US 2016O194388A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0194388 A1 Hallahan et al. (43) Pub. Date: Jul. 7, 2016

(54) MONOCLONAL ANTIBODIES TO HUMAN Publication Classification 14-3-3 EPSILON AND HUMAN 14-3-3 EPSILON SV (51) Int. Cl. C07K 6/8 (2006.01) (71) Applicant: Washington University, St. Louis,MO A6IN5/10 (2006.01) (US) A615 L/It (2006.01) A647/48 (2006.01) (72) Inventors: Dennis E. Hallahan, St. Louis, MO C07K 6/30 (2006.01) (US); Heping Yan, St. Louis, MO (US) (52) U.S. Cl. CPC ...... C07K 16/18 (2013.01); A61K 47/48569 (21) Appl. No.: 15/054,691 (2013.01); C07K 16/30 (2013.01); A61 K 51/1045 (2013.01); A61N 5/10 (2013.01); (22) Filed: Feb. 26, 2016 C07K 231 7/565 (2013.01); C07K 2317/567 (2013.01); C07K 2317/51 (2013.01); C07K Related U.S. Application Data 2317/515 (2013.01); A61N 2005/1098 (63) Continuation-in-part of application No. PCT/US2014/ (2013.01) 053207, filed on Aug. 28, 2014. (57) ABSTRACT (60) Provisional application No. 61/871,115, filed on Aug. The present invention provides isolated antibodies that bind 28, 2013, provisional application No. 61/907,677, to 14-3-3 epsilon that are useful in the recognition of tumor filed on Nov. 22, 2013. cells and tumor specific delivery of drugs and therapies. Patent Application Publication Jul. 7, 2016 Sheet 1 of 20 US 2016/O194388A1

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MONOCLONAL ANTIBODIES TO HUMAN 0005 Exposure of tumor cells to ionizing radiation (IR) is 14-3-3 EPSILON AND HUMAN 14-3-3 widely known to induce a number of cellular changes. One EPSILON SV way that IR can affect tumor cells is through the development of neoantigens which are new molecules that tumor cells CROSS REFERENCE TO RELATED express at the cell membrane following some insult or change APPLICATIONS to the cell. There have been numerous reports in the literature 0001. This application claims the benefit of PCT applica of changes in both tumor and tumor vasculature cell Surface tion number PCT/US2014/053207, filed Aug. 28, 2014, molecule expression following treatment with IR. The use which claims the benefit of U.S. provisional application No. fulness of neoantigens for imaging and therapeutic applica 61/871,115, filed Aug. 28, 2013, and U.S. provisional appli tions lies in the fact that they are differentially expressed on cation No. 61/907,677, filed Nov. 22, 2013, each of the dis the Surface of irradiated tumor cells to a greater extent than on closures of which are hereby incorporated by reference in normal tissues. This differential expression provides a mechanism by which tumor cells can be “marked by radia their entirety. tion for further targeting. Drug delivery vehicles or imaging GOVERNMENT SUPPORT agents conjugated to ligands that recognize and interact with the neoantigens can help to improve tumor-specific targeting 0002 This invention was made with government support and reduce systemic toxicity with cancer drugs. under 5R01 CA125757-06 awarded by the NIH. The govern ment has certain rights in the invention. SUMMARY OF THE INVENTION FIELD OF THE INVENTION 0006. In an aspect, the present invention encompasses cell 0003. The invention encompasses antibodies useful in the line that expresses an antibody comprising an amino acid recognition of tumor cells and tumor specific delivery of sequence selected from the group consisting of SEQID NO:3 drugs and therapies. and SEQ ID NO:4, wherein the antibody specifically binds 14-3-3 epsilon. BACKGROUND OF THE INVENTION 0007. In another aspect, the present invention encom 0004 14-3-3 proteins are a group of highly conserved passes an isolated antibody that specifically binds 14-3-3 proteins that are involved in many vital cellular processes epsilon and comprises a light chain CDR3 comprising the such as metabolism, protein trafficking, signal transduction, amino acid sequence of SEQIDNO:7 with zero to two amino apoptosis and cell cycle regulation. 14-3-3 proteins are phos acid Substitutions. pho-serine/phospho-threonine binding proteins that have a 0008. In still another aspect, the present invention encom diverse array of partners including transcription factors, bio passes an isolated antibody that specifically binds 14-3-3 synthetic enzymes, cytoskeletal proteins, signaling mol epsilon and comprises a heavy chain CDR3 comprising the ecules, apoptosis factors and tumor Suppressors. The 14-3-3 amino acid sequence of SEQ ID NO:10 with zero to two family consists of 7 isoforms; beta, gamma, epsilon, sigma, amino acid Substitutions. Zeta, tau and eta. 14-3-3 proteins are ubiquitously expressed 0009. In yet still another aspect, the present invention and self-assemble into homo- and heterodimers, with the encompasses a method of detecting a tumor in a Subject. The exception of 14-3-3 sigma, which exclusively forms method comprises exposing a target area of the Subject where homodimers and is found in cells of epithelial origin only. the presence of a tumor is Suspected to ionizing radiation; Each monomer contains an independent ligand-binding site, administering to the Subject a composition to detect the pres thus the 14-3-3 dimer can interact with two target proteins ence of 14-3-3 epsilon in the target area, wherein the compo simultaneously. 14-3-3 proteins are highly rigid structures sition comprises one or more targeting antibodies, wherein and ligand binding can induce conformational changes that each targeting antibody specifically binds to 14-3-3 epsilon alter the stability and/or catalytic activity of the ligand. Fur exposed on an irradiated cell and is conjugated to a detectable thermore, 14-3-3 protein binding can physically occlude label; and detecting the detectable label to detect the presence sequence-specific or structural motifs on the target that pre of 14-3-3 epsilon, wherein the presence of 14-3-3 epsilon vent molecular interactions and/or modulate the accessibility indicates the presence of a tumor in the target area of the of a target protein to modifying enzymes such as kinases, Subject. phosphatases and proteases. In addition, 14-3-3 proteins can 0010. In a different aspect, the present invention encom act as a scaffold molecule to anchor target proteins within passes a method of enhancing radiotherapy in a Subject. The close proximity of one another. 