Compositions and methods for the detection and / or treatment of cancer

Abstract

Provided are isolated, genetically engineered or synthetic Wade polypeptide and polynucleotide compositions and compositions and methods for treating a cancer using a Wade inhibitor and compositions and methods for detecting a cancer through detecting a Wade polypeptide or a Wade polynucleotide.

Description

Compositions and Methods for the Detection and / or Treatment of CancerCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U. S. Provisional Patent Application No. 63 / 715,064, filed November 1, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The sequence listing submitted on October 31, 2025 as an. XML file entitled ‘T0504-102W01-ST26.xmr’ created on October 30, 2025 and having a file size of 22268 bytes is hereby incorporated by reference pursuant to 37 C. F. R. § 1.52(e)(5).BACKGROUND OF THE INVENTION

[0003] Chemotherapy, and specifically cytotoxic chemotherapy, has been a standard treatment for cancer for decades. However, traditional cytotoxic chemotherapy kills noncancer cells along with cancer cells and can therefore be associated with numerous severe side effects, lire immediate effects of chemotherapy can be observed on skin, hair, bone marrow, blood, gastrointestinal tract and the kidneys. (Schirmacher, V., From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment, Int. J. Oncol. 2019 Feb; 54(2): 407-409) Other essential organs such as the heart, lungs and brain can also be affected. (Id.) Neurotoxicity from traditional chemotherapy can induce paralysis and coma. (Id.)

[0004] Accordingly, there is a need for the identification and elucidation of cancer cell specific biomarkers that can be used to develop therapies to target cancer cells and to reduce the deleterious effects of those therapies on non -cancer cells,BRIEF DESCRIPTION OF FIGURES

[0005] Fig. l(A-B). (A) Schematic of a 44 amino acid Wade polypeptide and its signal peptide. Boxes denote 3 exons (El, E2 and E3) of the Wade mRNA isoform, and the arrow represents the open reading frame encoding the 70-amino acid Wade polypeptide and (B) schematic of an Alpha Fold prediction of the secreted de polypeptide; SP = signal peptide.

[0006] Fig. 2. Schematic representation of the expression and function of Wade in cancer cells. Also shown is one of the signaling pathway involving activated API transcription factor that drives Wade mRNA expression in cells, and the changes in the production ofextracellular inflammatory cytokines and other proteins upon Wade knockdown in cancer cells.

[0007] Fig. 3. A graph showing Wade mRNA expression levels in cancer cell lines representing multiple tissue types, with some examples of lung adenocarcinoma, hepatocellular carcinoma, and glioblastoma highlighted by lines. Three examples of glioblastoma cell lines including U87MG are also highlighted as solid circles. Only a subset of cancer cell lines with Wade expression higher than 100 TPM (transcript per million) are shown. Median Wade expression in healthy tissues in Genotype-Tissue Expression (GTEx) is either undetected or less than 1 TPM (data not shown).

[0008] Fig. 4. A graph showing Wade expression is positively correlated with tire activation of the RAS-RAF-MEK-ERK pathway. Data represent changes in Wade expression upon cellular perturbations (e.g. treatment with inhibitors, over-expression of proteins, etc.) where each circle represents one perturbation experiment from published datasets. Filled circles represent some examples of perturbation experiments implicating RAS-RAF-MEK-ERK axis in Wade expression. Expression results were mined from the European Molecular Biology Laboratory (EMBL) expression atlas, an open public repository of gene expression data under different biological conditions,

[0009] Fig. 5(A-C). Graphs showing Wade is expressed in glioblastoma and ovarian cancer lines in a MEK1 / 2 dependent manner. (A) Wade expression in human cancer cells - glioblastoma cell line U87, ovarian cancer cell line ES2, THP-1 monocytes and HEK- 293T epithelial cells, (B) Expression of Wade in U87 cells treated with MEK1 / 2 inhibitor, U0126, and (C) RT-qPCR Ct value of the mouse homolog of Wade in mouse glioblastoma cells GL261.

[0010] Fig, 6(A-B). Graphs showing Wade knockdown leads to reduction in cell proliferation. (A) Flow cytometry of control or Wade shRNA expressing U87 cells where cells were labelled with cell trace violet dye and cultured for 4 days (high signal: low proliferation) and (B) The median fluorescence intensity (MFI) of the cells is shown on the graph. Wade shl refers to use of SEQ ID NO:3 and SEQ ID NO:4. Wade sh2 refers to use of SEQ ID NO:6 and SEQ ID NO:7. shLUC targets luciferase and refers to use of SEQ ID NO: 12 and SEQ ID NO: 13

[0011] Fig, 7(A-C) shows that Wade knockdown leads to impaired colony formation, (A) Schematic of the colony formation assay of ovarian cancer cell line ES2 expressing control or Wade shRNAs, (B) Photograph showing crystal violet staining of the colonies formed after 9 days of seeding, and (C) Graph showing absorbance of the crystal violetstain at 590 nm in control versus Wade knockdown. Wade shl refers to SEQ ID NO:3 and SEQ ID NO:4. Wade sh2 refers to SEQ ID NO:6 and SEQ ID NO:7. shLUC refers to SEQ ID NO: 12 and SEQ ID NO: 13. ES2 cells expressing either control (shLUC) or Wade shRNAs were seeded at a density of one thousand cells per well in triplicate in a 6-well plate. The culture medium was replaced every' 2-3 days. After 9 days, cells were washed with PBS and fixed with methanol. Following fixation, methanol was removed, and colonies were stained with 0.5% crystal violet prepared in 25% methanol. Excess stain 'as washed away, and the plates were air-dried. To quantify colony formation, 33% acetic acid w7as added to each w'ell, and the plates were shaken for 10 minutes at room temperature. The solubilized dye was transferred to a 96-well plate, and absorbance was measured at 590 nm.

[0012] Fig. 8(A-B) show's that Wade knockdown leads to impaired cell migration. (A) Photographs of scratch assay of U87 cells expressing control or Wade targeting shRNAs and (B) Graph showing the percentage of wound healing (area covered by migrated cells) in control versus Wade knockdown. Wade shl refers to SEQ ID NO:3 and SEQ ID NO:4. Wade sh2 refers to SEQ ID NO:6 and SEQ ID NO:7. shLUC refers to SEQ ID NO: 12 and SEQ ID NO: 13. Cells were seeded in 12-well plates in triplicates and following overnight culture scratched with a 1ml pipete tip and imaged at 0 hour and 24 hours.

