the test sample is compared to that control expression product level as a criterion for outcome prediction. As demonstrated by the following data, the method of the present invention is not limited to using a specific cut-off point when comparing the level of the expression product in the test sample to the control.

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

[0040] The term "immune cell" 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 precursors, monocytes) are the "big eaters" of the immune system. These cells, which come in various guises 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. At the time of tissue injury or infection, monocytes are rapidly recruited to the tissue, where they differentiate into tissue macrophages. Macrophages are remarkably plastic and can change their functional phenotype in response to environmental cues they receive. Through their ability to eliminate pathogens and instruct other immune cells, these cells play a central role in protecting the host, but 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 and secrete high levels of IL-12 and low levels of IL-10. M2 is the phenotype of resident tissue macrophages and can be further increased by IL-4. M2 macrophages produce high levels of IL-10, TGFβ, and low levels of IL-12. Tumor-associated macrophages are mainly macrophages of the M2 phenotype and appear to actively promote tumor growth.

[0042] Myeloid-derived suppressor cells (MDSCs) are an essential part of the myeloid cell lineage and are a heterogeneous population composed of progenitor cells of myeloid cells as well as granulocytes, macrophages, and dendritic cell precursors. MDSCs are defined by their myeloid origin, immature state, and ability to potently suppress T cell responses. MDSCs control immune responses and tissue repair in healthy individuals, and their population rapidly expands during inflammation, infection, and cancer. MDSCs are one of the major components of the tumor microenvironment. The main feature of these cells is their potent immunosuppressive activity. MDSCs are generated in the bone marrow and migrate to peripheral lymphoid organs and tumors in tumor-bearing hosts to contribute to the formation of the tumor microenvironment. This process is controlled by a set of defined chemokines that are upregulated in many cancers. Hypoxia seems to play an important role in the regulation of MDSC differentiation and function in tumors. Currently, therapeutic strategies targeting MDSCs are being developed to promote antitumor 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, mucosa, and lymphoid tissues. The main function of dendritic cells is to process antigens and present the processed antigens to T cells to promote immunity against foreign antigens and tolerance to self-antigens. Dendritic cells also secrete cytokines to control immune responses.

[0044] Conventional T cells, also known as Tconv or Teff, have effector functions (e.g., cytokine secretion, cytotoxic activity, self-recognition suppression, 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. "Naive Tcon" is a CD4 + T cell that has differentiated in the bone marrow and successfully undergone positive and negative thymic selection processes 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, naive Tcon is considered to be in a quiescent and non-dividing state and requires interleukin-7 (IL-7) and interleukin-15 (IL-15) for constant survival (see at least WO 2010 / 101870). In the context of immune response suppression, 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 "immunological therapy" refers to any treatment that uses certain parts of a subject's immune system to fight diseases such as cancer. For this purpose, one or more drugs are administered or not, and the subject's own immune system is stimulated (or suppressed). Immunotherapy designed to induce or increase an immune response is called "activating immunotherapy". Immunotherapy designed to reduce or suppress an immune response is called "suppressive immunotherapy". Any drug that is thought to have an effect on the immune system against transplanted genetically modified cancer cells can be assayed to determine whether the drug is an immunotherapy and whether a given genetic modification has an effect on the regulation of the immune response. In some embodiments, the immunotherapy is cancer cell specific. In some embodiments, the immunotherapy can be "non-targeted", which refers to the administration of drugs that do not selectively interact with immune system cells but still regulate immune system function. Representative examples of non-targeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0046] Immunotherapy is a form of targeted therapy that can 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 oncolytic viruses potentially useful in cancer therapy. The replication of oncolytic viruses destroys and promotes the growth of tumor cells and amplifies the virus titer at the tumor site. Oncolytic viruses can also act as vectors for anti-cancer genes and specifically deliver those anti-cancer genes to the tumor site. The immunotherapy can include passive immunity for short-term protection of the host, which is achieved by administration of pre-formed antibodies against cancer antigens or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens conjugated with chemotherapeutic agents or toxins if desired). For example, anti-VEGF inhibitors and anti-mTOR inhibitors are known to be effective in the treatment of renal cell carcinoma. Immunotherapy can also focus on the utilization of cytotoxic lymphocyte recognition epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc. can be used to selectively regulate biomolecules related to the occurrence, progression, and / or pathological conditions of tumors or cancers.

[0047] The immunotherapy can include passive immunity for short-term protection of the host, which is achieved by administration of pre-formed antibodies against cancer antigens or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens conjugated with chemotherapeutic agents or toxins if desired). Immunotherapy can also focus on the utilization of cytotoxic lymphocyte recognition epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc. can be used to selectively regulate biomolecules related to the occurrence, progression, and / or pathological conditions of tumors or cancers.

[0048] In some embodiments, the immunotherapy comprises one or more inhibitors of immune checkpoint(s). 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 anti-tumor immune response. Immune checkpoint proteins are well known in the art, and examples of such 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, butyrophilin, and A2aR (see, e.g., WO 2012 / 177624). The term further encompasses biologically active proteins as well as nucleic acids encoding full-length immune checkpoint proteins and biologically active fragments thereof. In some embodiments, the term further encompasses any fragment that follows the complementary description presented herein. In one embodiment, the immune checkpoint is PD-1.

[0049] "Immune checkpoint inhibition therapy" refers to the use of agents that inhibit immune checkpoint nucleic acids and / or proteins. To more effectively treat cancer, inhibitory signal transduction can be disrupted or otherwise neutralized by inhibiting one or more immune checkpoints, thereby upregulating the immune response. Examples of agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands that can bind and / or inactivate or inhibit immune checkpoint proteins or fragments thereof, as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and / or activity of immune checkpoint nucleic acids or fragments thereof. Examples of agents for upregulating the immune response include antibodies against those proteins that interfere with the interaction between one or more immune checkpoint proteins and their natural receptors, inactivated forms of one or more 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 natural receptors, fusion proteins that bind to the natural receptor (e.g., the extracellular portion of an immune checkpoint inhibitory protein fused to the Fc portion of an antibody or immunoglobulin), nucleic acid molecules that interfere with the transcription or translation of immune checkpoint nucleic acids, etc. Such agents can directly interfere with the interaction between the one or more immune checkpoints and their natural receptors (e.g., antibodies) to prevent inhibitory signal transduction and upregulate the immune response. Alternatively, the agent can indirectly interfere with the interaction between one or more immune checkpoint proteins and their natural receptors to prevent inhibitory signal transduction and upregulate the immune response. 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 that binds to the appropriate ligand. In one embodiment, anti-PD-1 antibodies, anti-PD-L1 antibodies, and / or anti-PD-L2 antibodies are used alone or in combination to inhibit immune checkpoints.These embodiments are also applicable to specific therapies for specific immune checkpoints such as the PD-1 pathway (e.g., anti-PD-1 pathway therapy, also known as PD-1 pathway inhibitor therapy).

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

[0051] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to generate an immune response against a tumor or cancer in its subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0052] The term "inhibit" includes, for example, a decrease, reduction, limitation, and / or interference with a particular action, function, and / or interaction. In some embodiments, the interaction between two molecules is "inhibited" if the interaction is reduced, disrupted, perturbed, 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, retarded, or prevented.

[0054] The term "interaction," when referring to the interaction between two molecules, refers to their mutual physical contact (e.g., binding) of those molecules. As a result of such an interaction, activation (resulting in a biological effect) of one or both of those molecules generally occurs.

[0055] "Isolated protein" refers to a protein that is substantially free of other proteins, other cellular materials, separation media, and culture media when isolated from cells or produced by recombinant DNA technology, or a protein that is substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or a biologically active portion thereof is substantially free of cellular materials or other contaminating proteins that originate from the cells or tissues from which the antibody, polypeptide, peptide, or fusion protein is derived, or is substantially free of chemical precursors or other chemicals when chemically synthesized. The phrase "substantially free of cellular materials" includes preparations in which the biomarker polypeptide or fragment thereof has been separated from the cellular components of the cells from which it is isolated or produced by recombinant techniques. In one embodiment, the phrase "substantially free of cellular materials" includes preparations of a biomarker protein or fragment thereof having less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as "contaminating protein"), more preferably less than about 20% of non-biomarker protein, even more preferably less than about 10% of non-biomarker protein, and most preferably less than about 5% of non-biomarker protein. When an antibody, polypeptide, peptide, or fusion protein, or a fragment thereof, e.g., a biologically active fragment thereof, is produced by recombinant techniques, it is also preferred that the biologically active fragment be substantially free of culture media, i.e., the culture media is 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 antibody classes (e.g., IgM, IgG1, IgG2C, etc.) encoded by heavy chain constant region genes.

[0057] The expression of a biomarker at the "normal" level is the expression level of that biomarker in cells of a subject not suffering from cancer, such as cells of a human patient. "Overexpression" or "significantly high level of expression" of a biomarker refers to the expression level in a test sample that is greater than the standard error of the assay used for the evaluation of 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 derived from a healthy subject not having a disease associated with that biomarker), more preferably at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or higher than the average expression level of that biomarker in several control samples. "Significantly low level of expression" of a biomarker refers to the expression level in a test sample that is preferably at least 10% lower than the expression level of that biomarker in a control sample (e.g., a sample derived from a healthy subject not having a disease associated with that biomarker), more preferably at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or lower than the average expression level of that biomarker in several control samples.

[0058] "Overexpression" or "significantly high level of expression" of a biomarker refers to the expression level in a test sample that is greater than the standard error of the assay used to evaluate the 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 derived from a healthy subject without the disease associated with that biomarker), more preferably 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or higher. "Significantly low level of expression" of a biomarker refers to the expression level in a test sample that is preferably at least 10% lower than the expression level of that biomarker in a control sample (e.g., a sample derived from a healthy subject without the disease associated with that biomarker), more preferably 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or lower.

[0059] The term "predict" includes using the status of biomarker nucleic acids and / or biomarker proteins before, during, or after treatment, such as increased or decreased tumor activity, appearance, expression, proliferation, quiescence, recurrence, or resistance, to determine a possible response of cancer to a cancer vaccine alone or in combination with immunotherapy and / or cancer therapy. Such use of said biomarkers for prediction is, for example, (1) an increase or decrease in copy number in, for example, more than about 5%, more than about 6%, more than about 7%, more than about 8%, more than about 9%, more than about 10%, more than about 11%, more than about 12%, more than about 13%, more than about 14%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 95%, more than about 100%, or more than that proportion of samples of the assayed human cancer type or cancer sample (e.g., by FISH, FISH plus SKY, single molecule sequencing as described, for example, in at least J. Biotechnol. Vol. 86: 289-301, or by qPCR), overexpression or underexpression of the biomarker nucleic acid (e.g., by ISH, Northern blot, or qPCR), an increase or decrease in the biomarker protein (e.g., by IHC), or an increase or decrease in activity, (2) the absolute or relative presence or absence of said biomarker in a biological sample, such as a sample containing a subject suffering from cancer, such as human tissue, whole blood, serum, plasma, buccal swab, saliva, cerebrospinal fluid, urine, feces, or bone marrow, (3) may be confirmed by the absolute or relative presence or absence of said biomarker in a small clinical population of cancer patients (e.g., a small population of patients responsive to a cancer vaccine alone or in combination with immunotherapy and / or cancer therapy, or patients developing resistance to that treatment).

[0060] The terms "prevent", "preventing", "prevention", "preventive treatment", etc. refer to reducing the probability of a subject developing a disease, disorder, or symptom, who does not have the subject's disease, disorder, or symptom but is at risk of developing it or is likely to develop it.

[0061] The terms "cancer response", "response to immunotherapy", or "response to modifiers of T cell-mediated cytotoxicity / immunotherapy combination therapy" refer to any response of a hyperproliferative disorder (e.g., cancer) to modifiers of T cell-mediated cytotoxicity and anti-cancer agents such as immunotherapy, preferably referring to changes in tumor mass and / or tumor volume after the initiation of neoadjuvant therapy or adjuvant therapy. The hyperproliferative disorder response may be evaluated, for example, with respect to efficacy or in the context of neoadjuvant therapy or adjuvant therapy, and the size of the tumor after systemic intervention can be compared to the initial size and dimensions measured by CT, PET, mammography, ultrasound, or palpation. The response can also be evaluated by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. The response can be recorded quantitatively, such as by the rate of change in tumor volume, or qualitatively, such as by "pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical disease stability" (cSD), "clinical disease progression" (cPD), or other qualitative criteria. Evaluation of the hyperproliferative disorder response can be performed early after the initiation of neoadjuvant therapy or adjuvant therapy, for example, after several hours, days, weeks, or preferably months. A typical endpoint for response evaluation is at the end of neoadjuvant chemotherapy or at the time of surgical removal of residual tumor cells and / or the tumor bed. This is typically 3 months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the treatment regimens described herein can be determined by evaluation of 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 stability (SD) at a time point at least 6 months after the end of treatment. A simplified notation of 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 a higher percentage.Other criteria for evaluating response to cancer therapy are related to "survival" and include the survival period to death, also known as overall survival (where the death can be any cause of death or tumor-related death), "recurrence-free survival" (where the term recurrence includes both local and distant recurrence), metastasis-free survival, and disease-free survival (where the term disease includes cancer and diseases associated with cancer). The survival period can be calculated by referring to a predetermined starting point (e.g., at diagnosis or at the start of treatment) and an end point (e.g., death, recurrence, or metastasis). Also, the criteria for treatment effectiveness can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence. For example, a specific cancer treatment plan can be implemented for a population of subjects to determine appropriate thresholds, and the outcome can be correlated with biomarker measurements determined prior to the implementation of any cancer therapy. The evaluation item can be the pathological response to treatment implemented in the context of neoadjuvant therapy. Alternatively, for subjects after cancer therapy with known biomarker measurements, evaluation items such as overall survival and disease-free survival may be monitored over a certain period. In certain embodiments, the dose administered is the standard dose known in the art for cancer therapeutics. The period for which the subject is monitored can vary. For example, the subject can be monitored for at least 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 14 months, 16 months, 18 months, 20 months, 25 months, 30 months, 35 months, 40 months, 45 months, 50 months, 55 months, or 60 months. The biomarker measurement threshold correlated with the outcome of cancer therapy can be determined using methods well-known in the art, such as the methods described in the examples.

[0062] The term "resistance" refers to acquired or natural resistance (i.e., being non-responsive to treatment or having a suppressive or limited response) of a cancer sample or mammal to a cancer therapy, for example, a response reduced by a rate of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or higher, for example, by a factor of 2, 3, 4, 5, 10, 15, 20, or higher, or any rate within a range including both ends. The reduction in the response can be evaluated by comparison with the same cancer sample or mammal before the resistance is 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 "multi-drug resistance". Multi-drug resistance may involve P-glycoprotein or other mechanisms, or may occur when a mammal is infected with multi-drug resistant microorganisms or a mixed population of microorganisms. Determination of resistance to treatment is routine in the art and within the skill of a skilled physician, and can be evaluated, for example, by cell proliferation assays and cell death assays as described herein as "sensitive". 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 at 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 taken when the untreated tumor is logarithmically growing.

[0063] The terms "response" or "responsiveness" refer to cancer response in the sense of a decrease in tumor size or inhibition of tumor growth. These terms can also refer to an improvement in prognosis, as reflected, for example, by an increase in the time to recurrence, which is the period until the first recurrence excluding the second primary cancer or death without evidence of recurrence as the first event, or an increase in overall survival, which is the period until death from any cause after treatment. Responding or having a response means that there is a beneficial endpoint achieved when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated upon exposure to the stimulus. It should be understood that assessing the likelihood that a tumor or subject will exhibit 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 non-responsive).