14-3-3 proteins represent an method comprises administering a pharmacologically effec integration point for proliferative, Survival, apoptotic and tive amount of an isolated anti-14-3-3 epsilon antibody of stress signalling pathways. Members of the 14-3-3 protein claim 7 or claim 9 to the subject, such that radiotherapy is family enhance the activity of many proteins with prolifera enhanced. tive and/or Survival functions, such as Raf kinases, and antagonize the activity of proteins that promote cell death and 0011. In other aspects, the present invention encompasses senescence. Such as Bad, Bim and Bax. Because many 14-3-3 a method of delivering atherapeutic agent to a cell expressing interactions are phosphorylation dependent, 14-3-3 proteins 14-3-3 epsilon in a subject. The method comprises exposing have been integrated into the core regulatory pathways that a target area of the Subject where the presence of a tumor is are crucial for normal growth and development. 14-3-3 pro Suspected to ionizing radiation; and administering an isolated teins are directly involved in cellular processes such as anti-14-3-3 epsilon antibody of claim 7 or claim 9 to the cytokinesis, cell-contact inhibition, anchorage-independent Subject. growth and cell adhesion, and it is these pathways that often 0012. In still other aspects, the present invention encom become dysregulated in disease states such as cancer. passes a method of detecting 14-3-3 epsilon in a sample. The US 2016/0194388 A1 Jul. 7, 2016

method comprises obtaining a sample, and detecting and/or Three clones, 7D4, 8B4 and 5D2, remained positive in the measuring the amount of 14-3-3 epsilon in the sample using Dot-blot assay and 4D3 completely lost the ability to secrete an antibody of the invention. antibody. 0022 FIG. 9 depicts an immunoblot showing that McAb BRIEF DESCRIPTION OF THE FIGURES 7D4 reacted with H23 cell lysate. The results indicated that 0013 The application file contains at least one photograph the staining increased in response to irradiation of H23 tumor. executed in color. Copies of this patent application publica The protein bands were located at around 27 kDa. The inten tion with color photographs will be provided by the Office sity levels of protein bands constantly increased from sham upon request and payment of the necessary fee. 0Gy to 3Gyx 1 with the highest level observed in 3Gyx3 0014 FIG. 1 shows the amino acid sequence of human treatment. 14-3-3 epsilon isoform 1 (SEQ ID NO:11). The underlined 0023 FIG. 10 depicts and immunoblot showing analysis sequence identifies the binding epitope of antibody 7D4. of IP purified protein from H23 cell lysates and reacted with 0015 FIG. 2 shows the amino acid sequence of human anti 14-3-3 epsilon antibody 7D4. The result indicated that 14-3-3 epsilon isoform SV (SEQID NO:12). The underlined the protein was captured by the antibody affinity beads. The sequence identifies the binding epitope of antibody 7D4. molecular weight of this protein band is around 27 kDa. The 0016 FIG. 3A depicts IHC results showing that the com protein density showing on gel was the highest in 3Gyx3 mercial anti 14-3-3 epsilon antibody strongly reacted with treated H23 human lung cancer cells compared to the others 3Gyx3 treated H23 lung cancer cells as well as 3Gyx1. The of 5Gyx1, 3Gy 1 and sham 0Gy treated. intensity in 3Gyx 1 was much lower than 3Gyx3 treated H23 (0024 FIG. 11A depicts IHC results showing that 7D4 cells. Additionally, sham 0Gy treated H23 still had intense monoclonal antibody strongly reacts with 3Gyx3 treated H23 staining on tumor cells. Controls included normal rabbit IgG tumor cells and slightly or negative with sham 0Gy treated and second antibody. FIG. 3B shows anti-14-3-3 epsilon anti H23 tumor cells in brown color (10x40). FIG. 11B depicts IF body detecting protein on H23 (NSCLC) treated with 3Gyx3 results showing similar results, high intensity staining was and sham OGy. 7D4 antibody binds to irradiated H23 cells but observed on tumor cells treated with 3Gyx3 and less intense not non-irradiated cells. The commercial antibody shows staining was observed on 3Gyx 1 treated tumor cells. Sham binding to non-irradiated cells. 0Gy cells had minimal staining. Controls of normal mouse 0017 FIG. 4A and FIG.4B show anti-14-3-3 epsilon anti IgG or secondary antibodies also had minimal staining on body reacting with human normal tissues. (FIG. 4A) Colon, treated tumor cells (10x40). liver, lung, pancreas, (FIG. 4B) kidney and tongue tissue was 0025 FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D depict evaluated. The 7D4 mouse monoclonal antibody to 14-3-3 images showing that the 7D4 mAb selectively binds to an epsilon showed no binding to colon, liver, pancreas, kidney or irradiated H23 tumor in an in vivo nude mouse model for at tongue. There was minimal binding to the lung. least 10 days. H23 cells were injected into both lower 0018 FIG. 5 shows flow cytometry of antibody binding to extremities of nude mice. The left lower limb was irradiated human lung cancer cell model H23. with 3Gyx3 in a triplicate regimen (9Gy total). The right 0019 FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D show that lower limb was shielded by lead and OGy sham irradiated. the 7D4 antibody activates phagocytosis by dendritic cells NIR intensity in the left hindlimbs was high and lasted 10 and monocytic cells. Murine dendritic cells (mDCs) are days. Right hind-limbs with OGy treated tumors were slightly attracted by 3Gyx3 irradiated H23 cells coated with McAb. positive and diminished completely at 240 hours after injec FIG. 6A shows examples of phagocytic events (accumulation tion (FIG. 12A). In the 5Gy treatment group, the intensity of of red signal within mDCs; extension of pseudopodia; adhe fluorescence was lower than the 3Gyx3 group (FIG. 12B). sion of mDCs to H23 cells). FIG. 6B and FIG. 6C controls Sham 0Gy treated mice administered 7D4 antibody and 5Gy show minimal phagocytic events. FIG. 6D shows a graph treated mice administered a normal mouse IgG control anti depicting the results of counting 50 random high-powerfields body conjugated to AF-750 were negative for fluorescence on to tally the number of phagocytic events. either IR treated or sham 0Gy treated tumors at 10 days (FIG. 0020 FIG. 7A and FIG. 7B show the ELISPOT IFN-y 12C, FIG. 12D). level test in H23 cells. FIG. 7A shows the production of interferon-gamma in cells treated with dendritic cells. Group DETAILED DESCRIPTION 