[0013] Fig. 9(A-D) showing that knockdown of Wade alters the cancer cell transcriptome.(A) Schematic of the RNA-seq and Luminex cytokine analysis performed on U87 cells expressing control or Wade shRNAs, (B) Graph showing number of genes differentially expressed in shWade versus control, (C) Graph showing gene ontology analyses of the molecular functions, and (D) Graph showing cellular components enriched for Wade- regulated differentially expressed genes.

[0014] Fig. 10(A-B). Wade knockdown leads to significantly reduced production of extracellular proteins. Results are shown in two heatmaps (A and B) for highly abundant proteins (A) and the rest that are produced at relatively low er quantities (B) in U87 cells expressing control (shLUC; SEQ ID NO: 12 and SEQ ID NO: 13) or Wade shRNAs (SEQ ID NO: 15 and SEQ ID NO: 16).

[0015] Fig. 1 l(A-C). Ectopic expression of Wade protein promotes cancer cell proliferation. (A) Schematic of cell proliferation assay w ith murine melanoma PTEN Braf CL24 cells expressing empty' vector (EV) or full-length wild type Wade using cell trace violet dye, (B) Graph showing flow' cytometry analysis of the cells on Day 5, and (C) Graph showing the median fluorescence intensity (MFI) of the cells.

[0016] Fig. 12(A-B). Wade protein is secreted out of cells. (A) Immunofluorescence images showing HeLa cells were transfected with plasmid expressing Wade-Hise (SEQ ID NO: 14) or empty vector (EV) and further treated with Brefeldin A for 6 hours. Cells were fixed with 4% formaldehyde and stained with anti-Hiss antibody for immunofluorescence and (B) A graph showing detection of secreted, mature Wade protein (SEQ ID NO: 1) in the culture supernatant of HEK293T cells stably expressing Wade-Hiss or control by Direct ELISA against His6-tag.

[0017] Fig. 13(A-C). Ectopic expression of Wade in shWade U87 cells restores their migratory ability similar to control cells. (A) A schematic showing the Wade wildtype and Wade mutants Wade T26R (SEQ ID NO: 10) (lacking cleavage of the signal peptide and secretion of mature Wade protein) and Met 1( SEQ ID NO: 11) (lacking protein translation due to mutation in tire translation-initiating Methionine), (B) A schematic of cell migration assay of shWade U87 cells transfected with plasmids expressing empty vector control, wild type Wade or a Wade mutant, and (C) A graph shows the number of cells migrated into the wells from the trans well inserts. Wade shl refers to use of SEQ ID NO: 3 and SEQ ID NO:4. Wade sh2 refers to use of SEQ ID NO:6 and SEQ ID NO:7. shLUC refers to use of SEQ ID NO: 12 and SEQ ID NO: 13

[0018] Fig. 14(A-B). Wade signal peptide is sufficient to drive protein secretion. (A) Schematic of Wade signal peptide fused to enhanced green fluorescent protein (EGFP) and (B) Immunofluorescence images showing HeLa cells transfected with plasmid expressing EGFP or SP-EGFP fusion protein.

[0019] Fig. 15(A-B). Brefeldin A treatment leads to intracellular retention of enhanced green fluorescent protein (EGFP) protein fused with the signal peptide of Wade (SP- EGFP). (A) A schematic of protein extraction to detect EGFP or SP-EGFP (B) A Western blot showing detection of SP-EGFP in Brefeldin A treated HeLa cells (highlighted by arrow).

[0020] Fig. 16(A-C), Inhibition of protein secretion with Brefeldin A treatment leads to intracellular retention of SP-EGFP fusion protein. (A) A schematic showing measurement of fluorescence by flow cytometry of HEK293T cells transfected with EGFP or SP-EGFP expressing plasmids, followed by Brefeldin A treatment, (B) a graph showing fluorescence intensity of the cells detected by flow cytometry, and (C) A graph showing MFI of the cells.

[0021] Fig. 17(A-B). Enhanced green fluorescent protein (EGFP) is secreted out from cells when fused with the signal peptide of Wade (SP-EGFP). (A) A schematic of theexperiment performed in HEK 293T cells and (B) A Western blot showing EGFP in the supernatant of HEK293T cells transfected with plasmid expressing SP-EGFP.

[0022] Fig, 18(A-C). Wade knockdown Suppresses Tumor Growth in NUDE Mouse Xenografts. (A) A schematic of the xenograft experiment in NUDE mice using human ES2 cells expressing control (shLUC; SEQ ID NO: 12 and SEQ ID NO: 13)or shWade (Wade shRNA2 SEQ ID NO:6 and SEQ ID NO:7), (B) Images of tumors isolated from respective groups at the end of the experiment on day 17, and (C) A chart showing tumor volumes at different days post injection. P== 0.006. 2way ANOVA. Ten female NUDE mice (Nu / J; 7-8 weeks old) were randomly assigned to either the control or experimental group. The control group was inoculated with human ES2 ovarian cancer cells expressing a non-targeting shRNA (shControl; Luciferase), whereas the experimental group received ES2 cells expressing shRNA targeting Wade (shRNA2; shWade). A total of 5 million cells were suspended in 200ul were injected subcutaneously into both the left and right flanks of each mouse. Animals were monitored regularly for general health, and subcutaneous tumor volumes were measured every 2-3 days over a 19-day period.

[0023] Fig. 19. Secreted mature form of Wade protein (aa27-70) (SEQ ID NO:1) binds on the cancer cell membrane. Representative images of melanoma cells incubated with biotinylated Wade and stained with Streptavidin-PE-Cy7. Fluorescent signal localizes to the cell membrane. Details: CL2.4 melanoma cells (a derivative of murine B16FI0 melanoma cells) were incubated with 100 ng of biotiny lated Wade protein in the presence of a 5-fold excess of BSA for 30 minutes at 4 °C. After washing, cells were stained with Streptavidin - PE-Cy 7 (1:1000) for 1 hour on ice, washed, and imaged using the ImageStream system. Wade protein (SEQ ID NO: 1) was custom made using solid phase peptide synthesis at a commercial vendor (Genscript).DETAILED DESCRIPTION

[0024] Provided herein are compositions and methods for detecting and treating a cancer.Described herein for the first time is a polypeptide, referred to herein as Wade, that is predominantly expressed in cancer cells. Accordingly, the methods of detecting a cancer included herein comprise detecting a Wade mRNA polynucleotide or polypeptide in a subject, wherein the detection indicates the presence of a cancer in the subject. Also disclosed herein is the surprising finding that Wade expression is driven by oncogenic MAPK / AP-1 activity, and functional studies demonstrate that Wade promotes cancer-cell proliferation, migration, and secretion of inflammatory' cytokines. Wade encodes a 70- amino-acid precursor containing a 26-amino-acid signal peptide, which is cleaved togenerate a 44-amino-acid secreted protein (SEQ ID NO: 1).. The methods described herein therefore also include methods of treating a cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a Wade inhibitor. Further provided herein are compositions and kits for the detection of Wade and / or treatment of cancer through the targeting of Wade.