[0064] As used herein, "RNA interfering agent" is defined as any agent that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, RNA molecules homologous to the target biomarker gene of the invention or fragments of those molecules, short interfering RNAs (siRNAs), and nucleic acid molecules comprising small molecules that interfere with or inhibit the expression of the target biomarker nucleic acid by RNA interference (RNAi).

[0065] "RNA interference (RNAi)" is an evolutionarily conserved process in which sequence-specific degradation or sequence-specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from a target biomarker nucleic acid occurs by the expression or introduction of RNA having a sequence identical or very similar to the target biomarker nucleic acid (see Coburn and Cullen, J. Virol. (2002), Vol. 76:9225), 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 plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by Dicer, a dsRNA-specific endonuclease, which promotes the processing cleavage of long dsRNA into double-stranded fragments called small interfering RNAs (siRNAs). The 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 suppress 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 decrease in the expression or protein activity or level of the target biomarker nucleic acid or the protein encoded by the target biomarker nucleic acid. The decrease can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more as compared to the expression of the target biomarker nucleic acid not targeted by the RNA interference agent or the activity or level of the protein encoded by the target biomarker nucleic acid.

[0066] In addition to RNAi, genome editing can be used to modify the copy number of a biomarker of interest or the gene sequence, for example, to introduce successive or inducible knockouts or mutations of the biomarker of interest. For example, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g., for the creation of non-functional mutations or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes can be expressed. For example, a vector containing only this guide RNA can be administered to a Cas9 enzyme transgenic animal or cell. Similar strategies (e.g., designer zinc fingers, transcription activator-like effectors (TALE), or homing meganucleases) may be used. Such systems are well known in the art (see, for example, U.S. Patent No. 8,697,359, Sander and Joung, (2014) Nat. Biotech., Vol. 32:347-355, Hale et al., (2009) Cell, Vol. 139:945-956, Karginov and Hannon, (2010) Mol. Cell, Vol. 37:7, U.S. Patent Application Publication Nos. 2014 / 0087426 and 2012 / 0178169, Boch et al., (2011) Nat. Biotech., Vol. 29:135-136, Boch et al., (2009) Science, Vol. 326:1509-1512, Moscou and Bogdanove, (2009) Science, Vol. 326:1501, Weber et al., (2011) PLoS One, Vol. 6:e19722, Li et al., (2011) Nucl. Acids Res., Vol. 39:6315-6325, Zhang et al., (2011) Nat. Biotech., Vol. 29:149-153, Miller et al., (2011) Nat. Biotech., Vol. 29:143-148, Lin et al., (2014) Nucl. Acids Res., Vol. 42:e47). Such genetic strategies can use 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 their interaction with Piwi proteins. These piRNA complexes are associated with both epigenetic silencing and post-transcriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly retrotransposons and other genetic elements during spermatogenesis. piRNAs differ from microRNAs (miRNAs) in terms of size (rather than 21 - 24 nt but rather 26 - 31 nt), lack of sequence conservation, and increased complexity. However, like other short RNAs, piRNAs are thought to be involved in gene silencing, specifically the silencing of transposons. Most piRNAs are antisense to transposon sequences, suggesting that transposons are the targets of piRNAs. In mammals, the activity of piRNAs in transposon silencing seems to be most important during embryonic development, and in Caenorhabditis 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 molecule or a peptide molecule that binds to a specific target molecule. A "nucleic acid aptamer" is a nucleic acid designed by repeating in vitro selection to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, or by an equivalent SELEX (systematic evolution of ligands by exponential enrichment). A "peptide aptamer" is an artificial protein selected or designed to bind to a specific target molecule. These proteins are composed of one or more peptide loops consisting of variable sequences presented by a protein scaffold. These aptamers are typically isolated from a combinatorial library and are often improved by direct mutagenesis or multiple rounds of variable region mutagenesis and selection. An "affimer protein", which is an evolved form of a peptide aptamer, is a very stable small protein designed to present a peptide loop that provides a high-affinity binding surface for a specific target protein. Affimer proteins are low molecular weight proteins of 12 - 14 kDa derived from the cystatin cysteine protease inhibitor family. Aptamers are useful in biotechnological and therapeutic applications because they provide molecular recognition properties comparable to those of antibodies, which are commonly used biomolecules. Aptamers offer advantages over antibodies in addition to fine discrimination because they can be fully engineered in vitro, are easily produced by chemical synthesis, have desirable storage properties, and induce little or no immunogenicity in therapeutic applications.

[0069] As used herein, the term "intracellular immunoglobulin molecule" refers to a complete immunoglobulin that is the same as a naturally secreted immunoglobulin but remains inside the cell after synthesis. "Intracellular immunoglobulin fragment" refers to any fragment that includes a single-chain fragment of an intracellular immunoglobulin molecule. Thus, an intracellular immunoglobulin molecule or fragment thereof is not secreted or expressed on the outer surface of the cell. A single-chain intracellular immunoglobulin fragment is referred to herein as a "single-chain immunoglobulin." As used herein, the term "intracellular immunoglobulin molecule or fragment thereof" is understood to encompass "intracellular immunoglobulin," "single-chain intracellular immunoglobulin" (or fragment thereof), "intracellular immunoglobulin fragment," "intracellular antibody" (or fragment thereof), and "intracellular antibody" (or fragment thereof). Thus, the terms "intracellular immunoglobulin," "intracellular Ig," "intracellular antibody," and "intracellular antibody" may be used interchangeably herein and all are encompassed by the general definition of "intracellular immunoglobulin molecule, or fragment thereof." In some embodiments, the intracellular immunoglobulin molecule or fragment thereof of the invention may include two or more subunit polypeptides, such as 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" that includes only a single polypeptide. As used herein, a "single-chain intracellular immunoglobulin" is defined as any single fragment having a desired activity, such as intracellular binding to an antigen. Thus, single-chain intracellular immunoglobulins include single-chain intracellular immunoglobulins that include a heavy-chain variable region and a light-chain variable region that act together to bind an antigen, as well as single-chain intracellular immunoglobulins having only a single variable region that binds an antigen, such as a "camelized" heavy-chain variable region as described herein. An intracellular immunoglobulin or Ig fragment can be expressed substantially anywhere within the cell, such as in the cytoplasm, on the inner surface of the cell membrane, or within an intracellular compartment (also referred to as a cellular sub-compartment or cell compartment) such as the cell nucleus, Golgi apparatus, endoplasmic reticulum, endosome, mitochondria, etc.Other cell compartments include those described herein and well known in the art.

[0070] The term "sample" used for detecting or determining the presence or level of at least one biomarker typically includes whole blood, plasma, serum, saliva, urine, feces (e.g., stool), tears, and any other body fluid (e.g., body fluid as described in the definition of "body fluid" above), or tissue samples such as bone marrow samples and bone samples (e.g., biopsy samples), or surgically excised tissue. In certain instances, the methods of the invention further comprise obtaining the sample from an individual prior to detecting or determining the presence or level of at least one marker in the sample.

[0071] The term "sensitize" means to alter cancer cells or tumor cells such that an associated cancer is more effectively treated by cancer therapy (e.g., immune checkpoint inhibition therapy, chemotherapy, and / or radiation therapy). In some embodiments, normal cells are not acted upon to the extent that those normal cells are overly damaged by their treatment. The increase or decrease in sensitivity to treatment is measured according to methods known in the art for the particular treatments and methods described herein below, which methods include cell proliferation assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res, 1982, Vol. 42:2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res, 1984, Vol. 94:161-173, Weisenthal L M, Lippman M E, Cancer Treat Rep, 1985, Vol. 69:615-632, Kaspers G J L, Pieters R, Edited by Twentyman P R, Weisenthal L M, Veerman A J P, Drug Resistance in Leukemia and Lymphoma, Langhorne, Pennsylvania, Harwood Academic Publishers, 1993, pages 415 - 432, including the work of Weisenthal L M, written by Weisenthal L M, Contrib Gynecol Obstet, 1994, Vol. 19: 82 - 90), but not limited thereto. The sensitivity or resistance can also be evaluated in animals by measuring the tumor size over a certain period, for example, for humans over 6 months. A composition or method is sensitive to the treatment if the increase in treatment sensitivity or the decrease in treatment resistance is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a higher percentage, such as 2 - fold, 3 - fold, 4 - fold, 5 - fold, 10 - fold, 15 - fold, 20 - fold, or a higher multiple, or any rate within the range including both ends, compared to the treatment sensitivity or treatment resistance when such a composition or method does not exist. The determination of sensitivity or resistance to treatment is routine in the art and within the skill of a skilled physician. It should be understood that any method described herein for enhancing the efficacy of cancer therapy is equally applicable to methods for sensitizing hyperproliferative cells or otherwise cancerous cells (e.g., resistant cells) to that cancer therapy.

[0072] Short interfering RNA (siRNA) is also referred to herein as "small interfering RNA" and is defined as a factor that functions, for example, to inhibit the expression of a target biomarker nucleic acid by RNA interference (RNAi). siRNA can be chemically synthesized, produced by in vitro transcription, or produced within a host cell. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule that is about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides in length, more preferably about 19 to about 25 nucleotides in length, and even more preferably about 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length, and may include 3' and / or 5' overhangs of about 0 nucleotides, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, or 5 nucleotides in length on each strand. 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. The siRNA preferably 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 short hairpin (also called stem-loop) RNA (shRNA). In one embodiment, these shRNAs are composed of a short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and a similar sense strand. Alternatively, the sense strand may be present before the nucleotide loop structure and the antisense strand may follow it. These shRNAs may be contained within plasmids, retroviruses, and lentiviruses and may be expressed, for example, from a pol III U6 promoter or another promoter (see, e.g., Stewart et al., (2003) RNA, April; Vol. 9(4):493-501, which is incorporated herein by reference).

[0074] RNA interference agents, such as siRNA molecules, can be administered to a patient having cancer or at risk of having cancer to inhibit the expression of a biomarker gene that is overexpressed in the cancer, thereby treating, preventing, or suppressing the cancer in the subject.

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

[0076] The term "specific binding" refers to an antibody that binds to a given antigen. Typically, this antibody has an affinity (K -7 ) of less than about 10 D M, such as less than about 10 -8 M, 10 -9 M, or 10 -10binds with an affinity less than M or even higher, and binds to the predetermined antigen with an affinity that is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10.0-fold or more higher than the affinity for non-specific antigens other than the predetermined antigen or related antigens (e.g., BSA, casein). The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen". Selective binding is a relative term that refers 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 the term "patient".

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

[0079] The term "synergistic effect" refers to the fact that the combined effect of two or more anti-cancer agents (e.g., cancer vaccine combined with immunotherapy) can be higher than the sum of the effects of those anti-cancer agents / treatments alone.

[0080] The term "T cell" includes CD4 + T cells and CD8 + T cells. The term "T cell" also includes both type 1 T helper cells and type 2 T helper cells. The term "antigen-presenting cell" includes professional 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 local or systemic effect in an animal, specifically a mammal, more specifically a human, caused by a pharmacologically active substance. Thus, this term means any substance intended to be used in the diagnosis, care, alleviation, treatment, or prevention of disease in an animal or human, or in the enhancement of a desirable physical or mental development and condition. The phrase "therapeutically effective amount" means an amount of such a substance that produces some desirable local or systemic effect at a reasonable risk-benefit ratio applicable to any treatment. In certain embodiments, the therapeutically effective amount of a compound depends on its therapeutic index, solubility, etc. For example, a particular compound discovered by the methods of the present invention can be administered in an amount sufficient to produce a reasonable risk-benefit ratio applicable to such treatment.

[0082] As used herein, the terms "therapeutically effective amount" and "effective amount" mean an amount of a compound, material, or composition that includes a compound of the present invention that is effective to produce some desirable therapeutic effect in at least a small population of cells of an animal at a reasonable risk-benefit ratio applicable to any medical treatment. The toxicity and therapeutic efficacy of the subject compound can be determined by standard pharmacological techniques in cell culture or experimental animals, e.g., LD 50 and ED 50 techniques for determination. Compositions showing a high therapeutic index are preferred. In some embodiments, LD 50(Lethal dose) is measurable and can be reduced by the agent, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or a higher percentage compared to not administering the agent. Similarly, ED 50 (i.e., the concentration that achieves half-maximal suppression of symptoms) is measurable and can be increased by the agent, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or a higher percentage compared to not administering the agent. Also, similarly, IC 50 (i.e., the concentration that achieves half-maximal cytotoxic or cytostatic effect on cancer cells) is measurable and can be increased by the agent, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or a higher percentage compared to not administering the agent. In some embodiments, the growth of cancer cells 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, 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% reduction 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 are separated from chemical precursors or other chemicals involved in the synthesis of the protein. In one embodiment, the phrase "substantially free of chemical precursors or other chemicals" means having less than about 30% (on a dry weight basis) of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein, more preferably having less than about 20% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein, even more preferably having less than about 10% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein, and most preferably having less than about 5% of chemical precursors or non-antibody, polypeptide, peptide, or fusion protein, and includes preparations of antibodies, polypeptides, peptides, or fusion proteins.

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

[0085] The term "host cell" is intended to refer to a cell into which a nucleic acid within the scope of the present invention, e.g., a recombinant expression vector within the scope 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 the particular subject cell but also to the progeny of such a cell or cells that might be progeny. Because certain changes may occur in progeny due to either mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term as used herein.

[0086] The term "vector" refers to a nucleic acid that can transport another nucleic acid to which it is ligated. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can ligate additional DNA segments. Another type of vector is a viral vector, to which additional DNA segments can be ligated into its viral genome. Certain vectors are capable of autonomous replication in the introduced host cell (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and are thereby replicated with the host genome. Further, certain vectors can express genes that are ligated so as 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 vectors, the terms "plasmid" and "vector" may be used interchangeably herein. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0087] As used herein, the term "non-responsiveness" includes the refractive power of cancer cells to a treatment or stimulus, such as the refractive power of immune cells, e.g., to a stimulus via an activating receptor or cytokine. Non-responsiveness can occur, for example, due to exposure to an immunosuppressive agent or exposure to a high dose of an antigen. As used herein, the terms "unresponsiveness" or "tolerance" include the refractive power to an activating receptor-mediated stimulus. Such refractive power is antigen-specific and generally persists after the exposure to the tolerizing antigen has ended. For example, T cell unresponsiveness (in contrast to non-responsiveness) is characterized by a lack of cytokine production, e.g., IL-2 production. T cell unresponsiveness occurs when a T cell is exposed to an antigen and receives a first signal (a T cell receptor or CD-3-mediated signal) in the absence of a second signal (a co-stimulatory signal). Re-exposure of the cell to the same antigen under these conditions results in an inability to produce cytokines (even if re-exposure is performed in the presence of a co-stimulatory polypeptide) and thus an inability to proliferate. However, unresponsive T cells can proliferate when cultured with a cytokine, e.g., IL-2. For example, T cell unresponsiveness can also be observed by a lack of IL-2 production by T lymphocytes as evaluated by ELISA or a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct can be used. For example, unresponsive T cells are unable to initiate transcription of the IL-2 gene induced by a heterologous promoter or a multimeric AP1 sequence (Kang et al., (1992) Science, Vol. 257:1134) found within the enhancer of the 5' IL-2 gene enhancer.