1: A, B, C, well 1-6: Group 2: D, E, F, well 1-6: Group 3: G, 0026. The present invention provides antibodies that spe H, well 1-6 & A 7-12: Group 4: B, C, D, well 7-12: Group 5: cifically bind 14-3-3 epsilon. Advantageously, these antibod E, F, well 7-12. FIG.7B shows the quantification of interferon ies specifically bind to epitopes exposed on irradiated tumor production. Group 1: 3Gyx3+7D4+mouse DC: Group 2: cells and not normal cells. More specifically, these antibodies 3Gyx3+7D4+0; Group 3: 3Gyx3+NMIgG+MDC: Group 4: specifically bind to epitopes exposed on irradiated human 0Gy+7D4+MDC: Group 5: 0Gy+0+0. lung cancer cells. The antibodies may be used to provide 0021 FIG. 8A depicts an image showing that clone of 7D4 tumor specific delivery, for instance, of drugs or therapeutic showed high IgG binding on IR 3Gyx3 treated tumor cells agents, as well as enhancing the efficacy of radiotherapy. and negative IgM binding either on 3Gyx3 or sham 0Gy (0027. As used herein, the term “14-3-3 epsilon” refers to treated tumor cells. Also, there is a 6 fold increase in binding human 14-3-3 epsilon and to orthologs from others species. on 3Gyx3 treated tumor cells relative to sham 0Gy treated Such orthologs are known in the art and include, but are not cells. FIG. 8B depicts dot-blot images indicating that hybri limited to the following NCBI Reference Sequences: doma Supernatants were strongly positive on 3Gyx3 treated XP 515815.4 (Pan troglodytes), NP 033562.3 (Mus muscu tumor cells and much lower on sham 0Gy treated tumor cells lus), NP 113791.1 (Rattus norvegicus), NP 77.6916.1 (Bos using the Dot-blot assay. Additionally, the bottom dot-blot taurus), XP 854358.1 (Canis familiaris), NP_001006219.1 shows four positive clones against 3Gyx3 treated tumor cells (Gallus gallus), BC045025.1 (Xenopus laevis), ECU89595.1 that were picked up from assays for hybridoma Sub-cloning. (Xenopus tropicalis), BC045325.1 (Danio rerio), US 2016/0194388 A1 Jul. 7, 2016

AY370883.1 (Oncorhynchus mykiss), AB037679.1 (Ciona is derived from a single copy or clone, including e.g., any intestinalis), NP 732309.1 (Drosophila melanogaster), eukaryotic, prokaryotic, or phage clone. "Monoclonal anti XP 322009.2 (Anopheles gambiae), NP 010384.1 (Saccha body' is not limited to antibodies produced through hybri romyces cerevisiae), NP 594167.1 (Schizosaccharomyces doma technology. Monoclonal antibodies can be produced pombe), NP 564451.2 (Arabidopsis thaliana), CF51152.1 using e.g., hybridoma techniques well known in the art, as (Vitis vinifera), AJ276594.1 (Oryza sativa), Y14200.1 (Hor well as recombinant technologies, phage display technolo deum vulgare), AY 1 10486.1 (Zea mays), and AF548740.1 gies, synthetic technologies or combinations of Such tech (Triticum aestivum). Further, the term “14-3-3 epsilon” nologies and other technologies readily known in the art. encompasses all 14-3-3 epsilon splice variants. For example, Furthermore, the monoclonal antibody may be labeled with a human 14-3-3 epsilon is encoded by the gene YWHAE (En detectable label, immobilized on a solid phase and/or conju trez, Gene ID 7531). Alternative splicing produces two iso gated with a heterologous compound (e.g., an enzyme or forms isoform 1 (SEQID NO:11) and isoform SV (SEQID toxin) according to methods known in the art. NO:12). 0035. Further by “antibody' is meant a functional mono 0028. In an aspect, anti-14-3-3 epsilon antibodies of the clonal antibody, or an immunologically effective fragment invention may be used to detect 14-3-3 epsilon in vitro or in thereof; such as an Fab, Fab', or F(ab')2 fragment thereof. In vivo. In another aspect, anti-14-3-3 epsilon antibodies useful Some contexts herein, fragments will be mentioned specifi herein also include antibodies that bind to specific regions of cally for emphasis; nevertheless, it will be understood that 14-3-3 epsilon, isoforms of 14-3-3 epsilon and to other forms regardless of whether fragments are specified, the term “anti of 14-3-3 epsilon. Specific regions of 14-3-3 epsilon include, body' includes such fragments as well as single-chain forms. but are not limited to, the C-terminal, the N-terminal, and The term “antibody” also includes bispecific monoclonal other central domains. Isoforms of 14-3-3 epsilon include antibodies (i.e. a protein that comprises fragments of two 14-3-3 epsilon isoform 1 (SEQID NO:11) and 14-3-3 epsilon different monoclonal antibodies and consequently binds two isoform SV (SEQID NO:12), as well as splice variants from different antigens). A specific example of a bispecific mono other species. Other forms of 14-3-3 epsilon include but are clonal antibody may be a Bi-specific T-cell engager (BiTE) not limited to truncated, modified, soluble, insoluble, intrac which is a fusion protein consisting of two single-chain vari ellular, extracellular, and dimerized or otherwise oligomer able fragments (sclvs) of different antibodies. In certain ized forms, as well as 14-3-3 epsilon complexed with other embodiments, BiTEs from a link between T cells and tumor proteins or molecules. cells. Accordingly, one sclv is a specific for 14-3-3 epsilon 0029. In another aspect, anti-14-3-3 epsilon antibodies and one scFv binds a T cell. Additionally, an antibody of the useful herein include those which are isolated, characterized, invention may be a chimeric antigen receptor (CAR), also purified, functional and have been recovered (obtained) from referred to as an artificial T cell receptor, a chimeric T cell a process for their preparation and thus available for use receptor, or a chimeric immunoreceptor. CARs are engi herein inauseful form in an amount Sufficient for conjugation neered receptors, which graft an arbitrary specificity onto an to a drug, drug delivery vehicles or imaging agent. immune effector cell. Typically, these receptors are used to 0030. In another aspect, antibodies useful herein include graft the specificity of a monoclonal antibody onto a T cell. those antibodies which have been isolated, characterized, Also included within the definition “antibody' for example purified, are functional and have been recovered (obtained) are single chain forms, generally designated Fv, regions, of from a process for their preparation and thus available for use antibodies with this specificity. These schvs are comprised of herein in a useful form in an amount Sufficient