[0025] Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicants desire that the following terms be given the particular definition as defined below.

[0026] Terminology

[0027] As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0028] The terms "about" and "approximately" are defined as being ‘"close to” as understood by one of ordinary' skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0029] The term “administering” refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-artenole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrastemal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. In some embodiments, the administration is intravenous.

[0030] The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g,, bispecific antibodies) and nanobodies.Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity'. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH)followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.

[0031] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments. The phrase "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full- length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one which can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FCERI AS used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')?. fragments. An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non- co valent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL. dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv composing only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding.

[0032] The term “monoclonal antibody’’ as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.

[0033] "Humanized" forms of non-human (e.g. murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin template chosen.

[0034] A “binding agent’’ may be any compound or complex of compounds which is capable of binding a target substance such as a Wade polypeptide or polynucleotide. Preferably, the binding agent is capable of specifically binding the target substance. Suitable binding agents may be obtained by screening a binding agent library in order to identify / obtain binding agents that bind to the target substance. In some embodiments, a binding agent is an antibody that specifically binds a Wade polypeptide.

[0035] A "composition" is intended to include a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0036] As used herein, the term "comprising" is intended to mean that tire compositions and methods include the recited elements but not excluding others, "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.

[0037] A "control" is an alternative subject or sample used in an experiment for comparison puipose, A control can be "positive" or "negative." For example, in some embodiments, a negative control is an individual or study population that does receive the treatment.

[0038] As used herein, the term “expression"’ refers to either or both “gene expression” and “protein expression.” “Gene expression” refers to the process by which polynucleotides are transcribed into mRNA and “protein expression” refers to the process by which mRNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. “Gene overexpression” refers to the overproduction of the mRNA transcribed from the gene, at a level that is at least about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample, “Protein overexpression” includes the overproduction of the protein product encoded by a gene at a level that is at least about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample.

[0039] The terms “genetically engineered” and “synthetic” refer to non-naturally occurring polynucleotides and polypeptides, e.g., materials produced by means of a technical process.

[0040] As used herein “surface expression” refers to the process by which polypeptides are translocated to the surface of a cell such that at least a portion of the polypeptide is located at the exterior of the cell surface. “Surface overexpression” includes an increase in the amount of a particular polypeptide at the exterior surface of a cell, at a level that is 2.5 times higher, 5 times higher, or 10 times higher than the surface expression level detected in a control sample.

[0041] The term "identity" shall be construed to mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the bases or residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary’ to achieve the maximum percent identity' for the entire sequence, and not considering any conservative substitutions as part of tire sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned over their full lengths, that percentage of bases (or amino acids) are the same in comparing tire two sequences. As described below, the preferred algorithms can account for gaps and the like.

[0042] Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identi cal to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0043] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, testand reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0044] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query' sequence, which either match or satisfy some positive-valued threshold score T when aligned with a w ord of the same length in a database sequence. T is referred to as the neighborhood w ord score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) andN (penalty score for mismatching residues; always. <0).

[0045] For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below', due to tire accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M==5, N= -4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Hemkoff (1989) Proc, Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0046] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0047] The term "isolated" means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. In one aspect of this invention, an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated with in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, a "concentrated", "separated" or "diluted" polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated" or less than "separated" than that of its naturally occurring counterpart. A polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, which differs from the naturally occurring counterpart in its primary sequence or for example, by its glycosylation pattern, need not be present in its isolated form since it is distinguishable from its naturally occurring counterpart by its primary sequence, or alternatively, by another characteristic such as glycosylation pattern. Although not explicitly stated for each of the inventions disclosed herein, it is to be understood that all the above embodiments for each of the compositions disclosed below and under the appropriate conditions, are provided by this invention. Thus, a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide. A protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.

[0048] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

[0049] The terms “specific binding.” “specifically binds,” “selective binding,” and “selectively binds” mean that a binding agent exhibits appreciable affinity for a particular binding partner polypeptide such as a Wade polypeptide or polynucleotide. Appreciable binding affinity includes binding with an affinity of at least 106M'1, specifically at least 107M’1, more specifically at least 10sM’1, yet more specifically at least 109M’1, or even yet more specifically at least 10’° M1A binding affinity can also be indicated as a range of affinities, for example, 106M"1to 1010M"1, specifically 107M"1to 1010M'1, more specifically 108M-1to 1010M'1. Specific binding can be determined according to any art- recognized means for determining such binding. In some embodiments, specific binding is determined according to Scatchard analysis and / or competitive binding assays,

[0050] The terms “pharmaceutically effective amount”, “therapeutically effective amount” or “therapeutically effective dose” refer to tire amount of a compound such as a Wade polypeptide inhibitor or Wade polypeptide or polynucleotide binding agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. In some embodiments, a desired response is treatment of a cancer. In some instances, a desired biological or medical response is achieved following admini stration of multiple dosages of the composition to tire subject over a period of days, weeks, or years. The terms “pharmaceutically effective amount”, “therapeutically effective amount” or “therapeutically effective dose” include that amount of a compound such as a Wade protein inhibitor or Wade polypeptide or polynucleotide binding agent that, -when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated such as a cancer. The therapeutically effective amount will vary- depending on the compound such as a Wade protein inhibitor or Wade polypeptide or polynucleotide binding agent, the disorder or conditions and its se verity, the route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and / or diet of the subject to be treated. In the context of the present method, a pharmaceutically or therapeutically effecti ve amount or dose of a Wade protein inhibitor or Wade polypeptide or polynucleotide binding agent includes an amount that is sufficient to reduce the size or numbers of, or reduces the metastasis of, a solid tumor.

[0051] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeuticcomposition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents.