[0088] The term "TGFβ-Smad / p63 signaling pathway" refers to a sub-pathway 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, cell homeostasis, and other cellular functions in both adult and developing embryos. In some embodiments, a TGFβ superfamily ligand (e.g., TGFβ1, TGFβ2, and / or TGFβ3) binds to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then binds to receptor-regulated SMADs (R-SMADs; e.g., SMAD1, SMAD2, SMAD3, SMAD5, or SMAD9), which can then bind to a coSMAD (e.g., SMAD4). The R-SMAD / coSMAD complex accumulates in the nucleus, where the complex acts as a transcription factor and is involved in regulating the expression of target genes. In the 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, the R-SMAD is Smad2. Activation of the TGFβ-Smad / p63 signaling pathway can be evaluated by analyzing, for example, phosphorylation of Smad2, nuclear translocation of Smad2, association of Smad2 with p63, and / or activation of TGFβ-Smad / p63 signature genes. The TGFβ-Smad / p63 signature can include, but is not limited to, upregulation of ICOS-L, PYCARD, SFN, PERP, RIPK3, and / or SESN1 and downregulation of KSR1, EIF4EBP1, ITGA5, EMILIN1, CD200, and / or CSF1.

[0089] In some embodiments, TGFβ promotes the formation of a heterodimer of TGFBRII and TGFBR1 on the cell plasma membrane upon binding to its receptor. Subsequently, R-Smads (such as Smad2 and Smad3), which are cytoplasmic signaling molecules, are phosphorylated by activated TGFBRI. These activated R-Smads form a complex with Co-Smad (such as Smad4) and translocate into the cell nucleus. As demonstrated herein, by combining with p63 (or other p53 family members such as p53 or p73), this Smad / p63 transcription complex upregulates pro-inflammatory genes (such as Icosl, Nfkbib, Tnfaip3, Pik3r1, and Perp), and downregulates oncogenic genes (such as Cd200, Cxcl5, Csf1, Pdgfrb, Fgfr1, Vegfa). Therefore, tumor cells with an activated TGFβ-Smad / p63 signature present a strong "eat-me" signal to the immune system and trigger an anti-tumor immune response by mobilizing antigen-presenting cells (such as dendritic cells). Dendritic cells (DCs) capture tumor-specific antigens and promote tumor-specific effector responses and memory T cell responses to provide the host with complete protection against tumors. By regulating the signaling molecules involved in this pathway, the TGFβ-Smad / p63 signaling pathway can be activated. 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 regulated to activate the TGFβ-Smad / p63 signaling pathway in the compositions and methods within the scope of the present invention.

[0090] The TGFβ-Smad / p63 signaling pathway can be activated by providing a TGFβ superfamily ligand or an agonist of the TGFβ signaling pathway. This signaling pathway can also be controllable, and / or at the levels of Smad and p63. 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 described in Table 1, and / or small molecules, peptides, and nucleic acids that can reduce the copy number, amount, and / or activity of one or more biomarkers or fragments thereof described in Table 2. Agents useful for activating the TGFβ-Smad / p63 signaling pathway or other biomarkers described herein also include TGFβ superfamily ligands.

[0091] In one embodiment, suitable agonists include natural agonists of said TGFβ superfamily member or fragments and variants thereof. For example, agonists of TGFβ signaling may include soluble endoglin. See, for example, U.S. Patent Nos. 5,719,120, 5,830,847, and 6,015,693, each of which is incorporated herein by reference in its entirety. In another embodiment, suitable agonists may include inhibitors of natural TGFβ antagonists. A number of natural modifiers that control TGFβ signaling have been identified. For example, the access of TGFβ ligand to the receptor is inhibited by LAP, decorin, and α2-macroglobulin, which are soluble proteins that bind to and sequester the ligand (Balemans and Van Hul, (2002) Dev. Biol., Vol. 250:231-250). The access of TGFβ ligand to the receptor is also controlled by membrane-bound receptors. BAMBI acts as a decoy receptor and competes with type I receptors (Onichtchouk et al., (1999) Nature, Vol. 401:480-485), and β-glycan (TGFβ type II receptor) increases the binding of TGFβ to its type II receptor (Brown et al., (1999) Science, Vol. 283:2080-2082; Massague, (1998) Annu. Rev. Biochem., Vol. 67:753-791; del Re et al., (2004) J. Biol. Chem., Vol. 279:22765-22772), and endoglin increases the binding of TGFβ to ALK1 in endothelial cells (Marchuk, (1998) Curr. Opin. 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 coreceptor, interfering with activin signaling while enhancing the binding of TGFβ ligands nodal, Vg1, and GDF1 to activin receptors (Cheng et al., (2003) Genes Dev. 17:31-36; Shen and Schier, (2000) Trends Genet. 16:303-309). Suitable agonists include synthetic or human recombinant compounds. Classes of molecules that can function as agonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof such as Fab fragments, Fab’2 fragments, and scFvs), and peptidomimetics.

[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 includes more than 30 members, most notably activin, inhibin, transforming growth factor β (TGFβ), growth differentiation factor (GDF), bone morphogenetic protein (BMP), and Mullerian inhibiting substance (MIS). All of these molecules are peptide growth factors that are structurally related to TGFβ. All of these molecules have a common motif called the cysteine knot, which is composed of particularly conserved seven cysteine residues organized within a rigid structure (Massague, (1998) Annu. Rev. Biochem. 67:753-791). Unlike classical hormones, members of the TGFβ superfamily are multifunctional proteins that have effects that are as dependent on the type and stage of the target cell as on the growth factor itself.

[0093] Suitable TGFβ superfamily members for use during the implementation of 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 member is a member of the TGFβ family, and members thereof include, but are not limited to, LAP, TGFβ1, TGFβ2, TGFβ3, and TGFβ5. In another embodiment, the TGFβ superfamily member is a member of the activin family, and members thereof include, but are 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 member is a member of the BMP (bone morphogenetic protein) family, and members thereof include, but are 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 decapentaplegic / DPP. In yet another embodiment, the TGFβ superfamily member is a member of the GDNF family, and members thereof include, but are not limited to, artemin, GDNF, neurturin, and persephin. Other TGFβ superfamily members include lefty A, lefty B, MIS / AMH, nodal, and SCUBE3.

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

[0095] The term "TGFβ1" or "transforming growth factor β1" refers to a secreted ligand of the TGFβ superfamily of proteins. Ligands of this family bind to various TGFβ receptors, causing the recruitment and activation of SMAD family transcription factors that control gene expression. The encoded preproprotein undergoes proteolytic processing to generate a latency-associated peptide (LAP) and a mature peptide, and is found in either an inactive latent form or an active form consisting only of mature peptide homodimers, LAP homodimers, and latent TGFβ binding proteins or mature peptide homodimers composed only of these. 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 chain and the TGFβ1 chain remain non-covalently bound and are stored in the extracellular matrix while keeping TGFβ1 inactive. At the same time, the LAP chain interacts with LTBP1, LRRC32 / GARP, and LRRC33 / NRROS, which are "environmental molecules" that control the activation of TGFβ1 and keep it latent while it is stored in the extracellular environment. TGFβ1 is dissociated from the LAP by integrins. Binding of integrins to the LAP stabilizes an alternative conformation of the LAP called the bow-tie form, causing a twist in the LAP chain and subsequent dissociation of the activated TGFβ1. Once activated after dissociation of the LAP, TGFβ1 acts by binding to TGFβ receptors and those receptors transmit signals. In preferred embodiments, the term "TGFβ1" refers to the activated form of TGFβ1.

[0096] TGFβ1 controls cell proliferation, differentiation, and growth and can regulate the expression and activation of other growth factors, including interferon γ and tumor necrosis factor α. TGFβ1 plays an important role in bone remodeling. TGFβ1 acts as a potent stimulator of bone formation by osteoblasts, causing chemotaxis, proliferation, and differentiation of the involved osteoblasts. TGFβ1 can promote differentiation into either the T helper 17 cell (Th17) or regulatory T cell (Treg) lineage in a concentration-dependent manner. At high concentrations, TGFβ1 induces FOXP3-mediated suppression of RORC and downregulation of IL-17 expression, supporting the generation of Treg cells. At low concentrations, TGFβ1, in conjunction with IL-6 and IL-21, induces the expression of the IL-17 receptor and IL-23 receptor, supporting differentiation into Th17 cells. TGFβ1 stimulates the sustained production of collagen through the activation of CREB3L1 by regulated intramembrane proteolysis (RIP). TGFβ1 establishes SMAD2 / 3 activation by inducing its phosphorylation and subsequent nuclear translocation (Hwangbo et al., (2016) Oncogene, Vol. 35:389-401). TGFβ1 can also induce epithelial-mesenchymal transition (EMT) and cell migration of various cell types (Hwangbo et al., (2016) Oncogene, Vol. 35:389-401). TGFβ1 is frequently upregulated in tumor cells, and mutations within this gene cause Camurati-Engelmann disease.

[0097] The term "TGFβ1" is meant to include its fragments, variants (e.g., allelic variants), and derivatives. Representative cDNA sequences of human TGFβ1 and protein sequences of human TGFβ1 are well known in the art and their protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). For example, one type of human TGFβ1 isoform is known. The human TGFβ1 transcript (NM_000660.7) encodes the TGFβ1 proprotein preproprotein (NP_000651.3). Nucleic acid sequences and polypeptide sequences of TGFβ1 orthologs in organisms other than humans are well known and include, 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), dog TGFβ1 (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 term "TGFβ2" or "transforming growth factor β2" refers to a secreted ligand of the TGFβ superfamily of proteins. As described herein, ligands of this family bind to various TGFβ receptors and cause the recruitment and activation of SMAD family transcription factors that regulate gene expression. The encoded preproprotein undergoes proteolytic processing to generate a latency-associated peptide (LAP) and a mature peptide, and this preproprotein is found in either the latent form, composed of the mature peptide homodimer, the LAP homodimer, and the latent TGFβ binding protein, or the active form, composed only of the mature or latent peptide homodimers. This mature peptide can also form heterodimers with other TGFβ family members. Activation to the mature form involves various steps. After cleavage of the proprotein within the Golgi apparatus, the LAP chain and the TGFβ2 chain remain non-covalently associated and are stored in the extracellular matrix while keeping TGFβ2 inactive. At the same time, the LAP chain interacts with "environmental molecules" such as LTBP1 and LRRC32 / GARP that control the activation of TGFβ2 and keep it latent while it is stored in the extracellular environment. Once activated after dissociation of the LAP, TGFβ2 acts through binding to TGFβ receptors, and those receptors transmit signals. In a preferred embodiment, the term "TGFβ2" refers to the activated form of TGFβ2. Disruption of the TGFβ / SMAD pathway has been shown in various human cancers. TGFβ2 controls various processes such as angiogenesis and heart development (Boileau et al., (2012) Nat. Genet., Vol. 44:916 - 921; Lindsay et al., (2012) Nat. Genet., Vol. 44:922 - 927). Chromosomal translocations involving the TGFβ2 gene are associated with Peters anomaly, a congenital defect of the anterior chamber of the eye. Mutations within the TGFβ2 gene may be associated with Rothmund - Thomson syndrome.

[0099] The term "TGFβ2" is meant to include its fragments, variants (e.g., allelic variants), and derivatives. Representative cDNA sequences and protein sequences of human TGFβ2 are well known in the art and 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 (NP_003238.2) is shorter than isoform 1. Both isoforms may undergo similar proteolytic processing. Nucleic acid sequences and polypeptide sequences of TGFβ2 orthologs in organisms other than humans are well known and include, 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), dog TGFβ2 (XM_005640824.2 and XP_005640881.1, XM_545713.6 and XP_545713.2, and 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 term "TGFβ3" or "transforming growth factor beta 3" refers to a secreted ligand of the TGFβ superfamily of proteins. As described herein, ligands of this family bind to various TGFβ receptors and cause the recruitment and activation of SMAD family transcription factors that control gene expression. The encoded preproprotein undergoes proteolytic processing to generate a latency-associated peptide (LAP) and a mature peptide, and this preproprotein is found in either a latent form or an active form consisting only of mature peptide homodimers, LAP homodimers, and latent or mature peptide homodimers composed of latent TGFβ-binding proteins. This mature peptide can also form heterodimers with other TGFβ family members. The activation of TGFβ3 to its mature form involves several steps. After cleavage of the proprotein within the Golgi apparatus, the LAP chain and the TGFβ3 chain remain non-covalently bound and are stored in the extracellular matrix while keeping TGFβ3 inactive. At the same time, the LAP chain interacts with "environmental molecules" such as LTBP1 and LRRC32 / GARP that control the activation of TGFβ3 and keep it in a latent state while it is stored in the extracellular environment. TGFβ3 is dissociated from the LAP by integrins. The binding of integrins causes a kink in the LAP chain, after which the activated form of TGFβ3 dissociates. Once activated after dissociation of the LAP, TGFβ-3 acts by binding to TGFβ receptors, and those receptors transmit signals. In a preferred embodiment, the term "TGFβ3" refers to the activated form of TGFβ3.

[0101] TGFβ3 is involved in embryonic development and cell differentiation and can play a role in wound healing. TGFβ3 is required for various processes such as secondary palate development. Mutations within the TGFβ3 gene are the cause of aortic aneurysm and aortic dissection, as well as the cause of familial arrhythmogenic right ventricular dysplasia 1.

[0102] The term "TGFβ3" is meant to include its fragments, variants (e.g., allelic variants), and derivatives. Representative cDNA sequences and protein sequences of human TGFβ3 are well known in the art and their 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 in the 5'UTR compared to variant 1 and encodes the same isoform as variant 1 (NP_001316868.1). TGFβ3 transcript variant 3 (NM_001329938.2) lacks some exons and its 3' terminal exon extends beyond the splicing site used by variant 1. This results in an earlier stop codon and a new 3'UTR compared to variant 1. The encoded isoform 2 (NP_001316867.1) has a shorter C-terminus than isoform 1. Nucleic acid sequences and polypeptide sequences of TGFβ3 orthologs in organisms other than human are well known and include, 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_001257475.1 and NP_001244404.1), dog 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 signal transduction transcription factors that transmit signals from TGFβ family receptors. Members of the Smad family of proteins have been identified based on their homology to the Drosophila gene mothers against dpp (mad), which encodes an essential element of the Drosophila dpp signal transduction pathway (Sekelsky et al., Genetics, Vol. 139: 1347-1358 (1995); Newfeld et al., Development, Vol. 122: 2099-2108 (1996)). Smad proteins are characterized by a highly conserved amino-terminal domain and a carboxy-terminal domain separated by a proline-rich linker. The amino-terminal domain (MH1 domain) mediates binding to DNA, and the carboxy-terminal domain (MH2 domain) binds to the receptor.

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

[0105] Smads 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 for TGFβ signaling (Heldin et al., (1997) Nature, Vol. 390: 465-471). These activated Smad2 and Smad3 interact with a common mediator, Smad4, translocate into the nucleus, and activate a series of specific genes in the nucleus (Heldin et al., (1997) Nature, Vol. 390: 465-471). This TGFβ pathway activates Smad2 and / or Smad3 and also uses inhibitory signaling proteins, Smad6 and Smad7, to balance the net output of signaling.

[0106] Smad2 and Smad3 have intrinsic transactivation activity as transcription factors (Zawel et al., (1998) Mol. Cell., Vol. 1: 611-617), and it has been shown from studies that they activate the expression of specific genes through specific interactions with other nuclear factors (Derynck et al., (1998) Cell, Vol. 95: 737-740). By activating specific Smad proteins, specific TGFβ-mediated effects on a given cell type can be achieved, resulting in changes in the expression of specific genes. Particularly interesting Smad proteins include Smad2, etc. (Nakao et al., (1997) J. Biol. Chem., Vol. 272: 2896-2900).