for an assay to the heavy and light chain variable regions connected by a detect and measure the amount of 14-3-3 epsilon in a biologi linker. Methods of making and using scFVs are known in the cal sample. art. Additionally, included within the definition “antibody' 0031. In another aspect, anti-14-3-3 epsilon antibodies are single-domain antibodies, generally designated SdAb. useful herein include those which are isolated, characterized, which is an antibody fragment consisting of a single mono purified, functional and have been recovered (obtained) from meric variable antibody domain. A saAb antibody may be a process for their preparation and thus available for use derived from camelids (VH fragments) or cartilaginous herein in a useful form in a therapeutically and medicinally fishes (VA fragments). As long as the protein retains the Sufficient amount. ability specifically to bind its intended target, it is included 0032. The antibodies and methods of their use are within the term “antibody.” described in further detail below. 0036 Preferably, but not necessarily, the antibodies useful in the discovery are produced recombinantly, as manipulation I. Anti-14-3-3 Epsilon Antibodies of the typically murine or other non-human antibodies with 0033. In an aspect, anti-14-3-3 epsilon antibodies useful the appropriate specificity is required in order to convert them herein include all antibodies that specifically bind an epitope to humanized form. Antibodies may or may not be glycosy within 14-3-3 epsilon. Generally speaking, the epitope is lated, though glycosylated antibodies are preferred. Antibod detectable on the surface of a tumor cell following irradiation. ies are properly cross-linked via disulfide bonds, as is known. The epitope may or may not be detectable on the cell surface 0037. The basic antibody structural unit of an antibody in the absence of irradiation. Alternatively, an epitope may be useful herein comprises a tetramer. Each tetramer is com detectable on the surface of a tumor cell bothin the absence of posed of two identical pairs of polypeptide chains, each pair irradiation and following irradiation, though the detectable having one “light (about 25 kDa) and one “heavy chain signal is greater following irradiation. In a specific embodi (about 50-70kDa). The amino-terminal portion of each chain ment, the epitope is selected from the group consisting of includes a variable region of about 100 to 110 or more amino SEQID NO:13, SEQID NO:14 and SEQID NO:15. acid sequences primarily responsible for antigen recognition. 0034. The term “antibody includes the term “monoclonal The carboxy-terminal portion of each chain defines a constant antibody.” “Monoclonal antibody' refers to an antibody that region primarily responsible for effector function. US 2016/0194388 A1 Jul. 7, 2016

0.038 Light chains are classified as gamma, mu, alpha, and are produced in genetically modified mice whose immune lambda. Heavy chains are classified as gamma, mu, alpha, systems have been altered to correspond to human immune delta, or epsilon, and define the antibody's isotype as IgC). systems. As mentioned above, it is sufficient for use in the IgM, IgA, Ig) and IgE, respectively. Within light and heavy methods of this discovery, to employ an immunologically chains, the variable and constant regions are joined by a “J” specific fragment of the antibody, including fragments repre region of about 12 or more amino acid sequences, with the senting single chain forms. heavy chain also including a “D” region of about 10 more 0042. Further, as used herein the term “humanized anti amino acid sequences. body” refers to an anti-14-3-3 epsilon antibody comprising a 0039. The variable regions of each light/heavy chain pair human framework, at least one CDR from a nonhuman anti form the antibody binding site. Thus, an intact antibody has body, and in which any constant region present is Substan two binding sites. The chains exhibit the same general struc tially identical to a human immunoglobulin constant region, ture of relatively conserved framework regions (FR) joined i.e., at least about 85-90%, preferably at least 95% identical. by three hyperVariable regions, also called complementarity Hence, all parts of a humanized antibody, except possibly the determining regions (hereinafter referred to as “CDRs.) The CDRs, are substantially identical to corresponding pairs of CDRs from the two chains are aligned by the framework one or more native human immunoglobulin sequences. regions, enabling binding to a specific epitope. From N-ter 0043. If desired, the design of humanized immunoglobu minal to C-terminal, both light and heavy chains comprise the lins may be carried out as follows. When an amino acid domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequence falls under the following category, the framework respectively. The assignment of amino acid sequences to each amino acid sequence of a human immunoglobulin to be used domain is in accordance with known conventions (See, Kabat (acceptor immunoglobulin) is replaced by a framework “Sequences of Proteins of Immunological Interest National amino acid sequence from a CDR-providing nonhuman Institutes of Health, Bethesda, Md., 1987 and 1991; Chothia, immunoglobulin (donor immunoglobulin): (a) the amino acid etal, J. Mol. Bio. (1987) 196:901-917: Chothia, et al., Nature sequence in the human framework region of the acceptor (1989) 342:878-883). immunoglobulin is unusual for human immunoglobulin at 0040. In an aspect, monoclonal anti-14-3-3 epsilon anti that position, whereas the corresponding amino acid bodies are generated with appropriate specificity by standard sequence in the donor immunoglobulin is typical for human techniques of immunization of mammals, forming hybrido immunoglobulin at that position; (b) the position of the amino mas from the antibody-producing cells of said mammals or acid sequence is immediately adjacent to one of the CDRS; or otherwise immortalizing them, and culturing the hybridomas (c) any side chain atom of a framework amino acid sequence or immortalized cells to assess them for the appropriate speci is within about 5-6 angstroms (center-to-center) of any atom ficity. In the present case, such antibodies could be generated of a CDR amino acid sequence in a three dimensional immu by immunizing a human, rabbit, rat or mouse, for