[0052] As used herein, the term “earner” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. Ihe preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington’s Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2.005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN1M(ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0053] As used herein, “reduce” or “decrease” means to decrease by a statistically significant amount. In some embodiments, the reduction or decrease is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70, 80%, or 90% as compared to a control. A therapeutically significant reduction in a symptom is, e.g. at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject. Measured or measurable parameters include clinically detectable markers ofdisease, for example, elevated or depressed levels of a biological marker, such as decreased levels of Wade in blood and / or tissue, as well as parameters related to a clinically accepted scale of symptoms or markers for a disease or disorder (e.g., reduction in tumor size, severity or metastasis). It will be understood, that the total daily usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. Tire exact amount required will vary' depending on factors such as the type of disease being treated.

[0054] The term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.

[0055] Tire terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mi tigating or impeding one or more causes of a disorder or condition.Treatments according to the invention may be applied preventively, prophylactically, pallatively or remedially. Treatments can be administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established developmen t of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a cancer.

[0056] In some instances, the terms “treat”, “treating”, “treatment” and grammatical variations thereof, include partially or completely reducing a cancer in a subject, reducing the size of a tumor, reducing the number of tumors, and / or reducing tlie metastasis of a tumor as compared with prior to treatment of the subject or as compared with the incidence of such symptom in an untreated general or study population.

[0057] "Vector" used herein means, in respect to a nucleic acid sequence, a nucleic acid sequence comprising a regulatory nucleic acid sequence that controls the replication of an expressible Wade inhibitor. A vector may be either a self-replicating, extrachromosomal vector or a vector which integrates into a host genome. Alternatively, a vector may also be a vehicle comprising a Wade inhibitor such as a shRNA. In some embodiments, the vector comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 and / or SEQ ID NO:7. A vector may be a plasmid, bacteriophage, viral particle (isolated, attenuated, recombinant, etc,), A vector may comprise a double-stranded or single-stranded DNA, RNA, or hybrid DNA / RNA sequence comprising double-stranded and / or single -stranded nucleotides. In some embodiments, the vector is a viral vector that comprises a nucleic acid sequence thatis a viral packaging sequence responsible for packaging one or a plurality of nucleic acid sequences that encode one or a plurality of polypeptides. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral particle. In some embodiments, the vector is viral vector with a natural and / or an engineered capsid. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the vector is an adenoviral vector.

[0058] Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia vims, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma vims vectors; (g) papilloma vims vectors; (h) picomavirus vectors; (i) pox vims vectors such as an orthopox, e.g., vaccinia vims vectors or avipox, e.g. canarypox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. Tire constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of an RNA will generally require regulatory- elements, e.g., promoters, enhancers, etc., to ensure the expression of a Wade inhibitor or a functional fragment thereof in target cells. Other aspects to consider for vectors and constructs are further described below.

[0059] Accordingly, in some embodiments, the vector can be a viral vector. "Viral vector" as disclosed herein means, in respect to a vehicle, any vims, virus-like particle, virion, viral particle, or pseudotyped vims that comprises a nucleic acid sequence that directs packaging of a nucleic acid sequence in the vims, vims-like particle, virion, viral particle, or pseudotyped vims. In some embodiments, the vims, vims-like particle, virion, viral particle, or pseudotyped virus is capable of transferring a vector (such as a nucleic acid vector) into and / or between host cells. In some embodiments, the vims, virus-like particle, virion, viral particle, or pseudotyped virus is capable of transferring a vector (such as a nucleic acid vector) into and / or between target cells, such as a hepatocyte in the liver of a subject. Importantly, in some embodiments, the vims, vims-like particle, virion, viral particle, or pseudotyped vims is capable of transporting into cytoplasm and / or a nucleus of a target cell (e.g., a cancer cell). The term “viral vector” is also meant to refer to those forms described more fully- in U. S. Patent Application Publication U. S. 2018 / 0057839,which is incorporated herein by reference for all purposes, in some embodiments, the viral vector is a lentiviral vector.

[0060] In some embodiments, the Wade inhibitor composition comprises one or more viral vectors that contain nucleic acid sequences encoding a Wade inhibitor. For example, the composition can comprise a retroviral vector. These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994). Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110- 114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U. S. Pat, Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

[0061] Vectors useful for the delivery of a Wade inhibitor can include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the Wade inhibitor in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated / inducible expression.

[0062] Methods of Treating

[0063] Provided herein are methods of treating a cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a Wade inhibitor. The tern “Wade inhibitor” encompasses all compositions and compounds that reduce expression of a Wade polypeptide, reduce secretion of a Wade polypeptide, reduce binding of a Wade polypeptide to a receptor, and / or increase an immune response to a Wade polypeptide, or a fragment thereof. The term “Wade polypeptide” includes a polypeptide comprising the sequence provided in SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the Wade polypeptide comprises SEQ ID NO: 1. In some embodiments, the Wade polypeptide comprises a sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1. In some embodiments, the Wade polypeptide comprises a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the Wade polypeptide comprises a sequence having at least 95% identity with SEQ ID NO:1. In some embodiments, the Wade polypeptide comprises SEQ IDNON. In some embodiments, the Wade polypeptide comprises a sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NON, or a polypeptide comprising a portion of SEQ ID NON. In some embodiments, the Wade polypeptide comprises a sequence having at least 90% identity with SEQ ID NON. In some embodiments, the Wade polypeptide comprises a sequence having at least 95% identity with SEQ ID NON.

[0064] In some embodiments, the ‘Wade inhibitor” refers to a composition that reduces expression of a Wade polypeptide and / or reduces secretion of a Wade polypeptide. The reduction is a statistically significant amount as compared to a control. In some embodiments, the reduction of expression, secretion, an / or binding is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70, at least about 80%, or at least about 90% of a reduction as compared to a control. In some embodiments, the control is an untreated sample,

[0065] In certain aspects, a Wade inhibitor comprises a polynucleotide that reduces expression of a Wade polypeptide as compared to a control. The polynucleotide can be an RNAi (an RNA interference molecule). In some embodiments, the RNAi is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). Exemplary short hairpin RNAs that are Wade inhibitors are provided in SEQ ID NON, SEQ ID NON, SEQ ID NO:6, and SEQ ID NO:7. In some embodiments, the polynucleotide that reduces expression of a Wade poly peptide as compared to a control comprises SEQ ID NO:3. In some embodiments, the polynucleotide that reduces expression of a Wade polypeptide as compared to a control comprises SEQ ID NON. In some embodiments, the polynucleotide that reduces expression of a Wade polypeptide as compared to a control comprises SEQ ID NO:6. In some embodiments, the polynucleotide that reduces expression of a Wade poly peptide as compared to a control comprises SEQ ID NON. In some embodiments, the polynucleotides that reduce expression of a Wade polypeptide as compared to a control comprise SEQ ID NON and SEQ ID NON, respectively. In some embodiments, the polynucleotides that reduce expression of a Wade polypeptide as compared to a control comprise SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0066] In some embodiments, the polynucleotide or polynucleotides that reduce expression of a Wadepolypeptide bind specifically to or target a polynucleotide sequence comprising SEQ ID NON. In other or further embodiments, the polynucleotide orpolynucleotides that reduce expression of a Wade polypeptide bind specifically to or target a polynucleotide sequence comprising SEQ ID NO:8.