[0107] The term "SMAD2" refers to SMAD family member 2, which belongs to the SMAD family of proteins that are similar to the gene products of the Drosophila gene "Mothers Against Decapentaplegic" (Mad) and the C. elegans gene Sma. SMAD proteins are signal transduction factors and transcriptional modifiers that mediate multiple signal transduction pathways. SMAD2 mediates the signal of TGFβ and thus controls multiple cellular processes such as cell proliferation, apoptosis, and differentiation. SMAD2 is recruited to the TGFβ receptor through its interaction with the SMAD anchor for receptor activation (SARA) protein. In response to the TGFβ signal, SMAD2 is phosphorylated by the TGFβ receptor. This phosphorylation induces the dissociation of SMAD2 from SARA and the binding of SMAD2 to its family member SMAD4. The binding to SMAD4 is important for the translocation of SMAD2 into the nucleus, where it binds to target promoters and forms a transcriptional repression complex with other cofactors (e.g., p63). SMAD2 binds to the TRE sequence within the promoter regions of many genes that are regulated by TGFβ. SMAD2 can also be phosphorylated by activin type I receptor kinase and mediate the signal from activin. SMAD2 can act as a tumor suppressor in colorectal cancer. SMAD2 positively regulates PDPK1 kinase activity by stimulating its 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 mass of 52306 Da.

[0108] The term "SMAD2" is meant to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative cDNA sequences of human SMAD2 and protein sequences of human SMAD2 are well known in the art and their 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) within 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 the generation of an isoform 2 (NP_001129409.1) that is shorter than isoform 1.Nucleic acid sequences and polypeptide sequences of SMAD2 orthologs in organisms other than humans 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_022421406.1 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), 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 presented in Table 1 below.

[0109] Anti-SMAD2 antibodies suitable for the detection of SMAD2 protein are well known in the art and include, 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), and the like. Also, reagents for detecting SMAD2 expression are well known. Furthermore, siRNA product numbers sc-38374 and sc-44338 and CRISPR product number sc-400475 from Santa Cruz Biotechnology, RNAi products SR320897, TG309255, TR309255, and TL309255 and CRISPR products KN404604 and KN516271 (Origene), as well as multiple CRISPR products from GenScript (Piscataway, New Jersey), and other multiple siRNA constructs, shRNA constructs, and CRISPR constructs for reducing SMAD2 expression can be found in the product lists of the companies referenced above. It should be noted that this term can be further used to refer to any combination of features 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 term "p63" or "TP63" refers to a member of the p53 family of transcription factors. The functional domains of the p53 family proteins include an N-terminal transactivation domain, a central DNA-binding domain, and an oligomerization domain. As a result of alternative splicing and the use of alternative promoters of the p63 gene, multiple transcript variants encoding different isoforms that vary in terms of functional characteristics are generated. These isoforms function during skin development and maintenance, adult stem cell / progenitor cell control, heart development, and premature aging. Some isoforms have been found to protect the germline by eliminating oocytes or testicular germ cells that have suffered DNA damage. Mutations within the p63 gene are associated with ectrodactyly-ectodermal dysplasia-cleft lip / palate syndrome 3 (EEC3), split hand / foot malformation 4 (SHFM4), ankyloblepharon-ectodermal dysplasia-cleft lip / palate, ADULT (limbs, skin, nails, lacrimal glands, teeth) syndrome, limbs-mammary syndrome, Rapp-Hodgkin syndrome (RHS), and cleft lip / palate 8. P63 acts as a sequence-specific DNA-binding transcriptional activator or repressor. These isoforms contain a set of variable transactivation domains and autoregulatory transactivation inhibitory domains and thus exhibit isoform-specific activities. Isoform 2 activates RIPK4 transcription. P63 may be required, together with TP73 / p73, for the initiation of p53 / TP53-dependent apoptosis in response to damage by genotoxic agents and the presence of activated oncogenes. P63 is involved in Notch signaling, perhaps by inducing JAG1 and JAG2. P63 plays a role in the control of epithelial morphogenesis. The ratio of ΔN-type isoforms and TA * type isoforms influences the maintenance of the epithelial stem cell compartment and may control the initiation of epithelial stratification from undifferentiated embryonic ectoderm. P63 is required for limb formation from the ectodermal apical ridge. P63 activates the transcription of the p21 promoter. In one embodiment, the human P63 protein has 680 amino acids and a molecular mass of 76785 Da.

[0111] The terms "p63" or "TP63" are meant to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative cDNA sequences of human p63 and protein sequences of human p63 are well known in the art and their 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 p63 isoform 1 (NP_003713.3), the longest isoform. p63 transcript variant 2 (NM_001114978.2) lacks exons 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 unique 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 unique 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 unique N-terminus. p63 transcript variant 5 (NM_001114981.2) differs from variant 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 a unique N-terminus and C-terminus. p63 transcript variant 6 (NM_001114982.2) differs from Variant 1 in the 5’UTR, coding region, 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 unique N-terminus and C-terminus. p63 transcript variant 7 (NM_001329144.2) lacks two exons in the 3’ coding region compared to Variant 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 unique C-terminus. p63 transcript variant 8 (NM_001329145.2) has multiple differences compared to Variant 1. As a result of these differences, an alternative start codon is used and a frameshift is introduced into 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 some 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 multiple differences compared to Variant 1. As a result of these differences, an alternative start codon is used and a frameshift is introduced into the 3’ coding region. The encoded isoform (Isoform 11) (NP_001316078.1) is shorter than Isoform 1 and has a unique N-terminus and C-terminus. p63 transcript variant 12 (NM_001329150.2) has multiple differences compared to variant 1. As a result of these differences, an alternative start codon is used, and a frameshift is introduced into the 3' coding region. The encoded isoform (isoform 12) (NP_001316079.1) is shorter than isoform 1 and has a unique N-terminus and C-terminus. p63 transcript variant 13 (NM_001329964.1) uses an alternative promoter and thus differs in the 5' UTR and 5' coding region compared to variant 1. This promoter and 5' terminal exon sequence are those of an endogenous retrovirus LTR (PMID: 21994760). The resulting isoform (isoform 13. NP_001316893.1, also known as GTAp63) is shorter than isoform 1 and has a unique N-terminus. The encoded protein is mainly expressed in testicular germ cells and eliminates germ cells that have received DNA damage. Nucleic acid sequences and polypeptide sequences of p63 orthologs in organisms other than humans 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), monkey p63 (XM_028843565.1 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, XM_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), dog 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_001120734.1, 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_001127341.1 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 presented in Table 1 below.

[0112] Anti-p63 antibodies suitable for the detection of p63 protein are well known in the art, and include, for example, TA323790 and CF811064 (OriGene Technologies, Rockville, Maryland), AF1916 (an antibody from Novus Biologicals, Littleton, Colorado), ab124762, ab53039, and ab735, ab97865 (antibodies from AbCam, Cambridge, Massachusetts), and the like. Also, reagents for detecting the expression of p63 are well known. Further, siRNA product numbers sc-36620 and sc-36621, RNAi products TR308688, TG308688, TL308688, and SR322466, and CRISPR products KN208013 and KN208013BN (Origene), and multiple CRISPR products from GenScript (Piscataway, New Jersey), etc., multiple siRNA constructs, shRNA constructs, and CRISPR constructs for reducing p63 expression can be found in the product lists of the companies referenced above. It should be noted that this term can be further used to refer to any combination of features 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 molecules included in the scope of the present invention.

[0113] The term "TP53" refers to tumor protein P53, a tumor suppressor protein that contains a transcriptional activation domain, a DNA-binding domain, and an oligomerization domain. The encoded protein controls the expression of target genes in response to various cellular stresses, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or metabolic changes. Mutations within this gene are associated with various human cancers, including hereditary cancers such as Li-Fraumeni syndrome. TP53 mutations are prevalent among multiple cancers. Defects in the tumor suppressor most often occur via large-scale deletion events such as frameshift mutations or premature stop codons. However, it has been found that many of the mutations observed in cancer in TP53 are single-base missense mutations. These mutations are widely distributed throughout the gene, but most are located within the DNA-binding domain. There is no single hotspot within the DNA-binding domain, and most of the mutations occur at amino acid positions 175, 245, 248, 273, and 282 (NM_000546). While a large proportion of cancer genome research has focused on somatic mutations, TP53 is also noted in the germline. Germline TP53 mutations are characteristic of Li-Fraumeni syndrome, and a number of (both germline and somatic) mutations have been found to have prognostic implications for patient prognosis. 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 control as a trans-activator that acts to negatively regulate this process by controlling a series of genes required for cell division. One of those genes that is activated is a cyclin-dependent kinase inhibitor. Apoptosis induction seems to occur either by stimulation of the expression of the BAX antigen and FAS antigen or by suppression of Bcl-2 expression. In cooperation with mitochondrial PPIF, TP53 is involved in the activation of oxidative stress-induced necrosis, but this function is almost independent of transcription. TP53 induces the transcription of long intergenic non-coding RNA-p21 (lincRNA-p21) and lincRNA-Mkln1. lincRNA-p21 is involved in TP53-dependent transcriptional repression that causes apoptosis and seems to have an effect on cell cycle control.TP53 is thought to be involved in the intersection of Notch signaling. TP53 binds to the CAK complex in response to DNA damage, suppressing CDK7 kinase activity and thus halting the progression of the cell cycle. Isoform 2 of TP53 enhances the transactivation activity from some, but not all, TP53-inducible promoters of isoform 1. Isoform 4 of TP53 suppresses transactivation activity, impairing growth suppression mediated by isoform 1. Isoform 7 of TP53 inhibits isoform 1-mediated apoptosis. TP53 controls the circadian clock by suppressing the CLOCK-ARNTL / BMAL1-mediated transcriptional activation of PER2 (Miki et al., (2013) Nat Commun, Vol. 4:2444). In some embodiments, the human TP53 protein has a molecular mass of 393 amino acids and 43,653 Da. Known binding partners of TP53 include AXIN1, ING4, YWHAZ, HIPK1, HIPK2, WWOX, GRK5, ANKRD2, RFFL, RNF34, and TP53INP1, among others.

[0114] The term "TP53" is meant to include its fragments, variants (e.g., allelic variants), and derivatives. Representative cDNA sequences of human TP53 and protein sequences of human TP53 are well known in the art and their protein sequences 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). 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). Nucleic acid sequences and polypeptide sequences of TP53 orthologs in organisms other than humans are well-known and include, 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 NP_001120705.1, and NM_011640.3 and NP_035770.2), rat TP53 (NM_030989.3 and NP_112251.2), Xenopus tropicalis 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 presented in Table 1 below.

[0115] Anti-TP53 antibodies suitable for detecting the TP53 protein are well known in the art and include, for example, TA502925 and CF502924 (Origene), NB200-103 and NB200-171 (Novus Biologicals, Littleton, CO), ab26 and ab1101 (AbCam, Cambridge, MA), 700439 (ThermoFisher Scientific), 33-856 (ProSci), and the like. Reagents for detecting TP53 are also well known. Multiple clinical tests for TP53 are available in the NIH Genetic Testing Registry (GTR®) (e.g., GTR Test ID: GTR000517320.2 provided by Fulgent Clinical Diagnostic Laboratories (Temple City, CA)). In addition, siRNA product numbers sc-29435 and sc-44218, and CRISPR product number sc-416469 from Santa Cruz Biotechnology, RNAi products SR322075 and TL320558V, and CRISPR product KN200003 (Origene), as well as multiple CRISPR products from GenScript (Piscataway, NJ) and other siRNA constructs, shRNA constructs, and CRISPR constructs for reducing TP53 expression can be found in the product lists of the companies referenced above. Chemical inhibitors of TP53, including cyclic pifithrin-α hydrobromide, RITA (TOCRIS, MN), etc., are also available. Note that this term may be further used to refer to any combination of features described herein with respect to the TP53 molecule. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc. can be used to describe the TP53 molecules included in the scope of the present invention. There is a well-known and unambiguous correspondence 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, there is a well-known and unambiguous correspondence 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, by which more than one nucleotide triplet code can be used for most of the amino acids used to construct proteins (as exemplified above). Thus, a number of different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered to be functionally equivalent because (although a particular organism may translate one sequence more efficiently than another) they will all produce the same amino acid sequence in all organisms. Furthermore, sometimes methylated variants of purines or pyrimidines may 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] In view of the foregoing, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any part 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 the polypeptide can be inferred from the genetic code (resulting in multiple nucleic acid sequences for any given amino acid sequence due to the degeneracy of the genetic code). Thus, it should be considered that the description and / or disclosure in this specification of a nucleotide sequence encoding a polypeptide also includes the description and / or disclosure of the amino acid sequence encoded by that nucleotide sequence. Similarly, it should be considered that the description and / or disclosure in this specification of the amino acid sequence of a polypeptide also includes 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 loci, biomarkers, and related biomarkers (e.g., the biomarkers described in Tables 1 and 2) included within the scope of the present invention are well known in the art and are readily available in public databases such as the National Center for Biotechnology Information (NCBI) of the United States. For example, exemplary nucleic acid sequences and amino acid sequences obtained from public sequence databases are listed below.

[0120] Table 1

Table 1

[0121] SEQ ID NO: 1: Human Smad2 transcript variant 2 mRNA sequence (NM_001003652.4, CDS: 127 - 1530)

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[0122] Nucleic acid molecules containing nucleic acid sequences 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 a higher percentage of identity to the DNA - binding domain - coding region or the full - length of the nucleic acid sequence of any of the accession numbers listed in Table 1 are included in Table 1. Such nucleic acid molecules can encode polypeptides having the function of the full - length polypeptide as further described herein.

[0123] Polypeptide molecules containing amino acid sequences 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 a higher percentage of identity to the DNA - binding domain or the full - length of the amino acid sequence of any of the accession numbers listed in Table 1 are included in Table 1. Such polypeptides can have the function of the full - length polypeptide as further described herein.

[0124] Table 2

Table 2

[0125] Array No. 177: Human Smad6 cDNA sequence (NM_005585.5, CDS: 1024 - 2514)

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[0126] A nucleic acid molecule comprising a nucleic 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 a higher percentage of identity to the DNA binding domain coding region or the full length of any of the nucleic acid sequences listed in Table 2 is included in Table 2. Such a nucleic acid molecule may encode a polypeptide having the function of the full-length polypeptide as further described herein.

[0127] A polypeptide molecule comprising 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 a higher percentage of identity to the amino acid sequence of any of the accession numbers listed in Table 2 is included in Table 2. Such a polypeptide may have the function of the full-length polypeptide as further described herein.

[0128] II. Cancer vaccine 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 cancer or blood cancer (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 caused by 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 cell. 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 additional genetic mutations. The cancer cells may be derived from the subject to be 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 to be treated with the cancer vaccine. The cancer cells may be derived from a different type of cancer than the cancer to be treated with the cancer vaccine. The cancer cells may be derived from a cancer having the same genetic mutation as the cancer to be treated with the cancer vaccine. The cancer cells may be derived from a cancer having a different genetic mutation than the cancer to be treated with the cancer vaccine.

[0129] Isolation and purification of cancer cells In some embodiments, the cancer cells are derived from a subject. Isolation and purification of tumor cells from various tumor tissues such as surgically resected tumor tissue, ascites, or cancerous pleural effusion are common processes for obtaining purified tumor cells. Cancer cells can be purified from fresh biopsy samples of 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 preferably has all viable cancer cells at a rate of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher, or any range therebetween, or any value therebetween. Numerous methods are available for purifying cancer cells from heterogeneous populations.