example, noglobulin model (Queen, et al., op.cit., and Co, ct al. Proc. with a peptide representing an epitope encompassing a region Natl. Acad. Sci. USA (1991) 88:2869). When each of the of the 14-3-3 epsilon protein coding sequence or an appro amino acid sequences in the human framework region of the priate Subregion thereof. Materials for recombinant manipu acceptor immunoglobulin and a corresponding amino acid lation can be obtained by retrieving the nucleotide sequences sequence in the donor immunoglobulin is unusual for human encoding the desired antibody from the hybridoma or other immunoglobulin at that position, Such an amino acid cell that produces it. These nucleotide sequences can then be sequence is replaced by an amino acid sequence typical for manipulated and isolated, characterized, purified and, recov human immunoglobulin at that position. ered to provide them in humanized form, for use herein if 0044. In all instances, an antibody of the invention specifi desired. cally binds 14-3-3 epsilon. In exemplary embodiments, an 0041 As used herein “humanized antibody' includes an antibody of the invention specifically binds to human 14-3-3 anti-14-3-3 epsilon antibody that is composed partially or epsilon isoform 1 (SEQ ID NO:11), human 14-3-3 epsilon fully of amino acid sequence sequences derived from a isoform SV (SEQ ID NO:12), or human 14-3-3 epsilon iso human antibody germline by altering the sequence of an form 1 and human 14-3-3 epsilon isoform SV. In other exem antibody having non-human complementarity determining plary embodiments, an antibody of the invention specifically regions (“CDR). The simplest such alteration may consist binds to an epitope on human 14-3-3 epsilon selected from the simply of Substituting the constant region of a human anti group consisting of SEQID NO:13, SEQID NO:14 and SEQ body for the murine constant region, thus resulting in a ID NO:15. The phrase “specifically binds' herein means anti human/murine chimera which may have sufficiently low bodies bind to the protein with an affinity constant or Affinity immunogenicity to be acceptable for pharmaceutical use. of interaction (KD) in the range of at least 0.1 mM to 1 pM, or Preferably, however, the variable region of the antibody and in the range of at least 0.1 pM to 10 nM, with a preferred range even the CDR is also humanized by techniques that are by being 0.1 pM to 1 nM. The sequence of 14-3-3 epsilon from now well known in the art. The framework regions of the a variety of species is known in the art, and methods of variable regions are Substituted by the corresponding human determining whether an antibody binds to 14-3-3 epsilon are framework regions leaving the non-human CDR Substantially known in the art. intact, or even replacing the CDR with sequences derived 0045. The antibodies of the present invention may also be from a human genome. CDRS may also be randomly mutated used as fusion proteins known as single chain variable frag such that binding activity and affinity for 14-3-3 epsilon is ments (sclv). These scFVs are comprised of the heavy and maintained or enhanced in the context of fully human germ light chain variable regions connected by a linker. In most line framework regions or framework regions that are Sub instances, but not all, the linker may be a peptide. A linker stantially human. Substantially human frameworks have at peptide is preferably from about 10 to 25 amino acids in least 90%. 95%, or 99% sequence identity with a known length. Preferably, a linker peptide is rich in glycine, as well human framework sequence. Fully useful human antibodies as serine or threonine. Sclvs can be used to facilitate phage US 2016/0194388 A1 Jul. 7, 2016

display or can be used for flow cytometry, immunohis 0050. In one embodiment, an antibody of the invention tochemistry, or as targeting domains. Methods of making and may be derived from the hybridoma 7D4, and may be using SchvS are known in the art. encoded by a nucleic acid sequence comprising 90,91, 92,93, 0046. In a preferred embodiment, the schvs of the present 94, 95, 96, 97,98, or 99% identity to the light chain variable invention are conjugated to a human constant domain. In region of SEQID NO:1, and/or may be encoded by a nucleic Some embodiments, the heavy constant domain is derived acid sequence comprising 90,91, 92,93, 94.95, 96.97, 98, or from an IgG domain, such as IgG1, IgG2, IgG3, or IgG4. In 99% identity to the heavy chain variable region of SEQ ID other embodiments, the heavy chain constant domain may be NO:2. In another embodiment, an antibody of the invention derived from IgA, IgM, or IgE. may be derived from the hybridoma 7D4, and may comprise 0047. The antibodies of the present invention, including ScFVs, may also be conjugated to a payload. Such as a thera an amino acid sequence with 90,91, 92,93, 94.95, 96, 97,98, peutic agent, a detectable, and/or a delivery device (including, or 99% identity to the light chain variable region of SEQID but not limited to, a liposome or a nanoparticle) containing the NO:3, and/or may comprise an amino acid sequence with 90, drug or detectable label. Methods of conjugating an antibody 91, 92,93, 94, 95, 96, 97,98, or 99% identity to the heavy to a therapeutic agent, a detectable label, a liposome, a nano chain variable region of SEQID NO:4. In each of the above particle or other delivery device are known in the art. Gener embodiments, the antibody may be humanized. ally speaking, the conjugation should not interfere with the 0051. In an exemplary embodiment of an antibody of the antibody recognizing its target, and should not interfere with invention that binds to anti-14-3-3 epsilon, the antibody com the active site of the target. In some instances, an antibody prises the light chain nucleic acid sequence of SEQID NO:1 may be generated with a cleavable linkage between the anti body and the payload. Such a linker may allow release of the and the heavy chain nucleic acid sequence of SEQID NO:2 payload at a specific cellular location. Suitable linkers i.e. the monoclonal antibody referred to herein as 7D4. In include, but are not limited to, amino acid chains and alkyl another exemplary embodiment of an antibody of the inven chains functionalized with reactive groups for conjugating to tion that binds to anti-14-3-3 epsilon, the antibody comprises both the antibody of the invention and the detectable label the light chain amino acid sequence of SEQID NO:3 and the and/or therapeutic agent. The therapeutic agent, detectable heavy chain amino acid sequence of SEQID NO:4 i.e. the label, and delivery device are described in further detail monoclonal antibody referred to herein as 7D4. below. 