[0067] Accordingly, in some embodiments, the method of treating a cancer in a subject comprises administering to the subject a pharmaceutically effective amount of an RNAi that reduces expression of a Wade polypeptide in a cell of the subject. In some embodiments, the method of treating a cancer in a subject comprises administering to the subject a pharmaceutically effective amount of an RNAi that reduces expression of a Wade polypeptide in a cell of the subject, wherein the RNAi is selected from the group consisting of a short hairpin RNA (shRNA), and a microRNA (miRNA). In some embodiments, the method of treating a cancer in a subject comprises administering to the subject a pharmaceutically effective amount of an shRNA that reduces expression of a Wade polypeptide in a cell of the subject. In some embodiments, the reduction of expression is about 10%, 20%, 30%, 40%, 50%, 60%, 70, 80%, or 90% as compared to a control.

[0068] In certain aspects, the Wade inhibitor is a composition that binds a Wade polypeptide and reduces binding of a Wade polypeptide to a receptor and / or increases an immune response to a Wade polypeptide, or a fragment thereof. The binding composition is preferably capable of specifically binding the Wade polypeptide, or fragment thereof.

[0069] In some embodiments, the Wade inhibitor is an antibody that specifically binds a Wade polypeptide, or fragment thereof. In certain aspects, a CAR T cell specific for Wade is also administered to the subject. In other or further embodiments, the Wade antibody is capable of inducing complement-dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC), which preferably results in lysis of the cancer cell.

[0070] In some embodiments, a Wade inhibitor is a chimeric antigen receptor (CAR). Tire CAR can be found on a T cell or other immune cell, and preferably, the CAR specifically binds a Wade polypeptide.

[0071] Accordingly, included in the present disclosure is a method of treating a cancer in a subject comprising administering to the subject a pharmaceutically effective amount of an antibody that specifically binds a Wade polypeptide. Also included herein is a method of treating a cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a CAR T cell wherein the CAR T cell receptor specifically binds a Wade polypeptide.

[0072] The cancers that can be treated using the present invention are any wherein the cancer cells express and / or secrete a Wade polypeptide. In some embodiments, a Wade polypeptide is expressed predominantly or even exclusively in the cancer cells. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: glioblastoma, ovarian cancer, melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal cavities breast carcinoma); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; prostate adenocarcinoma; ovarian carcinoma (ovarian epithelial carcinoma or surface epithelial- stromal tumor including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); esophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder carcinoma; carcinoma of the uterus (including endometrial adenocarcinoma, ocular, uterine papillary’ serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas, leiomyosarcomas, mixed mullerian tumors); glioma, glioblastoma, medulloblastoma, and other tumors of the brain; kidney cancers (including renal cell carcinoma, clear cell carcinoma, Wihn's tumor); cancer of the head and neck (including squamous cell carcinomas); cancer of the stomach (gastric cancers, stomach adenocarcinoma, gastrointestinal stromal tumor); testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schw annoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomysarcoma, skin, including melanoma, cervical, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, renal, bladder, soft tissue, adrenal gland, urethra, cancers of the penis, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignantfibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas, and cancers of the vagina among others.100731 In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a glioblastoma. In other embodiments, the cancer is an ovarian cancer.

[0074] The therapeutically effective amount of the Wade inhibitor compositions described herein can be determined by one of ordinary skill in the art and includes exemplar}' dosage amounts for a mammal of from about 0.5 to about 200 mg / kg of body weight of active composition per day, which can be administered in a single dose or in the form of individual divided doses, such as from I to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150 mg / kg of body weight of active composition per day, about 0.5 to 100 mg / kg of body weight of active compound per day, about 0.5 to about 75 mg / kg of body weight of active compound per day, about 0.5 to about 50 mg / kg of body weight of active composition per day, about 0.5 to about 25 mg / kg of body weight of active composition per day, about 1 to about 20 mg / kg of body weight of active composition per day, about 1 to about 10 mg / kg of body weight of active composition per day, about 20 mg / kg of body weight of active composition per day, about 10 mg / kg of body weight of active composition per day, or about 5 mg / kg of body weight of active composition per day.

[0075] Dosage forms for topical administration of the Wade inhibitor compositions described herein include ointments, gels, pastes, liquids, solutions, creams, oils, powders, aerosols, sprays and patches. Thickening agents, emollients, and stabilizers can be used to prepare the topical compositions of the present invention. Examples of thickening agents include petrolatum, beesw'ax, xantham gum, or polyethylene glycol, humectants such as sorbitol, emollients such as mineral oil, lanolin and its derivatives, or squalene. The Wade inhibitor compositions described herein are admixed under sterile conditions with apharmaceutically acceptable carrier and any preservatives, buffers, thickening agents, cosmetic agents, or propellants as can be required.

[0076] Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular conditi on,

[0077] Methods of Detecting

[0078] Also included herein are methods of detecting a cancer in a subject comprising obtaining a sample from the subject, detecting a Wade polypeptide or a Wade polynucleotide in the sample, wherein detection of the Wade polypeptide or the Wade polynucleotide in the sample indicates the presence of a cancer in the subject. In some embodiments, the Wade polynucleotide is an mRNA. Such detection can be for the purpose of diagnosing, staging, and / or prognosing a cancer, monitoring the susceptibility to a cancer treatment, or monitoring the success of a cancer treatment.