[0130] In one embodiment, laser microdissection is used for cancer cell isolation. The cancer cells of interest can be carefully excised from tissue sections prepared for microscopic observation. In this method, the tissue section is coated with a plastic film, and a focused near-infrared laser light pulse is irradiated onto the region containing the selected cells. This causes a small circle to melt out in the plastic film, and the cells underneath become attached. These captured cells are removed for other analyses. This technique is good 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 excised tissue and pituitary cells derived from heterogeneous cultured pituitary cell populations, thyroid cell populations, and carcinoid tumor cell populations, 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 the sorting and analysis of various cell populations. Cells having a target cell marker or other specific marker are tagged with an antibody or typically a mixture of antibodies that bind to those cell markers. Each antibody for a different marker is complexed to 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 their fluorochrome, and the presence of a particular labeled antibody is determined by the emission spectrum from those cells that are detected. Cells that exhibit different sets of cell markers are identified by the simultaneous detection of different fluorochromes, also called multicolor fluorescence cell sorting in the art, and can be isolated from other cells in the population. Cell selection based on size and viability is enabled by 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). FACS sorting and analysis of HSC and related lineage cells are well known in the art and are described, for example, in U.S. Patent Nos. 5,137,809; 5,750,397; 5,840,580; 6,465,249; Manz et al., Proc. Natl. Acad. Sci. U.S.A. 99:11872-11877 (2002); and Akashi et al., Nature 404:193-197 (2000). General guidelines for fluorescence-activated cell sorting are described, for example, in Shapiro, Practical Flow Cytometry, 4th Ed., Wiley-Liss (2003) and Ormerod, Flow Cytometry: A Practical Approach, 3rd Ed., Oxford University Press (2000).

[0132] Another method for isolating useful cell populations relates to solid or insoluble substrates to which an antibody or ligand that interacts with a specific cell surface marker is bound. In immunoadsorption methods, cells are contacted with a substrate containing the antibody (e.g., bead columns, flasks, magnetic particles, etc.), and any unbound cells are removed. Immunoadsorption methods can be scaled up to directly process large numbers of cells collected clinically. 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, the cells bound to those beads can be easily isolated by a magnetic separator (see, for example, Kato and Radbruch, Cytometry, Vol. 14:384-92 (1993)). Cell separation by affinity chromatography typically involves passing a cell suspension through a support bearing a selection ligand immobilized on its surface. The ligand interacts with its specific target molecule on the cell and is captured on the matrix. The bound cells are released by the addition of an eluent to the running buffer of the column, and the free cells are washed out of 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 may utilize non-specific adsorption. For example, adsorption to silica is a simple method for removing phagocytes from cell preparations. One of the most common applications of this technique is the use of an antibody against EpCAM, a cell surface glycoprotein that has been found to be well-expressed in epithelial cancers, to isolate circulating tumor cells (CTCs) from the blood of patients with breast cancer, NSCLC lung cancer, prostate cancer, and colon cancer.

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

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

[0135] According to a recently published paper (by Lin et al., Small (2015), Vol. 11: 4394 - 4402), fluorescent nanodiamonds (FNDs) are available for labeling and isolating low-proliferative / quiescent cancer stem cells, and according to the authors of that study, fluorescent nanodiamonds are difficult to isolate and track over the long term using traditional fluorescent markers. It was concluded that they do not cause nanoparticle DNA damage or interfere with cell proliferation and are superior to EdU fluorescent labeling and CFSE fluorescent labeling in terms of long-term tracking ability.

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

[0137] Modification and modification of cancer cells The cancer cells contained in the 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 modified with an agent that reduces the copy number, amount, and / or activity of PTEN and / or p53 to be PTEN-deficient and p53-deficient.

[0138] The agent that reduces the copy number, amount, and / or activity of PTEN and / or p53 may be a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interference agent, antisense oligonucleotide, peptide inhibitor or peptidomimetic inhibitor, aptamer, antibody, or intracellular antibody.

[0139] In one embodiment, a peptide or peptidomimetic can be used to attenuate the activity of PTEN and / or p53. In one embodiment, variants of PTEN and / or p53 that function as modulators for their respective full-length proteins can be identified by screening a combinatorial library of variants, such as 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. The diversified variant library can be generated, for example, by enzymatically ligating a mixture of synthetic oligonucleotides to a gene sequence such that a degenerate set of possible polypeptide sequences can be expressed as individual polypeptides that contain the polypeptide sequences of the set within them. There are various methods available for creating a library of polypeptide variants from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed on an automated DNA synthesizer, followed by ligation of the synthetic genes into an appropriate expression vector. Using a degenerate gene set provides all of the sequences that encode the possible polypeptide sequences of the desired set as a single mixture. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang, S. A., Tetrahedron (1983) 39:3; Itakura et al., Annu. Rev. Biochem. (1984) 53:323; Itakura et al., Science (1984) 198:1056; Ike et al., Nucleic Acid Res. (1983) 11:477).

[0140] Also, a library of fragments of a polypeptide coding sequence can be used to create a diversified population of polypeptide fragments for screening and subsequent selection of variants of a given polypeptide. In one embodiment, the library of coding sequence fragments is prepared by treating double-stranded PCR fragments of the polypeptide coding sequence with a nuclease under conditions where nick formation occurs only about once per polypeptide, denaturing the double-stranded DNA, regenerating the DNA to form double-stranded DNA that may contain sense / antisense pairs from the different nicked products, removing single-stranded portions by treatment with S1 nuclease from the reformed double-stranded, and ligating the resulting fragment library into an expression vector. By this method, an expression library encoding N-terminal fragments, C-terminal fragments, and internal fragments of the polypeptide of various sizes can be derived.

[0141] Several techniques are known in the art for screening gene products of combinatorial libraries created by point mutations or deletions, and for screening cDNA libraries for gene products having selected properties. Such techniques are applicable to the rapid screening of said gene libraries created by combinatorial mutagenesis of polypeptides. The most widely used technique suitable for screening large gene libraries and also applicable to high-throughput analysis is to clone said gene library into a replicable expression vector, transform appropriate cells with the resulting vector library, and express their combinatorial genes under conditions that facilitate isolation of the vector encoding the gene whose product was detected by detection of the desired activity. Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional variants in said library, can be used in combination with said screening assay to identify the variant of interest (Arkin and Youvan, (1992) Proc. Natl. Acad. Sci. USA, Vol. 89: 7811-7815, Delagrave et al., (1993) Protein Eng., Vol. 6 (No. 3): 327-331). In one embodiment, cell-based assays can be utilized for the analysis of diversified polypeptide libraries. For example, an expression vector library can be transfected into cell lines that normally synthesize PTEN and / or p53. Subsequently, full-length polypeptides and specific variant polypeptides are produced, and the transfected cells are cultured such that the effect of the expression of the variant on the activity of the full-length polypeptide in the cell supernatant can be detected by, for example, any of a number of functional assays. Subsequently, plasmid DNA can be recovered from cells 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 produce a more stable peptide by using tissue substitution of one or more amino acids in the polypeptide amino acid sequence with the same type of D-amino acid (for example, D-lysine instead of L-lysine). In addition, a constrained peptide containing the target polypeptide amino acid sequence or a substantially identical sequence variation can be produced by methods known in the art (Rizo and Gierasch, (1992) Annu. Rev. Biochem., Vol. 61: 387. Incorporated herein by reference), for example, by the addition of internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[0143] Those skilled in the art can create peptide sequences corresponding to polypeptides and their sequence variants based on the amino acid sequences disclosed herein. Such polypeptides can be produced in prokaryotic or eukaryotic host cells by the expression of polynucleotides encoding the peptide sequences, often as parts of larger polypeptides. 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 the art and are further described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd ed., Cold Spring Harbor, NY, Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (1987), Academic Press, San Diego, CA, Merrifield, J., (1969) J. Am. Chem. Soc., Vol. 91:501, Chaiken I. M., (1981) CRC Crit. Rev. Biochem., Vol. 11:255, Kaiser et al., (1989) Science, Vol. 243:187, Merrifield, B., (1986) Science, Vol. 232:342, Kent, S. B. H., (1988) Annu. Rev. Biochem., Vol. 57:957, and Offord, R. E., (1980) Semisynthetic Proteins, Wiley Publishing, and these references are hereby incorporated by reference into this specification.

[0144] Peptides can typically be made by direct chemical synthesis. Peptides can be made as modified peptides having non-peptide moieties attached by covalent bonds to the N-terminus and / or C-terminus. In certain preferred embodiments, either or both of the carboxy terminus or the amino terminus are chemically modified. The most common modifications of the terminal amino group and carboxyl group are acetylation and amidation, respectively. Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal modifications such as amidation, as well as other terminal modifications including cyclization, are applicable to various embodiments of the present invention. Certain amino-terminal modifications and / or carboxy-terminal modifications and / or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties such as increased characteristics, increased stability, increased titer and / or potency, resistance to serum proteases, desirable pharmacokinetic properties, and the like. The peptides disclosed herein can be used for therapeutic purposes, for example, to treat a disease by altering co-stimulation in a patient.

[0145] Peptidomimetics (Fauchere, J. L., Adv. Drug Res., 15:29 (1986); Veber, D. F. and Freidinger, R. M., TINS, 392 (1985); Evans, B. E. et al., J. Med. Chem., 30:1229 (1987), each of which is incorporated herein by reference) are commonly developed with the aid of computer molecular modeling. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce equivalent therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to typical polypeptides (i.e., polypeptides having biological or pharmacological activity), but are known in the art and are described in the following references, each of which is incorporated herein by reference: "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins", Weinstein, B., ed., Marcel Dekker, New York, p. 267 (1983), the work of Spatola, A. F. therein; Spatola, A. F., Vega Data, 1(3): "Peptide Backbone Modifications" (Review), Morley, J. S., Trends Pharm. Sci., 463 - 468 (1980) (Review); Hudson, D. et al., Int. J. Pept. Prot. Res., 14:177 - 185 (-CH2NH-, -CH2CH2-) (1979); Spatola, A. F. et al., Life Sci., 38:1243 - 1249 (-CH2-S) (1986); Hann, M. M., J. Chem. Soc. Perkin Trans. I, 307 - 314 (-CH=CH-, cis and trans) (1982); Almquist, R. G. et al., J. Med. Chem., 23:1392 - 1398 (-COCH2-) (1980); Jennings-White, C. et al., Tetrahedron Lett., 23:2533 (-COCH2-) (1982); Szelke, M. et al., European Appln. EP 45665 CA: Journal, Vol. 97: 39405 (-CH(OH)CH2-), by Holladay, M. W. et al., (1983) Tetrahedron Lett., Vol. 24: 4401 - 4404 (-C(OH)CH2-), and Hruby, V. J., (1982) Life Sci., Vol. 31: 189 - 199 (-CH2-S-), have one or more peptide bonds optionally substituted by a bond selected from the group consisting of -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. A particularly preferred non-peptide bond is -CH2NH-. Such peptidomimetics may have significant advantages over polypeptide embodiments, including, for example, higher production economy, higher chemical stability, enhanced pharmacological properties (half-life, absorption, titer, efficacy, etc.), alteration of specificity (e.g., broad range of biological activities), reduced antigenicity, and others. Labeling of peptidomimetics typically involves covalent attachment of one or more labels to non-interfering positions on the peptidomimetic, either directly or via a spacer (e.g., an amide group), as predicted by quantitative structure-activity data and / or molecular modeling. Such non-interfering positions are generally those that do not come into direct contact with the macropolypeptide to which the peptidomimetic binds to produce a therapeutic effect. Derivatization (e.g., labeling) of peptidomimetics needs to substantially not interfere with the desired biological or pharmacological activity of the peptidomimetic.

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

[0147] Examples of methods for molecular library synthesis are found in the art, 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, Carrell et al., (1994) Angew. Chem. Int. Ed. Engl., Vol. 33:2061, and Gallop et al., (1994) J. Med. Chem., Vol. 37:1233.

[0148] Compound libraries can be presented in solution (e.g., Houghten, Biotechniques, Vol. 13:412 - 421 (1992)), on beads (Lam, Nature, Vol. 354:82 - 84 (1991)), on chips (Fodor, Nature, Vol. 364:555 - 556 (1993)), on bacteria (Ladner U.S. Patent No. 5,223,409), on spores (Ladner U.S. Patent No. 5,223,409), on plasmids (Cull et al., Proc. Natl. Acad. Sci. USA, Vol. 89:1865 - 1869 (1992)), or on phage (Scott and Smith, Science, Vol. 249:386 - 390 (1990)), (Devlin, Science, Vol. 249:404 - 406 (1990)), (Cwirla et al., Proc. Natl. Acad. Sci. USA, Vol. 87:6378 - 6382 (1990)), (Felici, J. Mol. Biol., Vol. 222:301 - 310 (1991)), (Ladner, supra). Compounds can be screened in cell-based or cell-free assays. Compounds can be pooled (e.g., multiple compounds per test sample) for screening or screened as individual compounds.

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

[0150] In one embodiment, the binding can be by conventional base pair complementarity or, for example, in the case of binding to a DNA duplex, can be through specific interactions in the major groove of the double helix. In general, "antisense" refers to the art generally used technical scope and includes any process that depends on specific binding to an oligonucleotide sequence.

[0151] It is well known in the art that modifications can be made to the sequence of miRNA or pre-miRNA without impairing miRNA activity. As used herein, the term "functional variant" of an miRNA sequence refers to an oligonucleotide sequence that varies from the native miRNA sequence but retains one or more functional characteristics of that miRNA (e.g., inhibition of cancer cell proliferation, induction of cancer cell apoptosis, enhancement of the sensitivity of cancer cells to chemotherapeutic agents, inhibition of specific miRNA targets). In some embodiments, the functional variant of the miRNA sequence retains all of the functional characteristics of that miRNA. In certain embodiments, the functional variant of the miRNA has a nucleobase 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 that miRNA or its precursor over a region of about 5 bases, about 6 bases, about 7 bases, about 8 bases, about 9 bases, about 10 bases, about 11 bases, about 12 bases, about 13 bases, about 14 bases, about 15 bases, about 16 bases, about 17 bases, about 18 bases, about 19 bases, about 20 bases, about 21 bases, about 22 bases, about 23 bases, about 24 bases, about 25 bases, about 30 bases, about 35 bases, about 40 bases, about 45 bases, about 50 bases, about 55 bases, about 60 bases, about 65 bases, about 70 bases, about 75 bases, about 80 bases, about 85 bases, about 90 bases, about 95 bases, about 100 bases, or more nucleobases, or the functional variant hybridizes to the complement of the miRNA or its precursor under stringent hybridization conditions. Thus, in certain embodiments, the nucleobase sequence of the functional variant is capable of hybridizing to one or more target sequences of that 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 found on the World Wide Web at the address microrna.sanger.ac.uk. Entries in the miRBase sequence database show the predicted hairpin portion (stem-loop) of the miRNA transcript along with information on the location and sequence of its mature miRNA sequence. The miRNA stem-loop sequences in that database are not strictly precursor miRNAs (pre-miRNAs) and in some instances may include pre-miRNAs derived from the predicted primary transcript and some flanking sequences. The miRNA nucleobase sequences described herein encompass any form of that miRNA and include sequences described in Release 10.0 of the miRBase sequence database and sequences described in any release of the miRBase sequence database prior thereto. Depending on the release of the sequence database, a particular miRNA may be renamed. Depending on the release of the sequence database, variants of the mature miRNA sequence may occur.

[0153] In some embodiments, the miRNA sequences of the invention may bind to a second RNA sequence that may be located on the same RNA molecule as the miRNA sequence or on a different RNA molecule. In such cases, the miRNA sequence may be referred to as the active strand, while the second RNA sequence that is at least partially complementary to the miRNA sequence may be referred to as the complementary strand. Hybridization of their active and complementary strands produces a double-stranded RNA similar to the native miRNA precursor. The activity of the miRNA can be optimized by maximizing the incorporation of its active strand by the miRNA-protein complex that controls gene translation and minimizing the incorporation of its complementary strand. This optimization can be carried out by modification and / or design of the complementary strand.