0052. In one embodiment, an antibody of the invention 0.048. In an aspect, the present invention encompasses a may comprise a light chain CDR1, such as antibody 1 of Table cell line, the cell line expressing an antibody comprising an A. In another embodiment, an antibody of the invention may amino acid sequence selected from the group consisting of comprise a light chain CDR2, such as antibody 4 of Table A. SEQ ID NO:3 and SEQ ID NO:4, wherein the antibody In yet another embodiment, an antibody of the invention may specifically binds 14-3-3 epsilon. In another embodiment, the comprise a light chain CDR3, such as antibody 6 of Table A. present invention encompasses a cell line, the cell line In an alternative embodiment, an antibody of the invention expressing an antibody encoded by a nucleic acid sequence may comprise a combination of two or three light chain comprising a nucleic acid sequence selected from the group CDRs, such as the antibodies 2, 3 and 5 of Table A. consisting of SEQID NO:1 and SEQID NO:2, wherein the antibody specifically binds 14-3-3 epsilon. In some embodi 0053 Similarly, in one embodiment, an antibody of the ments, the cell line is an immortalized cell line. In preferred invention may comprise a heavy chain CDR1. Such as anti embodiments, the cell line is a hybridoma. Methods of gen body 7 of Table A. In another embodiment, an antibody of the erating hybridomas capable of producing antibodies are invention may comprise a heavy chain CDR2. Such as anti known in the art body 10 of Table A. In yet another embodiment, an antibody 0049. A preferred antibody is a humanized form of mouse of the invention may comprise a heavy chain CDR3, such as antibody derived from a hybridoma designated 7D4. As used antibody 12 of Table A. In an alternative embodiment, an herein, the term "derived from' means that the "derived antibody of the invention may comprise a combination of two antibody comprises at least one CDR region from the anti or three heavy chain CDRs, such as the antibodies 8, 9 and 11 body produced by 7D4. Stated another way, the "derived of Table A. antibody’ comprises at least one CDR region comprised of 0054 Alternatively, an antibody of the invention may the amino acid sequence selected from the group consisting comprise one or more light chain CDRS and one or more of SEQID NO:5, 6, 7, 8,9, and 10. heavy chain CDRs, such as the antibodies 13-48 of Table A. TABLE A Anti- Light Chain Heavy Chain body CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 SEQID NO: 5 SEQID NO: 5 SEQ ID NO: 6 SEQID NO: 5 SEQ ID NO: 6 SEQID NO: 7 SEQ ID NO: 6 SEQ ID NO: 6 SEQID NO: 7 SEQID NO: 7 SEQID NO: 8 SEQID NO: 8 SEQ ID NO: 9 SEQID NO: 8 SEQ ID NO:9 SEQID NO: 10 1 SEQ ID NO: 9 US 2016/0194388 A1 Jul. 7, 2016

TABLE A-continued Anti- Light Chain Heavy Chain body CDR CDR2 CDR3 CDR CDR2 CDR3 11 SEQ ID NO:9 SEQID NO: 10 12 SEQID NO: 10 13 SEOID NO:S SEQID NO: 8 14 SEOID NO:S SEQID NO: 8 SEQ ID NO: 9 15 SEOID NO:S SEQID NO: 8 SEQ ID NO:9 SEQID NO: 10 16 SEOID NO:S SEQ ID NO: 9 17 SEOID NO:S SEQ ID NO:9 SEQID NO: 10 18 SEOID NO:S SEQID NO: 10 19 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 8 20 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 8 SEQ ID NO: 9 21 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 8 SEQ ID NO:9 SEQID NO: 10 22 SEQ ID NO: 5 SEQID NO: 6 SEQ ID NO: 9 23 SEQ ID NO: 5 SEQID NO: 6 SEQ ID NO:9 SEQID NO: 10 24 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 10 25 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 7 SEQID NO: 8 26 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 7 SEQID NO: 8 SEQ ID NO: 9 27 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 7 SEQID NO: 8 SEQ ID NO:9 SEQID NO: 10 28 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 7 SEQ ID NO: 9 29 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 7 SEQ ID NO:9 SEQID NO: 10 30 SEQ ID NO: 5 SEQID NO: 6 SEQID NO: 7 SEQID NO: 10 31 SEQID NO: 6 SEQID NO: 8 32 SEQID NO: 6 SEQID NO: 8 SEQ ID NO: 9 33 SEQID NO: 6 SEQID NO: 8 SEQ ID NO:9 SEQID NO: 10 34 SEQID NO: 6 SEQ ID NO: 9 35 SEQID NO: 6 SEQ ID NO:9 SEQID NO: 10 36 SEQID NO: 6 SEQID NO: 10 37 SEQID NO: 6 SEQID NO: 7 SEQID NO: 8 38 SEQID NO: 6 SEQID NO: 7 SEQID NO: 8 SEQ ID NO: 9 39 SEQID NO: 6 SEQID NO: 7 SEQID NO: 8 SEQ ID NO:9 SEQID NO: 10 40 SEQID NO: 6 SEQID NO: 7 SEQID NO: 9 41 SEQID NO: 6 SEQID NO: 7 SEQ ID NO:9 SEQID NO: 10 42 SEQID NO: 6 SEQID NO: 7 SEQID NO: 10 43 SEQID NO: 7 SEQID NO: 8 44 SEQID NO: 7 SEQID NO: 8 SEQ ID NO: 9 45 SEQID NO: 7 SEQID NO: 8 SEQ ID NO:9 SEQID NO: 10 46 SEQID NO: 7 SEQ ID NO: 9 47 SEQID NO: 7 SEQ ID NO:9 SEQID NO: 10 48 SEQID NO: 7 SEQID NO: 10

0055. In various embodiments, an antibody of the inven embodiment, a humanized antibody of the invention may tion is humanized. For instance, in one embodiment, a comprise a light chain variable region comprising a CDR1 of humanized antibody of the invention may comprise a light amino acid sequence SEQID NO:5, a CDR2 of amino acid chain variable region comprising a CDR1 of amino acid sequence SEQ ID NO:6, a CDR3 of amino acid sequence sequence SEQID NO:5 with Zero to two amino acid substi SEQ ID NO:7, a heavy chain variable region comprising a tutions, a CDR2 of amino acid sequence SEQID NO:6 with CDR1 of amino acid sequence SEQ ID NO:8, a CDR2 of Zero to two amino acid substitutions, and a CDR3 of amino amino acid sequence SEQ ID NO:9, and a CDR3 of amino acid sequence SEQ ID NO:7 with zero to two amino acid acid sequence SEQ ID NO:10. The invention also encom Substitutions, and/or may comprise a heavy chain variable passes the corresponding nucleic acid sequences of SEQID region comprising a CDR1 of amino acid sequence SEQID NO:5, 6, 7,8,9, and 10, which can readily be determined by NO:8 with Zero to two amino acid substitutions, a CDR2 of one of skill in the art, and may be incorporated into a vector or amino acid sequence SEQID NO:9 with Zero to two amino other large DNA molecule, such as a chromosome, in order to acid substitutions, and a CDR3 of amino acid sequence SEQ express an antibody of the invention. ID