[0079] The Wade polypeptide can be any as described herein, including a polypeptide composing SEQ ID NO:1, SEQ ID NO:2, or a fragment thereof. The Wade polynucleotide can be any sequence that encodes a Wade polypeptide. In some embodiments, the Wade polynucleotide comprises SEQ ID NO:1. In some embodiments, the Wade polypeptide comprises a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the Wade polypeptide comprises a sequence having at least 95% identity with SEQ ID NO:1. In some embodiments, the Wade polynucleotide comprises SEQ ID NO:2. In some embodiments, the Wade polypeptide comprises a sequence having at least 90% identity with SEQ ID NO:2. In some embodiments, the Wade polypeptide comprises a sequence having at least 95% identity with SEQ ID NO:2.

[0080] It should be understood that detection of a Wade polynucleotide in the sample includes, for example, the creation of cDNA from mRNA in the sample, and detection of the cDNA. The sample can be any sample from the subject including a biopsy sample, a fluid sample, a tissue sample, a blood sample, or a plasma sample. In some embodiments, the sample is a blood sample.

[0081] The expression of a Wade polypeptide in a cancer tissue may be measured by a variety of techniques that are well known in the art. In addition to the use of arrays andmicroarrays, it is contemplated that a number of different assays could be employed to analyze Wade expression. Such assays include, but are not limited to, digital color-coded barcode technology analysis, microarray expression profiling, quantitative PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, endpoint PCR, multiplex end-point PCR, cold PCR, ice-cold PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA), branched DNA (bDNA) assay, rolling circle amplification (RCA), single molecule hybridization detection, invader assay, and / or Bridge Litigation Assay.

[0082] The cancer that is detected can be any wherein the cancer cells express a Wade polypeptide. In certain aspects, the cancer cells secrete a Wade polypeptide. In some embodiments, a Wade polypeptide is expressed predominantly or even exclusively on the cancer cells. A representative but non-limiting list of cancers that the disclosed compositions can be used to detect is the following: glioblastoma, ovarian cancer, melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal cavities breast carcinoma); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; prostate adenocarcinoma; ovarian carcinoma (ovarian epithelial carcinoma or surface epithelial-stromal tumor including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); esophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder carcinoma; carcinoma of the uterus (including endometrial adenocarcinoma, ocular, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas, leiomyosarcomas, mixed mullerian tumors); glioma, glioblastoma, medulloblastoma, and other tumors of the brain; kidney cancers (including renal cell carcinoma, clear cell carcinoma, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas); cancer of the stomach (gastric cancers, stomach adenocarcinoma, gastrointestinal stromal tumor); testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast, andother organs; signet ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma,leiomyosarcoma, skin, including melanoma, cervical, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, renal, bladder, soft tissue, adrenal gland, urethra, cancers of the penis, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas, and cancers of the vagina among others.

[0083] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a glioblastoma. In other embodiments, the cancer is an ovarian cancer.

[0084] In certain aspects of the invention, the method comprises detecting a cancer in a subject comprising obtaining a sample from the subject, detecting a Wade polypeptide or a Wade polynucleotide in the sample, wherein detection of the Wade polypeptide or the Wade polynucleotide in the sample indicates the presence of the cancer in the subject, followed by treatment of the cancer in the subject. The treatment can be any as described herein or any cancer treatment known to those of ordinary skill in the art.

[0085] Compositions and Kits

[0086] Further included herein are Wade compositions and compositions and kits for detection and / or treatment of cancer, and in particular, cancers that express a Wade polypeptide. In some embodiments, the present invention comprises an isolated, genetically engineered, or synthetic Wade polypeptide or polynucleotide. In certain aspects of the invention, the composition comprises an isolated Wade polypeptide comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment thereof. In certain aspects ofthe invention, the composition comprises an isolated Wade polypeptide comprising SEQ ID NO: 1. In certain aspects of the invention, the composition comprises an isolated Wade polypeptide comprising or SEQ ID NO:2. Also included herein are compositions comprising an isolated Wade polypeptide comprising SEQ ID NO: 1 or SEQ ID NO:2 or a sequence having at least 80%, 85% or 90% identity thereto. In some embodiments, an isolated Wade polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, an isolated Wade polypeptide comprises a sequence having at least 90% identity to SEQ ID NO:2. Included herein is an isolated Wade polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, a sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO:2, or a fragment thereof. In certain aspects of the invention, the composition comprises a genetically engineered or synthetic Wade polypeptide comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment thereof. In certain aspects of the invention, the composition comprises a genetically engineered or synthetic Wade polypeptide comprising SEQ ID NO: 1. In certain aspects of the invention, the composition comprises a genetically engineered or synthetic Wade polypeptide comprising SEQ ID NO:2. Also included herein are compositions comprising a genetically engineered or synthetic Wade polypeptide comprising SEQ ID NO: 1 or SEQ ID NO:2 or a sequence having at least 80%, 85% or 90% identity thereto. In some embodiments, a genetically engineered or synthetic Wade polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO:2 In some embodiments, a genetically engineered or synthetic Wade polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, a genetically engineered or synthetic Wade polypeptide comprises a sequence having at least 90% identity to SEQ ID NO:2. Included herein is a genetically engineered or synthetic Wade polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, a sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO:2, or a fragment thereof.

[0087] Further included herein is an isolated, genetically engineered, or synthetic Wade polynucleotide that encodes SEQ ID NO: 1 or SEQ ID NO:2 or a fragment thereof. In some embodiments, the isolated, genetically engineered, or synthetic Wade polynucleotide encodes SEQ ID NO:1. In some embodiments, the isolated, genetically engineered, or synthetic Wade polynucleotide encodes SEQ ID NO:2, The Wade protein is translated from the open reading frame (ORF) residing within one of the transcript variants (1475 nucleotides in length) of the LINC00973 gene, which is currently classified as non-coding RNAs. The full-length parent transcript, referred herein as Wade mRNA, is provided asSEQ ID NO: 18, and the Wade ORF is provided as SEQ ID NO: 17. Accordingly, in some embodiments, the isolated Wade polynucleotide comprises SEQ ID NO: 17 or SEQ ID NO: 18, a fragment thereof, or a cDNA corresponding to SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof. Included herein is an isolated Wade polynucleotide comprising a sequence encoding SEQ ID NO: 1, SEQ ID NO: 2, a sequence having at least 80%, 85% or 90% identity to SEQ ID NO: 1 or SEQ ID NO:2, or a fragment thereof. Also included herein is a genetically engineered or synthetic Wade polynucleotide comprising SEQ ID NO: 17, SEQ ID NO: 18 or a cDNA corresponding thereto. Included herein is an isolated Wade polynucleotide comprising a sequence encoding SEQ ID NO: 1, SEQ ID NO:2, a sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO:2, or a fragment thereof. Also included herein is a genetically engineered or synthetic Wade polynucleotide comprising SEQ ID NO: 17, SEQ ID NO: 18 or a cDNA corresponding thereto. In some embodiments, the composition comprises a genetically engineered or synthetic Wade polynucleotide comprising SEQ ID NO: 17 or a cDNA corresponding thereto. In some embodiments, the composition comprises a genetically engineered or synthetic Wade polynucleotide comprising SEQ ID NO: 18 or a cDNA corresponding thereto.