[0154] In some embodiments, the complementary strand is modified such that a chemical group other than a phosphate group or a hydroxyl group is present at its 5' end. The presence of such a 5' modification clearly excludes the uptake of the complementary strand and subsequently aids the uptake of the active strand by the miRNA-protein complex. The 5' modification can be any of a variety of molecules known in the art, including NH2, NHCOCH3, and biotin.

[0155] In another embodiment, the uptake of the complementary strand via the miRNA pathway is reduced by incorporating nucleotides having a modification on the sugar into the first 2-6 nucleotides of the complementary strand. It should be noted that such sugar modifications can be combined with the above 5' end modifications to further enhance miRNA activity.

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

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

[0158] Alternatively, the short-chain nucleic acid and / or antisense construct is an oligonucleotide probe that is prepared ex vivo and, when introduced into a cell, hybridizes with the cellular nucleic acid. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, such as exonucleases and / or endonucleases, and are thus stable in vivo. Examples of nucleic acid molecules used as short-chain nucleic acids and / or antisense oligonucleotides are phosphoramidate analogs of DNA, phosphorothioate analogs, and methylphosphonate analogs (see also U.S. Patent Nos. 5,176,996, 5,264,564, and 5,256,775). Also, general approaches to the construction of oligomers useful in antisense therapy are outlined, for example, in Van der Krol et al., (1988) BioTechniques, Vol. 6:958-976, and Stein et al., (1988) Cancer Res., Vol. 48:2659-2668.

[0159] The antisense approach can involve the design of an oligonucleotide (either DNA or RNA) that is complementary to the cellular nucleic acid (e.g., complementary to the PTEN gene and / or the p53 gene). Absolute complementarity is not required. Thus, in the case of a double-stranded antisense nucleic acid, one strand of the double-stranded DNA may be examined, and triple-strand formation may be analyzed. The hybridization ability depends on both the degree and length of complementarity of the antisense nucleic acid. The longer the hybridizing nucleic acid, the more likely it is to contain mismatches with more nucleic acids (e.g., RNA), and yet still be able to form a stable double-strand (or in some cases a triple-strand). One of ordinary skill in the art can confirm the degree of acceptable mismatches 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 up to the AUG start codon for example, should act most efficiently to inhibit translation. However, it has recently been shown that sequences complementary to the 3' untranslated sequence of the mRNA are similarly effective in inhibiting mRNA translation (Wagner, (1994) Nature, Vol. 372: 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 an antisense approach to inhibit the translation of endogenous mRNA. The oligonucleotide complementary to the 5' untranslated region of the mRNA may include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region are not very 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 a cellular mRNA, short-chain nucleic acid, and / or antisense nucleic acid should be at least 6 nucleotides in length and less than about 1000 nucleotides in length, less than about 900 nucleotides in length, less than about 800 nucleotides in length, less than about 700 nucleotides in length, less than about 600 nucleotides in length, less than about 500 nucleotides in length, less than about 400 nucleotides in length, less than about 300 nucleotides in length, less than about 200 nucleotides in length, less than about 100 nucleotides in length, less than about 50 nucleotides in length, less than about 40 nucleotides in length, less than about 30 nucleotides in length, less than about 25 nucleotides in length, less than about 24 nucleotides in length, less than about 23 nucleotides in length, less than about 22 nucleotides in length, less than about 21 nucleotides in length, less than about 20 nucleotides in length, less than about 19 nucleotides in length, less than about 18 nucleotides in length, less than about 17 nucleotides in length, less than about 16 nucleotides in length, less than about 15 nucleotides in length, or less than about 10 nucleotides in length.

[0161] To quantify the gene expression inhibitory ability of an antisense oligonucleotide, it is preferable to first conduct in vitro tests regardless of the selection of the target sequence. In one embodiment, these tests utilize a control that distinguishes between the antisense gene inhibition of the oligonucleotide and its non-specific biological effects. 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 contemplated to compare the results obtained using a control oligonucleotide with those obtained using an antisense oligonucleotide. The control oligonucleotide is preferably approximately the same length as the test oligonucleotide, and the nucleotide sequence of the oligonucleotide is not more different from the antisense sequence than necessary to prevent specific hybridization to the target sequence.

[0162] The short-chain nucleic acid and / or antisense oligonucleotide may be DNA or RNA or a chimeric mixture thereof or derivatives or modified forms thereof, and may be single-stranded or double-stranded. The short-chain nucleic acid and / or antisense oligonucleotide may have a modified base moiety, sugar moiety, or phosphate backbone, for example, to improve the stability or hybridization of the molecule, and may have other appended groups such as peptides (for example, for targeting receptors of host cells), cell membranes (for example, as described by Letsinger et al., (1989) Proc. Natl. Acad. Sci. U.S.A., Vol. 86: 6553-6556, Lemaitre et al., (1987) Proc. Natl. Acad. Sci. Agents that facilitate transport across the blood-brain barrier (see, e.g., U.S.A. Journal, Vol. 84: pp. 648-652, PCT International Publication No. 88 / 09810) or the blood-brain barrier (see, e.g., PCT International Publication No. 89 / 10134), hybridization-inducing cleavage agents (see, e.g., Krol et al., (1988) BioTech. Journal, Vol. 6: pp. 958-976), or intercalating agents (see, e.g., Zon, (1988) Pharm. Res. Journal, Vol. 5: pp. 539-549) may be included. For this purpose, short-chain nucleic acids and / or antisense oligonucleotides can be complexed with another molecule, such as a peptide, a hybridization-inducing cross-linking agent, a transport factor, a hybridization-inducing cleavage agent, etc.

[0163] The short-chain nucleic acid and / or antisense oligonucleotide may include at least one modified base moiety selected from the group consisting of, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxyethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. The short-chain nucleic acid and / or antisense oligonucleotide may also include at least one modified sugar moiety selected from the group consisting of, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0164] In certain embodiments, the compound comprises an oligonucleotide (e.g., miRNA or an oligonucleotide encoding miRNA) complexed to one or more moieties that enhance the activity, intracellular distribution, or cellular uptake of the oligonucleotide. In certain such embodiments, the moiety is a cholesterol moiety (e.g., antagomir), a lipid moiety, or a liposomal complex. Other moieties for complexation include carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In certain embodiments, a complexing group is directly attached to the oligonucleotide. In certain embodiments, the complexing group is attached to the oligonucleotide by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturated groups (e.g., double or triple bonds), 8-amino-3,6-dioxaoctanoic acid (ADO), N-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, the substituent is 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, for example, nuclease stability. Stabilizing groups include cap structures. These terminal modifications protect the oligonucleotide from exonuclease degradation and can aid in delivery and / or intracellular localization. The cap can be present at the 5'-end (5'-cap), at the 3'-end (3'-cap), or at both ends. Cap structures include, for example, inverted deoxyabasic caps.

[0166] Suitable cap structures include 4’,5’-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4’-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, phosphorodithioate linkages, threo-pentofuranosyl nucleotides, acyclic 3’,4’-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3’-3’-inverted nucleotide moieties, 3’-3’-inverted abasic moieties, 3’-2’-inverted nucleotide moieties, 3’-2’-inverted abasic moieties, 1,4-butanediol phosphate, 3’-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3’-phosphate, 3’-phosphorothioate, phosphorodithioate, bridged methylphosphonate moieties and non-bridged methylphosphonate moieties 5’-amino-alkyl phosphates, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 5’-5’-inverted nucleotide moieties, 5’-5’-inverted abasic moieties, 5’-phosphoramidate, 5’-phosphorothioate, 5’-amino, bridged and / or non-bridged 5’-phosphoramidate, phosphorothioate, and 5’-mercapto moieties.

[0167] Short-chain nucleic acids and / or antisense oligonucleotides can 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. U.S.A., Vol. 93:14670, and Eglom et al., (1993) Nature, Vol. 365:566. One advantage of PNA oligomers is their ability to bind complementarily essentially independently of the ionic strength of the medium because their DNA is a neutral backbone. In yet another embodiment, the short-chain nucleic acids and / or antisense oligonucleotides comprise at least one modified phosphate backbone selected from the group consisting of phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphorodiamidate, methylphosphonate, alkylphosphotriester, and formacetal or analogs thereof.

[0168] In other embodiments, the short-chain nucleic acids and / or antisense oligonucleotides are α-anomer oligonucleotides. α-Anomer oligonucleotides form specific double-stranded hybrids in which the strands extend parallel to each other, in contrast to normal b-units, with complementary RNA (Gautier et al., (1987) Nucl. Acids Res., Vol. 15:6625-6641). This oligonucleotide is a 2'-O-methyl ribonucleotide (Inoue et al., (1987) Nucl. Acids Res., Vol. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al., (1987) FEBS Lett., Vol. 215:327-330).

[0169] The short-chain nucleic acids and / or antisense oligonucleotides of the methods and compositions presented in this specification can be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (e.g., those commercially available from Biosearch, Applied Biosystems, etc.). For example, when phosphorothioate oligonucleotides are synthesized by the method described by Stein et al. (1988) Nucl. Acids Res., Vol. 16: 3209, or when methylphosphonate oligonucleotides are prepared by using a controlled pore glass polymer support (Sarin et al. (1988) Proc. Natl. Acad. Sci. U.S.A., Vol. 85: 7448-7451), etc. may be possible. 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 a conventional DNA / RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthetic reagents include, for example, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colorado, USA), Pierce Chemical (a part of Perbio Science, Rockford, Illinois, USA), Glen Research (Sterling, Virginia, USA), ChemGenes (Ashland, Massachusetts, USA), Cruachem (Glasgow, UK), and Exiqon (Vedbaek, 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 directly injected into tissue sites or modified antisense molecules (e.g., antisense linked to a peptide or antibody that specifically binds to a receptor or antigen expressed on the surface of target cells) designed to target desired cells can be systemically administered.

[0171] In one embodiment, the short-chain nucleic acid and / or antisense oligonucleotide may comprise double-stranded small interfering RNA (siRNA) that, when expressed intracellularly, effects degradation by a sequence that is fully complementary to the cellular nucleic acid (e.g., mRNA) or effects translational inhibition by a sequence that is incompletely complementary to the cellular nucleic acid (e.g., mRNA), or may result from such double-stranded small interfering RNAs. In another embodiment, the double-stranded siRNA 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) that is homologous in sequence to the gene to be silenced. In vivo, long dsRNA is cleaved by ribonuclease III to generate 21-nucleotide and 22-nucleotide siRNAs. 21-nucleotide siRNA duplexes have been shown to specifically inhibit the expression of endogenous heterologous genes in various mammalian cell lines, including human embryonic kidney (293) cells and HeLa cells (Elbashir et al., (2001) Nature, Vol. 411:494-498). Thus, translation of an intracellular gene can be inhibited by contacting the cell with a short double-stranded RNA having a length of about 15-30 nucleotides or about 18-21 nucleotides or about 19-21 nucleotides. Alternatively, a vector encoding such siRNA or short hairpin RNA (shRNA) that is metabolized into siRNA can be introduced into the target cell (see, for example, McManus et al., (2002) RNA, Vol. 8:842, Xia et al., (2002) Nature Biotechnology, Vol. 20:1006, and Brummelkamp et al., (2002) Science, Vol. 296:550). Vectors that can be used are commercially available, for example, from OligoEngine under the name pSuperRNAi system (trademark).

[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. WO 90 / 11364, published Oct. 4, 1990, Sarver et al., (1990) Science 247:1222-1225, and U.S. Pat. No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used for the destruction of cellular mRNA, it is preferred to use hammerhead ribozymes. Hammerhead ribozymes cleave mRNA at a position defined by an adjacent region that forms complementary base pairs with the target mRNA. The only requirement is that the target mRNA have the following two-base sequence, namely 5'-UG-3'. The construction and production of hammerhead ribozymes are well known in the art and are more fully described in Haseloff and Gerlach, (1988) Nature 334:585-591. The ribozyme can be modified such that the cleavage recognition site is located near the 5' end of the cellular mRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

[0173] The ribozymes of the methods presented herein include RNA endoribonucleases (hereinafter "Cech-type ribozymes"), such as the ribozyme (known as IVS or L-19 IVS RNA) that occurs naturally within Tetrahymena thermophila and has been extensively described by Thomas Cech and co-workers (Zaug et al., (1984) Science 224:574-578; Zaug et al., (1986) Science 231:470-475; Zaug et al., (1986) Nature 324:429-433; WO 88 / 04300; and Been et al., (1986) Cell 47:207-216). Cech-type ribozymes have an 8-base pair active site that hybridizes to a target RNA sequence and then causes cleavage of the target RNA. The methods and compositions presented herein include these Cech-type ribozymes that target the 8-base pair active site sequences present in cellular genes.

[0174] Like the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., to improve stability, targeting, etc.). Preferred delivery methods involve using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, either a pol III promoter or a pol II promoter, such that the transfected cells produce sufficient amounts of the ribozyme to disrupt endogenous cellular messages and inhibit translation. Since ribozymes are catalytic, unlike antisense molecules, the intracellular concentrations required for efficiency are lower.

[0175] The nucleic acid molecules used in the formation of triple helices 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 the Hoogsteen base pairing rules, which generally require a fairly large stretch of either purine or pyrimidine to be present in one of the strands of the double helix. The nucleotide sequence may be a pyrimidine base, whereby TAT triplets and CGC triplets will be present across the three associated strands of the resulting triple helix. These high-pyrimidine molecules achieve base complementarity in an orientation parallel to that strand, relative to the high-purine region of one of the strands of the double helix. Also, nucleic acid molecules that are high in purine, such as nucleic acid molecules containing G residue extensions, may be selected. These molecules form triple helices with GC pair-rich DNA double strands, in which most of the purine residues are located on one of the strands of the targeted double helix, whereby CGC triplets will be present across the three strands of the triple helix.

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

[0177] The short-chain nucleic acids (e.g., miRNA, pre-miRNA, pri-miRNA, miRNA) of the methods and compositions presented herein *, anti-miRNA, or miRNA binding site, 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, chemical synthesis techniques for oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as solid-phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro transcription and in vivo transcription of the DNA sequence encoding the antisense RNA molecule. 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, an antisense cDNA construct that synthesizes antisense RNA constitutively or inducibly depending on the promoter used can be stably introduced into a cell line.

[0178] Furthermore, various well-known modifications to nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioates or 2'O-methyls rather than phosphodiester linkages within the oligodeoxyribonucleotide backbone, but are not limited thereto. One of ordinary skill in the art can readily understand that polypeptides, short-chain nucleic acids, and antisense oligonucleotides can be further conjugated to another peptide or polypeptide (e.g., a heterologous peptide) that serves as a means of protein detection, for example. Non-limiting examples of labeled peptide moieties or labeled polypeptide moieties useful for detection in the present invention include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, epitope tags such as FLAG tag, MYC tag, HA tag, or HIS tag, fluorophores such as green fluorescent protein, dyes, radioisotopes, digoxigenin, biotin, antibodies, polymers, and others known in the art, for example, those in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (ed.), Plenum Press, 2nd Edition (July 1999), but are not limited thereto.