NO:10 with Zero to two amino acid substitutions. In a 0056. The invention also encompasses a vector compris preferred embodiment, a humanized antibody of the inven ing a nucleic acid sequence capable of encoding an antibody tion may comprise a light chain variable region comprising a of the invention. As used herein, a vector is defined as a CDR1 of amino acid sequence SEQID NO:5 with Zero to two nucleic acid molecule used as a vehicle to transfer genetic amino acid Substitutions, a CDR2 of amino acid sequence material. Vectors include but are not limited to, plasmids, SEQ ID NO:6 with Zero to two amino acid substitutions, a phasmids, cosmids, transposable elements, viruses (bacte CDR3 of amino acid sequence SEQID NO:7 with Zero to two riophage, animal viruses, and plant viruses), and artificial amino acid substitutions, a heavy chain variable region com chromosomes (e.g., YACs). Such as retroviral vectors (e.g. prising a CDR1 of amino acid sequence SEQID NO:8 with derived from Moloney murine leukemia virus vectors (Mo Zero to two amino acid substitutions, a CDR2 of amino acid MLV), MSCV. SFFV, MPSV, SNV etc), lentiviral vectors sequence SEQID NO:8 with Zero to two amino acid substi (e.g. derived from HIV-1, HIV-2, SIV, BIV. FIV etc.), aden tutions, and a CDR3 of amino acid sequence SEQID NO:10 Oviral (Ad) vectors including replication competent, replica with Zero to two amino acid Substitutions. In an exemplary tion deficient and gutless forms thereof, adeno-associated US 2016/0194388 A1 Jul. 7, 2016

viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine waves. Examples of signal transducing machines include, papillomavirus vectors, Epstein-Barr virus, herpesvirus vec without limitation, gamma cameras including SPECT/CT tors, vaccinia virus vectors, Harvey murine sarcoma virus devices, PET scanners, fluorimeters, and Magnetic Reso vectors, murine mammary tumor virus vectors, Rous sarcoma nance Imaging (MRI) machines. As such, the detectable label virus vectors. An expression vector encoding an antibody of comprises a label that can be detected using magnetic reso the invention may be delivered to the cell using a viral vector nance imaging, Scintigraphic imaging, ultrasound, or fluores or via a non-viral method of transfer. Viral vectors suitable for cence. In a specific embodiment, the detectable label com introducing nucleic acids into cells include retroviruses, prises a label that can be detected using positron emission adenoviruses, adeno-associated viruses, rhabdoviruses, and tomography, single photon emission computed tomography, herpes viruses. Non-viral methods of nucleic acid transfer gamma camera imaging, or rectilinear Scanning. include naked nucleic acid, liposomes, and protein/nucleic 0060 Suitable fluorophores include, but are not limited to, acid conjugates. An expression construct encoding an anti fluorescein isothiocyante (FITC), fluorescein thiosemicarba body of the invention that is introduced to the cell may be Zide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), linear or circular, may be single-stranded or double-stranded, Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; and may be DNA, RNA, or any modification or combination Alexa647), near infrared (NIR) (700-900 nm) fluorescent thereof. An expression vector encoding an antibody of the dyes, and carbocyanine and aminostyryl dyes. B or an ana invention may be administered to a subject as gene therapy. log thereof can be labeled for fluorescence detection by label ing the agent with a fluorophore using techniques well known (a) Detectable Label in the art (see, e.g., Lohse et al., Bioconj Chem 8:503-509 0057. In an aspect, an antibody of the invention may be (1997)). For example, many known dyes are capable of being conjugated to a detectable label. A detectable label may be coupled to NH-terminal groups. Alternatively, a fluoro directly conjugated to an antibody of the invention or may be chrome Such as fluorescein may be bound to a lysine residue indirectly conjugated to an antibody of the invention. In an of a peptide linker. In a specific embodiment, an alkyne modi embodiment, a detectable label may be complexed with a fied dye, Such an Alexa Fluor dye, may be clicked to an azido chelating agent that is conjugated to an antibody of the inven modified Busing, for example, Sharpless click chemistry tion. In another embodiment, a detectable label may be com (Kolb et al., Angew Chem Int Ed 2001; 40: 2004-2021, which plexed with a chelating agent that is conjugated to a linker that incorporated by reference in its entirety). is conjugated to an antibody of the invention. In still another 0061. A radionuclide may be a Y-emitting radionuclide, embodiment, a detectable label may be conjugated to a linker Auger-emitting radionuclide, f-emitting radionuclide, an that is conjugated to an antibody of the invention. In still yet C-emitting radionuclide, or a positron-emitting radionuclide. another embodiment, a detectable label may be indirectly A radionuclide may be a detectable label and/or a therapeutic attached to an antibody of the invention by the ability of the agent. Non-limiting examples of Suitable radionuclides may label to be specifically bound by a second molecule. One include carbon-11, nitrogen-13, oxygen-15, fluorine-18, example of this type of an indirectly attached label is a biotin fluorodeoxyglucose-18, phosphorous-32, Scandium-47, cop label that can be specifically bound by the second molecule, per-64, 65 and 67, gallium-67 and 68, bromine-75, 77 and streptavidin or other biotin binding protein. Single, dual or 80m, rubidium-82, strontium-89, zirconium-89, yttrium-86 multiple labeling may be advantageous. An isolated antibody and 90, ruthenium-95, 97, 103 and 105, rhenium-99m, 101, of the present invention may be conjugated to one, two, three, 105, 186 and 188, technetium-99m, rhodium-105, mercury four, or five types of detectable labels. 