[0088] In certain aspects of the compositions and / or kits for detection and / or treatment of a cancer, the Wade polypeptide is a genetically engineered or synthetic protein that comprises SEQ ID NO: 1, In some embodiments, the genetically engineered or synthetic Wade polypeptide comprises a sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1. In some embodiments, the genetically engineered or synthetic Wade polypeptide comprises a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the genetically engineered or synthetic Wade polypeptide comprises a sequence having at least 95% identity with SEQ ID NO: 1. In some embodiments, the genetically engineered or synthetic Wade polynucleotide comprises SEQ ID NO:2. In some embodiments, the genetically engineered or synthetic Wade polypeptide comprises a sequence having at least 90% identity with SEQ ID NO:2. In some embodiments, the genetically engineered or synthetic Wade polypeptide comprises a sequence having at least 95% identity’ with SEQ ID NO:2.

[0089] In some embodiments, the composition or kit for detection and / or treatment of a cancer comprises one or more polynucleotides that reduce expression of a Wade polypeptide in a cell. The one or more polynucleotides that reduce expression of the Wadepolypeptide can be an RNAi, including but not limited to, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). In some aspects, the one or more polynucleotides are a shRNA that comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7. In some aspects, the polynucleotide is a shRNA that comprises SEQ ID NO:3. In some aspects, the one or more polynucleotides are a shRNA that comprises SEQ ID NO:4, In some aspects, the one or more polynucleotides are a shRNA that comprises SEQ ID NO:6. In some aspects, the one or more polynucleotides are a shRNA that comprises SEQ ID NO:7. In some embodiments, the one or more polynucleotides that reduce expression of a Wade polypeptide comprise SEQ ID NO: 3 and SEQ ID NO:4, respectively. In some embodiments, the one or more polynucleotides that reduce expression of a Wade polypeptide comprise SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0090] In some aspects, the one or more polynucleotides are a shRNA that binds to or targets a sequence comprising SEQ ID NO:5. In some aspects, the one or more polynucleotides are a shRNA that binds to or targets a sequence comprising SEQ ID NO:8.

[0091] In other embodiments, the composition or kit comprises one or more polynucleotides or other composition that detects a Wade polynucleotide sequence. In some embodiments, the Wade polynucleotide is a Wade mRNA. These detection polynucleotides or other compositions can be suitable for use in arrays and microarrays, digital color-coded barcode technology analysis, microarray expression profiling, quantitative PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-point PCR, multiplex end-point PCR, cold PCR, ice-cold PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA), branched DNA (bDNA) assay, rolling circle amplification (RCA), single molecule hybridization detection, invader assay, and / or Bridged Ligation Assay.

[0092] In still other embodiments, the composition or kit comprises one or more binding agents that bind to or detect a Wade polypeptide. As discussed above, “binding agent” may be any compound or complex of compounds which is capable of binding a Wade polypeptide, or fragment thereof. Preferably, the binding agent is capable of specifically binding the Wade polypeptide or fragment thereof. Suitable binding agents may be obtained by screening a binding agent library in order to identify / obtain binding agents that bind to the Wade polypeptide or fragment thereof. In some embodiments, a binding agent is an antibody that specifically binds a Wade polypeptide or fragment thereof.Binding agents that bind to or detect a Wade polypeptide can be suitable for the detection of cancer in the subject.

[0093] In some embodiments, the kits described herein comprise any combination of an extraction buffer, a storage buffer, a washing buffer, a loading buffer, or a staining buffer. In some embodiments, the kit further comprises an agarose solution or a polyacrylamide solution.

[0094] It should be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims. All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes.

[0095] EXAMPLES

[0096] Example 1

[0097] The human genome contains thousands of orphan genes of unknown function.Understanding their biological role remains one of the major goals of modem biology. Reported herein is the phenotypic and molecular characterization of a novel cancerspecific secreted microprotein, referred to as Wade. Wade is expressed in a variety of cancers via RAF-MEK-ERK signaling but not in healthy cells or tissues. (See Figures 2 and 4.) Interestingly, Wade was abundantly expressed in the U87 glioblastoma cell line carrying BRAFV600E / TP53+ / - mutation, but was undetectable in the genetically comparable GL261 murine cells. (Figures 3 and 5.)

[0098] Wade encodes for a 70-aa protein with an N-terminal signal peptide (SEQ ID NO:2), which is cleaved into 44-aa secreted protein of 5 kDa (SEQ ID NO: 1). (See Figure 1 schematic.) Brefeldin A treatment led to intracellular retention of transfected Wade, and the secreted Wade was detected in the supernatant. (Figure 12.) shRNA knockdown of Wade led to impaired proliferation and migration of U87 and ES2 ovarian cancer, (Figures 6-8.) In rescue experiments, ectopic expression of wild type (WT) but not the Wade mutants lacking translation initiation (Metl)(SEQ ID NO: 11) or lacking signal peptide (WadeT26R; SEQ ID NO: 10) restored the migration of shWade U87 in transwell assays.(Figure 13.) Analysis of supernatants using 65-plex luminex reveals that shWade U87 showed significantly lower level of cytokines (IL-8, IL-6, LIF, GM-CSF), chemokines (CCL2, CXCL1) and growth factors (TGFA, VEGF). (Figure 9.) RNA-seq analysis showed greater than 600 differentially expressed genes (DEGs) in shWade U87 compared to control. (Figure 9.) GO and protein-network analyses of Wade -regulated differential gene expression analyses (DEGs) showed significant enrichment of genes involved in growth factor signaling, angiogenesis, EMT and IL-6 / STAT3 pathway. (Figure 10.) Collectively, these results unveil a critical role for Wade in driving cancer proliferation and migration.

[0099] Example 2

[0100] Wade knockdown Suppresses Tumor Growth in NUDE Mouse Xenografts.