[0179] The present invention also contemplates well-known methods for genetically modifying the genome of an organism or cell such that upon completion of the genetic modification, the expression and / or activity of PTEN and / or p53 is altered without contacting the organism or cell with a drug. For example, cancer cells can be genetically modified using recombinant techniques for the regulation of the expression and / or activity of PTEN and / or p53 such that it is not necessary to contact the cancer cells with a drug for the regulation of the expression and / or activity of PTEN and / or p53. For example, a target gene knockout method or a non-target gene knockout method can be used for ex vivo recombinant modification, for example, before injecting the target cancer cells into a subject. For example, target DNA in the genome can be manipulated by deletion, insertion, and / or mutation using a tissue-specific promoter, gene targeting, transposable elements, and / or any other method for the introduction of foreign DNA or the production of modified DNA / modified nuclear DNA, retro-viral insertion, artificial chromosome technology, gene insertion, random insertion. Other modification techniques include deletion of DNA sequences from the genome and / or alteration of nuclear DNA sequences. For example, nuclear DNA sequences can be altered by site-directed mutagenesis. Such methods generally use host cells into which the recombinant expression vectors 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 a particular target cell but also to progeny cells of such a cell or cells that may potentially be progeny. Because certain changes may occur in progeny due to either mutation or environmental influence, such progeny may not actually be identical to the parent cell, but 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 transfection techniques. As used herein, the terms "transformation" and "transfection" are meant to refer to a variety of techniques understood in the art for introducing foreign nucleic acid into a host cell, including co-precipitation with calcium phosphate or calcium chloride, DEAE dextran-mediated transfection, lipofection, or electroporation.Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., (supra), and other laboratory manuals. It is known that only a small percentage of cells can incorporate exogenous DNA into their genomes depending on the expression vector and transfection technique used for stable transfection of mammalian cells. To identify and select these inserts, a gene encoding a selectable marker (e.g., a marker for resistance to an antibiotic) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include markers that confer drug resistance such as G418, hygromycin, and methotrexate. Cells into which the introduced nucleic acid has been stably transfected can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene survive and other cells die).

[0180] Similarly, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g., for creating null mutations). In such embodiments, the CRISPR guide RNA and / or Cas enzyme can be expressed. For example, a vector containing only this guide RNA can be administered to a Cas9 enzyme transgenic animal or cell. Similar strategies (e.g., designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases) may be used. Such systems are well known in the art (see, e.g., U.S. Patent No. 8,697,359; Sander and Joung, (2014) Nat. Biotech., Vol. 32:347-355; Hale et al., (2009) Cell, Vol. 139:945-956; Karginov and Hannon, (2010) Mol. Cell, Vol. 37:7; U.S. Patent Application Publication Nos. 2014 / 0087426 and 2012 / 0178169; Boch et al., (2011) Nat. Biotech., Vol. 29:135-136; Boch et al., (2009) Science, Vol. 326:1509-1512; Moscou and Bogdanove, (2009) Science, Vol. 326:1501; Weber et al., (2011) PLoS One, Vol. 6:e19722; Li et al., (2011) Nucl. Acids Res., Vol. 39:6315-6325; Zhang et al., (2011) Nat. Biotech., Vol. 29:149-153; Miller et al., (2011) Nat. Biotech., Vol. 29:143-148; Lin et al., (2014) Nucl. Acids Res., Vol. 42:e47). Such genetic strategies can use 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 for administration of radiation (e.g., gamma irradiation and / or UV irradiation) and / or an agent that renders the cells unable to replicate (e.g., a cell membrane disruptor, a DNA replication inhibitor, an inhibitor of spindle formation during cell division, etc.). Typically, a minimum dose of about 3500 rad is sufficient, but doses up to about 30,000 rad are acceptable. In some embodiments, a sub-lethal dose of radiation may be used. For example, the cancer cells may be irradiated with radiation to suppress cell growth prior to administration of the cancer vaccine to reduce the risk of new neoplastic lesions occurring. It should be understood that radiation is just one way to make cells non-replicative, and other ways 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 encompassed by the present invention.

[0182] An agent that activates the TGFβ-Smad / p63 signaling pathway It is demonstrated herein that activation of the TGFβ-Smad / p63 axis in cancer cells promotes an immune response and ultimately controls the expression of multiple pathways that promote activation of cytotoxic T cells and immune memory. Accordingly, the cancer cells included within the scope of the present invention described herein are modified to activate the TGFβ-Smad / p63 signaling pathway. In one embodiment, the cancer cells are contacted with a TGFβ superfamily protein to activate the TGFβ-Smad / p63 signaling pathway. In another embodiment, the cancer cells are contacted with a modifier 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 tissues) can be cultured in 2D or 3D in vitro or ex vivo (e.g., can be cultured as tumor spheroids or tumor organoids).

[0183] In some embodiments, the cancer vaccine comprising the modified cancer cells described herein can be tested for certain desirable characteristics or functions prior to administration to a subject. In one embodiment, loss of PTEN and p53 is 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 a 2D or 3D culture system, activation of the TGFβ-Smad / p63 signature, such as upregulation of ICOS-L, 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, etc., and / or activation of T cells in the presence of DCs, such as an increase in the secretion of TNFα and / or IFNγ by T cells in the presence of DCs is tested for one or more of.

[0184] i. TGFβ superfamily proteins In one embodiment, the PTEN- and p53-deficient cancer cells described herein are contacted with a TGFβ superfamily protein to activate the TGFβ-Smad / p63 signaling pathway. This TGFβ superfamily protein can be any member of the TGFβ superfamily that can activate the TGFβ-Smad / p63 signaling pathway. The TGFβ superfamily protein may be derived from the TGFβ family, which includes, but is not limited to, LAP, TGFβ1, TGFβ2, TGFβ3, and TGFβ5. The TGFβ superfamily protein may 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 decapentaplegic / DPP. The TGFβ superfamily protein may be derived from the GDNF family and may be derived from artemin, GDNF, neurturin, and persephin. The TGFβ superfamily protein may be derived from a family other than the above families, which includes, but is not limited to, lefty A, lefty B, MIS / AMH, nodal, and SCUBE3. In certain embodiments, the TGFβ superfamily protein is TGFβ1, TGFβ2, and / or TGFβ3. In one embodiment, the cancer cells are contacted with a single TGFβ superfamily protein (e.g., TGFβ1, TGFβ2, or TGFβ3).In another embodiment, the cancer cells are contacted with a combination of TGFβ superfamily proteins (e.g., a combination of TGFβ1, TGFβ2, and TGFβ3).

[0185] The cancer cells can be contacted with TGFβ superfamily proteins only 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 TGFβ superfamily proteins are not administered in vivo to the subject, and the cancer cells are administered to the subject. In another embodiment, the cancer cells are administered to the subject, and 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 the cancer cells are administered to the subject. The TGFβ superfamily proteins can 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 the TGFβ superfamily proteins in vitro, in vivo, and / or ex vivo in combination with an immune checkpoint inhibitor. The immune checkpoint inhibitor can be administered to the subject before, after, and / or simultaneously with the administration of the cancer vaccine.

[0186] The dosage of the TGFβ superfamily proteins can vary to obtain a degree of TGFβ-Smad / p63 signaling pathway activation effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and to be non-toxic to that patient.

[0187] The selected dosage level will depend on various 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 rate of excretion or metabolism of the specific TGFβ superfamily protein being used, the treatment period, other drugs, compounds, and / or substances being used in combination with the specific TGFβ superfamily protein, the age, sex, weight, condition, general health, 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 contacted with a TGFβ superfamily protein at a dosage greater than 0.1 ng / ml, such as 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 contacted with a TGFβ superfamily protein at a dosage of about 0.1 ng / ml to about 100 ng / ml. In preferred embodiments, the cancer cells are contacted with a TGFβ superfamily protein at a dosage of about 1 ng / ml to about 10 ng / ml, such as about 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 contacted with a TGFβ superfamily protein for a period of time. That period can be from several minutes to 4 weeks, 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 a period of any value in between. The preferred range of that period is about 6 hours to about 21 days, about 12 hours to about 15 days, about 1 day to about 10 days, or about 3 days to about 7 days.

[0191] ii. An agent that increases the copy number, amount, and / or activity of at least one biomarker described in Table 1 In another embodiment, the PTNE- and p53-deficient cancer cells described herein are contacted with a modifier of the copy number, expression, and / or activity of one or more biomarkers described in Table 1, thereby activating the TGFβ-Smad / p63 signaling pathway. An agent that increases the copy number, expression, and / or activity of one or more biomarkers described in Table 1 can do so directly or indirectly.

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

[0193] In one embodiment, an isolated nucleic acid molecule that specifically hybridizes to one or more biomarkers described in Table 1 or their biologically active portions or encodes them. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (i.e., cDNA or genomic DNA), RNA molecules (i.e., mRNA), and analogs of DNA or RNA created using nucleotide analogs. The nucleic acid molecule can 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 that are present in the natural source of the nucleic acid. An "isolated" nucleic acid preferably does not contain sequences that are naturally adjacent to the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., the sequences located at the 5' end and 3' end of the nucleic acid). For example, in various embodiments, an isolated nucleic acid molecule corresponding to one or more biomarkers described in Table 1 may contain less than about 5 kb, less than about 4 kb, less than about 3 kb, less than about 2 kb, less than about 1 kb, less than about 0.5 kb, or less than about 0.1 kb of nucleotide sequences that are naturally adjacent to the nucleic acid molecule in the genomic DNA of the cell (i.e., lymphoma cell) from which the nucleic acid is derived. Further, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be free of other cellular material or culture medium when produced by recombinant techniques or free of chemical precursors or other chemicals when chemically synthesized.

[0194] Nucleic acid molecules within the scope of the present invention, such as the nucleotide sequences of one or more biomarkers described in Table 1, or the nucleotide sequences of one or more biomarkers described in Table 1 or a part thereof (i.e., 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 450 nucleotides, 500 nucleotides, or more nucleotides), having a nucleotide sequence that is at least about 50% identical, preferably at least about 60% identical, more preferably at least about 70% identical, even more preferably at least about 80% identical, even more preferably at least about 90% identical, and most preferably at least about 95% or more (e.g., about 98%) identical can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, human cDNA can be isolated from human cell lines (derived from Stratagene, La Jolla, CA or Clontech, Palo Alto, CA) using the entire or a portion of such nucleic acid molecule or fragment thereof as a hybridization probe and standard hybridization techniques (i.e., hybridization techniques as described in Sambrook, J., Fritsh, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Further, nucleic acid molecules encompassing the entire or a portion of a nucleotide sequence that is at least about 50% identical, preferably at least about 60% identical, more preferably at least about 70% identical, even more preferably at least about 80% identical, even more preferably at least about 90% identical, and most preferably at least about 95% or more identical to the nucleotide sequence of one or more biomarkers described in Table 1 or the nucleotide sequence or fragment thereof can be isolated by polymerase chain reaction using one or more biomarkers described in Table 1 or a fragment thereof or oligonucleotide primers designed based on said homologous nucleotide sequence.For example, mRNA can be isolated from muscle cells (i.e., isolated by the guanidinium thiocyanate extraction method described by Chirgwin et al. (1979) Biochemistry, Vol. 18: 5294-5299), and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for PCR amplification can be designed according to methods well known in the art. Nucleic acids included within the scope of the present invention can be amplified using cDNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. Nucleic acids amplified in this way can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to the nucleotide sequences of one or more biomarkers described 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 described in Table 1 can be used to detect or confirm the desired transcript or genomic sequence encoding the same or a homologous protein. In a preferred embodiment, the probe further comprises a label group attached thereto, i.e., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such a probe can be used as part of a diagnostic test kit for identifying cells or tissues expressing one or more biomarkers described in Table 1 by measuring the nucleic acid levels of one or more biomarkers described in Table 1 in a cell sample derived from a subject, i.e., detecting the mRNA levels of one or more biomarkers described in Table 1.

[0196] Nucleic acid molecules encoding proteins corresponding to one or more biomarkers described in Table 1 derived from various species are also contemplated. For example, rat or monkey cDNAs 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 molecules included within the scope of the present invention are proteins or portions thereof that contain an amino acid sequence that is sufficiently homologous such that one or more of the following biological activities, namely, (a) binding to its biomarker, (b) regulating the copy number of its biomarker, (c) regulating the expression level of its biomarker, and (d) regulating the activity level of its biomarker, are regulated (e.g., enhanced), and encode the protein or portion thereof that contains an amino acid sequence that is sufficiently homologous to the amino acid sequence of one or more biomarkers described in Table 1.

[0197] As used herein, the phrase "sufficiently homologous" refers to a protein or portion thereof that has an amino acid sequence containing the same or equivalent amino acid residues such that one or more of the following biological activities, namely, (a) binding to its biomarker, (b) regulating the copy number of its biomarker, (c) regulating the expression level of its biomarker, and (d) regulating the activity level of its biomarker, are regulated (e.g., enhanced), and contains a minimum number of amino acid residues that are the same or equivalent (e.g., amino acid residues having the same side chain as the amino acid residues in the biomarker or its fragment) to the amino acid sequence of one or more biomarkers described in Table 1 or its fragment.

[0198] In another embodiment, the protein is 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 higher homologous to the entire amino acid sequence of the biomarker or its fragment.

[0199] The portion of the protein encoded by the nucleic acid molecule of one or more biomarkers described in Table 1 is preferably the biologically active portion of the protein. As used herein, the term "biologically active portion" of one or more biomarkers described in Table 1 refers to a portion having one or more of the biological activities of the full-length protein, including, for example, 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, can be performed to determine the ability of the protein or its biologically active fragment to maintain the biological activity of the full-length protein.

[0201] The present invention further encompasses nucleic acid molecules that, due to the degeneracy of the genetic code, differ from the nucleotide sequence of one or more biomarkers described in Table 1 or fragments thereof, and thus encode the same protein as the protein encoded by the nucleotide sequence or fragment thereof. In another embodiment, the isolated nucleic acid molecule included within the scope of the present invention has a nucleotide sequence encoding a protein having the amino acid sequence of one or more biomarkers described in Table 1 or a fragment thereof, or an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identical to the amino acid sequence of one or more biomarkers described in Table 1 or a fragment thereof. In another embodiment, the nucleic acid encoding the polypeptide consists of a nucleic acid sequence encoding a portion of the full-length fragment of interest that is 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 amino acids in length.

[0202] One of ordinary skill in the art will understand that DNA sequence polymorphisms leading to changes in the amino acid sequences of one or more of the biomarkers described in Table 1 may exist within a population (e.g., a mammalian population and / or a human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules that include an open reading frame encoding one or more of the biomarkers described in Table 1, preferably a mammalian protein, e.g., a human protein. Such natural allelic variation typically can result in 1-5% mismatches within the nucleotide sequences of one or more of the biomarkers described in Table 1. All such nucleotide variations and the resulting amino acid polymorphisms within one or more of the biomarkers described in Table 1 that are the result of natural allelic variation and that do not alter the functional activity of one or more of the biomarkers described in Table 1 are intended to be within the scope of the present invention. Further, nucleic acid molecules encoding the proteins of one or more of the biomarkers described in Table 1 that are derived from other species.

[0203] In addition to the natural allelic variants of one or more of the biomarkers listed in Table 1 that may exist within the population, one of ordinary skill in the art further understands that mutations can introduce changes into the nucleotide sequence or fragments thereof, thereby causing changes in the amino acid sequence of one or more of the encoded biomarkers listed in Table 1 without altering the functional ability of one or more of the biomarkers listed in Table 1. For example, nucleotide substitutions that cause amino acid substitutions at "non-essential" amino acid residues can be made within the sequence or fragments thereof. A "non-essential" amino acid residue is a residue that can be changed from the wild-type sequence of one or more of the biomarkers listed in Table 1 without altering the activity of one or more of the biomarkers listed in Table 1, while an "essential" amino acid residue is necessary for the activity of one or more of the biomarkers listed in Table 1. However, other amino acid residues (e.g., amino acid residues that are not conserved or only semi-conserved between mouse and human) may not be essential for activity and thus may be alterable without changing the activity of one or more of the biomarkers listed in Table 1.