[0101] Ten female NUDE mice (Nu / J; 7-8 weeks) w’ere randomly divided into two groups: control and experimental. The control group received human ES2 cells expressing shControl and the experimental group received ES2 cells expressing shWade. 5 million cells suspended in 200 pl were subcutaneously injected on the left and right side of each mouse. Animals were monitored for general health, activity and subcutaneous tumor sizes were measured every’ two or three days for 19 days. Figure 18A shows a schematic of the xenograft experiment in NUDE mice using human ES2 cells expressing control or shWade. Figure 18B shows images of tumors isolated from respective groups at the end of the experiment on day 17, and Figure 18C is a chart showing tumor volumes at different days post injection. P= 0.006, 2way ANOVA.

[0102] Example 3

[0103] It is envisioned that the patient presents with a confirmed diagnosis of glioblastoma multifomie. The Wade inhibitor composition is prepared as a sterile, injectable suspension. The pharmaceutically acceptable carrier is a biocompatible lipid- nanocarrier system composed of phosphatidylcholine and cholesterol, providing selective accumulation within the tumor microenvironment and sustained release of active agents, trehalose (2%) to maintain nanoparticle integrity and prevent aggregation during storage and administration.

[0104] The composition is administered intravenously at a dose of 2 mg / kg body weight, diluted in isotonic saline to a total infusion volume of 100 mL. The infusion is delivered over 45 minutes under controlled temperature and monitoring conditions. Once administered, the Wade inhibitor circulates systemically and demonstrates preferential uptake in glioblastoma tissue via enhanced permeability and retention effects. Within thetumor, the Wade inhibitor interferes with Wade expression and cancer cell proliferation and / or metastasis.

[0105] Throughout treatment, laboratory monitoring includes measurements of Wade expression, serum lactate dehydrogenase (LDH), mitochondrial potential markers, and circulating tumor DNA (ctDNA) levels. Decreases in these values indicate effective suppression of tumor proliferation. Each treatment cycle consists of two infusions per week for six consecutive weeks. Magnetic resonance imaging (MRI) performed at four- week intervals demonstrates measurable reduction in contrast-enhancing tumor regions.

[0106] By the end of the second treatment cycle, tumor proliferation indices (Ki-67 expression) decrease by 40-60% relative to baseline. The therapy maintains minimal off- target toxicity and preserves surrounding neural tissue integrity. Clinical observation confirms stabilization of disease progression and partial regression of tumor mass without systemic adverse effects.

Claims

CLAIMS1. A method of treating a cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a Wade inhibitor.

2. The method of claim 1, wherein the cancer is a solid tumor.

3. The method of claim 1 or 2, wherein the Wade polypeptide comprises SEQ ID NO: 1 or a sequence having at least 80% identity with SEQ ID NO: 1.

4. The method of any one of claims 1-3, wherein the Wade inhibitor comprises a polynucleotide that reduces expression of the Wade polypeptide as compared to a control, 5. The method of claim 4, wherein the polynucleotide is a small interfering ribonucleic acid (RNA), a short hairpin RNA (shRNA), or a microRNA.

6. The method of claim 5, wherein the polynucleotide is a shRNA.

7. The method of claim 6, wherein the shRNA comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7.

8. The method of any one of claims 1-3, wherein the Wade inhibitor comprises an antibody.

9. The method of any one of claims 1-3, wherein the Wade inhibitor comprises a chimeric antigen receptor (CAR).

10. The method of claim 9, wherein a T cell comprises the CAR.

11. The method of any one of claims 1-10, wherein the cancer is a glioblastoma.

12. The method of any one of claims 1 - 10, wherein the cancer is an ovarian cancer,13. A method of detecting a cancer in a subject comprising obtaining a sample from the subject, detecting a Wade polypeptide or a Wade polynucleotide in the sample, wherein detection of the Wade polypeptide or the Wade polynucleotide indicates the cancer in the subject.

14. The method of claim 13, wherein the cancer is a solid tumor.

15. The method of claim 13 or 14, wherein the Wade polypeptide comprises SEQ ID NO:1 or a sequence having at least 80% identity with SEQ ID NO: 1.

16. The method of any one of claims 13-15, wherein the Wade polynucleotide is a messenger ribonucleic acid (mRNA).

17. The method of any one of claims 13-16, wherein the Wade polynucleotide encodes a polypeptide comprising SEQ ID NO: 1.

18. The method of any one of claims 13-17, wherein the cancer is a glioblastoma.

19. The method of any one of claims 13-17, wherein the cancer is an ovarian cancer.

20. The method of any one of claims 13-19, further comprising treating the cancer in the subject comprising administering to the subject a pharmaceutically effective amount of a Wade inhibitor.

21. The method of claim 20, wherein the Wade inhibitor comprises an inhibitor polynucleotide that reduces expression of the Wade polypeptide as compared to a control.

22. The method of claim 21, wherein the inhibitor polynucleotide is a small interfering ribonucleic acid (RNA), a short hairpin RNA (shRNA), or a microRNA.

23. The method of claim 22, wherein the inhibitor polynucleotide is a shRNA.

24. The method of claim 23, wherein the shRNA comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7.

25. The method of claim 20, wherein the Wade inhibitor comprises an antibody.

26. The method of claim 20, wherein the Wade inhibitor comprises a chimeric antigen receptor (CAR).

27. The method of claim 26, wherein a T cell comprises the CAR.

28. The method of any one of claims 1-27, wherein the subject is a human.

29. A composition comprising a polynucleotide that reduces expression of a Wade polypeptide in a cell,30. Tire composition of claim 29, wherein the Wade polypeptide comprises SEQ ID NO: 1 or a sequence having at least 80% identity with SEQ ID NO: 1.

31. The composition of claim 29 or claim 30, wherein the polynucleotide specifically binds to or targets a sequence comprising SEQ ID NO:5 or SEQ ID NO:8.

32. The composition of any one of claims 29-31, wherein the polynucleotide comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7.

33. A genetically engineered, synthetic or isolated Wade polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO:2, or a fragment thereof.

34. A genetically engineered, synthetic or isolated Wade polynucleotide comprising a sequence encoding SEQ ID NO: 1, SEQ ID NO:2, a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO:2, or a fragment thereof.

35. The genetically engineered, synthetic or isolated Wade polynucleotide of claim 34 comprising SEQ ID NO: 17, SEQ ID NO: 18 or a cDNA corresponding thereto.

36. A kit comprising the composition of any one of claims 29-35.

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