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

[0205] The comparison of sequences and the determination of the percent homology between two sequences can be accomplished using mathematical algorithms. It is preferred to perform the alignment using the Clustal method. Examples of multiple alignment parameters include gap penalty = 10, gap length penalty = 10. For DNA alignment, the pairwise alignment parameters can be H tuple = 2, gap penalty = 5, window = 4, and diagonal saved = 4. For protein alignment, the pairwise alignment parameters can be K tuple = 1, gap penalty = 3, window = 5, and diagonal saved = 5.

[0206] In a preferred embodiment, the percent identity between two amino acid sequences is determined using either the Blossom62 matrix or the PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6, and the Needleman and Wunsch algorithm (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program within the GCG software package (available online). In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix with a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6, and using the GAP program within the GCG software package (available online). In another embodiment, the percent identity between two amino acid sequences or between two nucleotide sequences is determined using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and the algorithm of E. Meyers and W. Miller (CABIOS 4:11-17 (1989)) incorporated into the ALIGN program (version 2.0) (available online).

[0207] An isolated nucleic acid molecule encoding a protein that is homologous to one or more biomarkers or fragments thereof described in Table 1 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence or a fragment thereof or a homologous nucleotide sequence such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. The mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are preferably made 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 with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, predicted non-essential amino acid residues in one or more biomarkers described in Table 1 are preferably replaced with another amino acid residue of the same side chain family. Alternatively, in another embodiment, mutations can be randomly introduced into all or part of the coding sequence of one or more biomarkers described in Table 1 by, for example, saturation mutagenesis, and the resulting mutants can be screened for the activities described herein to identify mutants that retain the desired activity. After mutagenesis, the encoded protein can be expressed by recombinant techniques according to methods well known in the art, and the activity of the protein can be determined, for example, using the assays described herein.

[0208] The level of one or more biomarkers described in Table 1 can be evaluated by any of a variety of well-known methods for detecting the expression of transcribed molecules or proteins. Non-limiting 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 a preferred embodiment, the level of one or more biomarkers described in Table 1 is confirmed by measurement of gene transcripts (e.g., mRNA), by measurement of the amount of translated protein, or by measurement of gene product activity. Expression levels can be monitored by a variety of methods including detection of mRNA levels, detection of protein levels, or detection of protein activity, all of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or can be a qualitative assessment of gene expression levels, particularly in comparison to control levels. The type of level detected will be apparent from the context.

[0210] In certain embodiments, the mRNA expression level can be determined in both in situ and in vitro formats in a biological sample using methods known in the art. The term "biological sample" is taken to include tissues, cells, biological fluids, and isolates thereof isolated from a subject, as well as tissues, cells, and body fluids present within a subject. A number of expression detection methods use isolated RNA. Any RNA isolation technique not selected for mRNA isolation is available for purification of RNA from cells for in vitro methods (e.g., Ausubel et al., Current Protocols in Molecular See Biology, John Wiley and Sons, New York, 1987 - 1999). Also, for example, a number of tissue samples can be readily 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. 4,843,155).

[0211] The isolated mRNA can be used in hybridization assays or amplification assays including, but not limited to, Southern analysis, 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) capable of hybridizing to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, at least 7 nucleotides in length, 15 nucleotides in length, 30 nucleotides in length, 50 nucleotides in length, 100 nucleotides in length, 250 nucleotides in length, or 500 nucleotides in length, and can be a full - length cDNA or a portion thereof, such as an oligonucleotide sufficient to specifically hybridize under stringent conditions to the mRNA or genomic DNA encoding one or more of the biomarkers listed in Table 1. Other suitable probes for use in the diagnostic assays encompassed by the present invention are described herein. Hybridization of the mRNA with the probe indicates that one or more of the biomarkers listed in Table 1 are expressed.

[0212] In one format, the mRNA is immobilized on a solid surface, for example, by electrophoresing the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose, and then contacting it with the probe. In another format, in a gene chip array, for example, an Affymetrix (trademark) gene chip array, the probe is immobilized on a solid surface and the mRNA is contacted with the probe. Those skilled in the art can readily apply known mRNA detection methods for detecting the expression levels of one or more of the biomarker mRNAs listed in Table 1.

[0213] Another method for determining the mRNA expression level in a sample involves nucleic acid amplification, such as RT-PCR (experimental embodiments shown in U.S. Patent No. 4,683,202 to Mullis, 1987), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, Vol. 88: 189-193), self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA, Vol. 87: 1874-1878), transcription amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA, Vol. 86: 1173-1177), Qβ replicase (Lizardi et al., 1988, Bio / Technology, Vol. 6: 1197), rolling circle replication (U.S. Patent No. 5,854,033 to Lizardi), or any other nucleic acid amplification method, followed by detection using techniques well known to those skilled in the art for the amplified molecules. These detection schemes are particularly useful for detecting such molecules when the nucleic acid molecules are present in very low numbers. As used herein, an amplification primer is defined as a pair of nucleic acid molecules that can anneal to the 5' or 3' region of a gene (the plus and minus strands, respectively, or vice versa) and can include a short region therebetween. Generally, amplification primers are about 10 to 30 nucleotides in length and are adjacent to a region about 50 to 200 nucleotides in length. 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 methods, it is not necessary to isolate mRNA from cells prior to detection. In such methods, cell samples or tissue samples are prepared / processed using known histological methods. The sample is then fixed on a support, typically a glass slide, and subsequently contacted with a probe that can hybridize to the mRNA of one or more biomarkers listed in Table 1.

[0215] As an alternative method for making a determination based on the absolute expression level, the determination can be made based on the normalized expression levels of one or more biomarkers described in Table 1. The expression level is normalized by correcting the absolute expression level by comparing its expression to the expression of a non-biomarker gene, for example, a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene or epithelial cell-specific genes. By this normalization, the expression level in one sample, for example, a subject sample, can be compared to the expression level in another sample, for example, a normal sample, or can be compared between samples derived from different origins.

[0216] The level or activity of the protein corresponding to one or more biomarkers described in Table 1 can also be detected and / or quantified by detection or quantification of the expressed polypeptide. This polypeptide can be detected and quantified by any of a number of means well known to those skilled in the art. These means can include analytical biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high diffusion chromatography, or various immunological methods such as liquid or gel precipitation reactions, immunodiffusion (mono- or bi-), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting. Those skilled in the art can readily apply known protein / antibody detection methods for use in determining whether a cell expresses the biomarker of interest.

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

[0218] In one aspect, the polypeptide may comprise the full-length amino acid sequence corresponding to one or more biomarkers described in Table 1 or a full-length amino acid sequence having 1 to about 20 conservative amino acid substitutions. Any amino acid sequence described herein is 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 sequence of one or more biomarkers described in Table 1, the full-length sequences well known in the art, or a fragment thereof. In another aspect, the invention contemplates a composition comprising an isolated polypeptide corresponding to a polypeptide of one or more biomarkers described in Table 1 and less than about 25%, or less than 15%, or less than 5% contaminating biological macromolecules or contaminating polypeptides.

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

[0220] An isolated polypeptide or a fragment thereof (or a nucleic acid encoding such a polypeptide) corresponding to one or more biomarkers described in Table 1 can be used as such for generating 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 antigenic peptide contains at least 8 amino acid residues and encompasses the epitope present in each full-length molecule such that the antibody generated against the peptide forms a specific immune complex with each full-length molecule. Preferably, the antigenic peptide contains at least 10 amino acid residues. In one embodiment, such an epitope can be specific for a given polypeptide molecule derived from one species, e.g., mouse or human (i.e., an antigenic peptide spanning a region of the polypeptide molecule that is not conserved across multiple species is used as the immunogen. Such non-conserved residues can 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 described in Table 1 and enhances its biological function. In another embodiment, an antibody, particularly an intracellular antibody, binds substantially specifically to a binding partner of one or more biomarkers described in Table 1 and enhances its biological function.

[0222] Antibodies used in accordance with the present invention can be generated according to methods well known in the art. For example, a polypeptide immunogen is typically used to prepare an antibody by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with an immunogen. Suitable immunogenic preparations can include recombinantly expressed or chemically synthesized molecules or fragments thereof that elicit an immune response. The preparation can further include an adjuvant such as Freund's complete adjuvant or Freund's incomplete adjuvant, or a similar immunostimulant. Immunization of a suitable subject with the immunogenic preparation induces a polyclonal antibody response against the antigenic peptide contained therein.

[0223] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide immunogen. The polypeptide antibody titer in the immunized subject can be monitored over time by standard techniques such as the use of an enzyme-linked immunosorbent assay (ELISA) using an immobilized polypeptide. If desired, the 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, e.g., when the antibody titer is highest, antibody-producing cells are obtained from the subject and used to prepare monoclonal antibodies by standard techniques such as hybridoma technology (originally described by Kohler and Milstein, Nature (1975) 256:495-497) (see also Brown et al., J. Immunol. (1981) 127:539-46, Brown et al., J. Biol. Chem. (1980) 255:4980-83, Yeh et al., Proc. Natl. Acad. Sci. (1976) 76:2927-31, Yeh et al., Int. J. Cancer (1982) 29:269-75), more recently human B cell hybridoma technology (Kozbor et al., Immunol. Today (1983) 4:72), EBV hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985) 77-96), or trioma technology. Techniques for generating monoclonal antibody hybridomas are well known (see generally the works of Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Press, New York, NY (1980), Lerner, E. A., Yale J. Biol. Med. (1981) 54:387-402, Gefter, M. L. et al., Somatic Cell Genet. (1977) 3:231-36).Briefly described, an immortal cell line (typically myeloma) is fused with lymphocytes (typically splenocytes) derived from a mammal immunized with the immunogen as described above, and the culture supernatant of the resulting hybridoma cells is screened to identify hybridomas that produce monoclonal antibodies that preferably bind specifically to the polypeptide antigen.

[0224] Any of a number of well-known protocols used to fuse lymphocytes with immortalized cell lines is applicable for the purpose of generating monoclonal antibodies against one or more of the biomarkers or fragments thereof listed in Table 1 (see, for example, Galfre, G. et al., (1977) Nature, Vol. 266:550 - 52, Gefter et al., (1977) supra, Lerner, (1981) supra, Kenneth, (1980) supra). Further, one of ordinary skill in the art will understand that there are numerous variations of such methods that are similarly useful. The immortalized cell line (e.g., a myeloma cell line) is typically derived from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be produced by fusing lymphocytes derived from a mouse immunized with an immunogenic preparation within the scope of the present invention with an immortalized mouse cell line. Preferred immortalized cell lines are mouse myeloma cell lines that are sensitive to a culture medium containing hypoxanthine, aminopterin, and thymidine (the "HAT medium"). Any of a number of 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 can be used. These myeloma lines are available from the American Type Culture Collection (ATCC) in Rockville, Maryland. HAT-sensitive mouse myeloma cells are typically fused to mouse spleen cells using polyethylene glycol ("PEG"). Subsequently, the resulting hybridoma cells from the fusion are selected using HAT medium, which kills unfused myeloma cells and myeloma cells that do not produce even after fusion (unfused spleen cells die after a few days as they are not transformed). Hybridoma cells that produce monoclonal antibodies within the scope of the present invention are detected by screening the hybridoma culture supernatant for antibodies that bind to a given polypeptide using standard ELISA assays and 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 an appropriate polypeptide, thereby isolating immunoglobulin library members that bind to the polypeptide, and a monoclonal specific for one of the above polypeptides can be identified and isolated. Kits for creating and screening phage display libraries are commercially available (e.g., Pharmacia's Recombinant Phage Antibody System, catalog number 27-9400-01, and Stratagene's SurfZAP™ Phage Display Kit, catalog number 240612). Also, examples of methods and reagents that can be used for creating and screening antibody display libraries are described, for example, in U.S. Patent No. 5,223,409 to Ladner et al., International Publication No. WO 92 / 18619 to Kang et al., International Publication No. WO 91 / 17271 to Dower et al., International Publication No. WO 92 / 20791 to Winter et al., International Publication No. WO 92 / 15679 to Markland et al., International Publication No. WO 93 / 01288 to Breitling et al., International Publication No. WO 92 / 01047 to McCafferty et al., International Publication No. WO 92 / 09690 to Garrard et al., International Publication No. WO 90 / 02809 to Ladner et al., Fuchs et al., (1991) Biotechnology (New York), Vol. 9:1369-1372, Hay et al., (1992) Hum. Antibod. Hybridomas, Vol. 3:81-85, Huse et al., (1989) Science, Vol. 246:1275-1281, Griffiths et al., (1993) EMBO J., Vol. 12:725-734, Hawkins et al., (1992) J. It can be found in 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. USA, Vol. 89: pp. 3576-3580, by Garrard et al. (1991); Biotechnology (New York), Vol. 9: pp. 1373-1377, by Hoogenboom et al. (1991); Nucleic Acids Res., Vol. 19: pp. 4133-4137, by Barbas et al. (1991); Proc. Natl. Acad. Sci. USA, Vol. 88: pp. 7978-7982, and by McCafferty et al. (1990); Nature, Vol. 348: pp. 552-554.

[0226] Since it is well known in the art that the CDR3 domains of the heavy and light chains of an antibody play a particularly important role in the binding specificity / affinity of the antibody for an antigen, the recombinant monoclonal antibodies included within the scope of the present invention prepared as shown above preferably contain the CDR3s 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 included within the scope of the present invention. The antibody may further contain the CDR1 of the variable region included within the scope of the present invention. In other embodiments, the antibody may contain any combination of the CDRs.

[0227] The CDR1 region, CDR2 region, and / or CDR3 region of the modified antibody described above may contain the exact amino acid sequences of those regions of the variable region included in the scope of the present invention. However, those skilled in the art will understand that there may be some deviations (e.g., conservative sequence modifications) from those exact CDR sequences while still retaining the ability of the antibody to efficiently bind to a target such as one or more biomarkers described in Table 1 and / or one or more natural binding partners. Thus, in another embodiment, the modified antibody may be composed 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 non-human antibodies or human antibodies (e.g., rat anti-mouse / anti-human antibodies) can be used to create structurally related human antibodies, particularly intrabodies, that retain at least one functional property of the antibodies included in the scope of the present invention, such as one or more biomarkers described in Table 1, the binding partners / substrates of one or more biomarkers described in Table 1, and / or binding to immune checkpoints. Another functional property includes inhibition of the 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 homologies are equivalent to introducing one, two, three, four, five, six, seven, eight, nine, ten, or more conservative amino acid substitutions within a given CDR, and can be achieved by such introduction.

[0230] Monoclonal antibodies within the scope of the present invention can include a heavy chain, the variable domain of which includes at least one CDR having a sequence selected from the group consisting of the heavy chain variable domain CDRs described herein, and said monoclonal antibody can include a light chain, the variable domain of which includes at least one CDR having a sequence selected from the group consisting of the light chain variable domain CDRs described herein.

[0231] Such monoclonal antibodies can include a light chain, the variable domain of which includes 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 said monoclonal antibody can include a heavy chain, the variable domain of which includes 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, said monoclonal antibody capable of binding to one or more biomarkers described in Table 1 includes 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 said monoclonal antibody within the scope of the present invention can include or consist of the vH amino acid sequence shown herein, and / or the light chain variable domain of said monoclonal antibody within the scope of the present invention can include or consist of the vκ amino acid sequence shown herein.

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

[0234] Other fragments of the monoclonal antibodies included within the scope of the present invention are also contemplated. For example, individual immunoglobulin heavy and / or light chains are provided in which the variable domain comprises at lea...

Claims

[Claim 1] The invention described in the specification.

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