107, palladium-109, indium-111, silver-111, indium-113m, 0058 As used herein, a “detectable label' is any type of lanthanide-114m, tin-117m, tellurium-121 m, 122m and label which, when attached to an antibody of the invention 125m, iodine-122, 123, 124, 125, 126, 131 and 133, renders the antibody detectable. A detectable label may also praseodymium-142, promethium-149, Samarium-153, gado be toxic to cells or cytotoxic. Accordingly, a detectable label linium-159, thulium-165, 167 and 168, dysprosium-165, hol may also be a therapeutic agent or cytotoxic agent. In general, mium-166, lutetium-177, rhenium-186 and 188, iridium-192, detectable labels may include luminescent molecules, chemi platinum-193 and 195m, gold-199, thallium-201, titanium luminescent molecules, fluorochromes, fluorophores, fluo 201, astatine-211, bismuth-212 and 213, lead-212, radium rescent quenching agents, colored molecules, radioisotopes, 223, actinium-225, and nitride or oxide forms derived there radionuclides, cintillants, massive labels such as a metal atom from. In a specific embodiment, a radionuclide is selected (for detection via mass changes), biotin, avidin, streptavidin, from the group consisting of copper-64, Zirconium-89, protein A, protein G, antibodies or fragments thereof, Grb2, yttrium-86, yttrium-90, technetium-99m, iodine-125, iodine polyhistidine, Ni", Flag tags, myc tags, heavy metals, 131, lutetium-177, rhenium-186 and rhenium-188. enzymes, alkaline phosphatase, peroxidase, luciferase, elec 0062) A variety of metal atoms may be used as a detectable tron donors/acceptors, acridinium esters, and colorimetric label. The metal atom may generally be selected from the substrates. The skilled artisan would readily recognize other group of metal atoms comprised of metals with an atomic useful labels that are not mentioned above, which may be number of twenty or greater. For instance, the metal atoms employed in the operation of the present invention. may be calcium atoms, Scandium atoms, titanium atoms, 0059 A detectable label emits a signal that can be detected Vanadium atoms, chromium atoms, manganese atoms, iron by a signal transducing machine. In some cases, the detect atoms, cobalt atoms, nickel atoms, copper atoms, Zinc atoms, able label can emit a signal spontaneously, such as when the gallium atoms, germanium atoms, arsenic atoms, selenium detectable label is a radionuclide. In other cases the detectable atoms, bromine atoms, krypton atoms, rubidium atoms, label emits a signal as a result of being stimulated by an strontium atoms, yttrium atoms, Zirconium atoms, niobium external field such as when the detectable label is a relaxivity atoms, molybdenum atoms, technetium atoms, ruthenium metal. Examples of signals include, without limitation, atoms, rhodium atoms, palladium atoms, silver atoms, cad gamma rays, X-rays, visible light, infrared energy, and radio mium atoms, indium atoms, tin atoms, antimony atoms, tel US 2016/0194388 A1 Jul. 7, 2016

lurium atoms, iodine atoms, Xenon atoms, cesium atoms, resulting in a biologically destructive nuclear reaction. In barium atoms, lanthanum atoms, hafnium atoms, tantalum certain embodiments, the non-radioactive isotope may be atoms, tungsten atoms, rhenium atoms, osmium atoms, iri boron-10 or gadolinium. dium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, (b) Therapeutic Agent radium atoms, actinium atoms, cerium atoms, praseodymium 0065. In an aspect, an antibody of the invention may be atoms, neodymium atoms, promethium atoms, Samarium conjugated to a therapeutic agent, Such that the therapeutic atoms, europium atoms, gadolinium atoms, terbium atoms, agent can be selectively targeted to a cell expressing 14-3-3 dysprosium atoms, holmium atoms, erbium atoms, thulium epsilon. In a specific embodiment, the therapeutic agent can atoms, ytterbium atoms, lutetium atoms, thorium atoms, pro be selectively targeted to an irradiated tumor cell expressing tactinium atoms, uranium atoms, neptunium atoms, pluto 14-3-3 epsilon. The therapeutic agent may be directly conju nium atoms, americium atoms, curium atoms, berkelium gated to an antibody of the invention or may be indirectly atoms, californium atoms, einsteinium atoms, fermium conjugated to an antibody of the invention. In an embodi atoms, mendelevium atoms, nobelium atoms, or lawrencium ment, the therapeutic agent may be complexed with a chelat atoms. In some embodiments, the metal atoms may be ing agent that is conjugated to an antibody of the invention. In selected from the group comprising alkali metals with an another embodiment, the therapeutic agent may be com atomic number greater than twenty. In other embodiments, plexed with a chelating agent that is conjugated to a linker that is conjugated to an antibody of the invention. In still another the metal atoms may be selected from the group comprising embodiment, the therapeutic agent may be conjugated to a alkaline earth metals with an atomic number greater than linker that is conjugated to an antibody of the invention. In twenty. In one embodiment, the metal atoms may be selected still yet another embodiment, the therapeutic agent may be from the group of metals comprising the lanthanides. In conjugated to a linker that is conjugated to a chelating agent another embodiment, the metal atoms may be selected from that is complexed with a detectable label and conjugated to an the group of metals comprising the actinides. In still another antibody of the invention. embodiment, the metal atoms may be selected from the group 0066. A “therapeutic agent' is any compound known in of metals comprising the transition metals. In yet another the art that is used in the detection, diagnosis, or treatment of embodiment, the metal atoms may be selected from the group a condition or disease. Such compounds may be naturally of metals comprising the poor metals. In other embodiments, occurring, modified, or synthetic. Non-limiting examples of the metal atoms may be selected from the group comprising therapeutic agents may include drugs, therapeutic com gold atoms, bismuth atoms, tantalum atoms, and gadolinium pounds, toxins, genetic materials, metals (such as radioactive atoms. In preferred embodiments, the metal atoms may be isotopes), proteins, peptides, carbohydrates, lipids, Steroids, selected from the group comprising metals with an atomic nucleic acid based materials, or derivatives, analogues, or number of 53 (i.e. iodine) to 83 (i.e. bismuth). In an alterna combinations thereof in their native form or derivatized with tive embodiment, the metal atoms may be atoms suitable for hydrophobic or charged moieties to enhance incorporation or magnetic resonance imaging. In another alternative embodi adsorption into a cell. Such therapeutic agents may be water ment, the metal atoms may be selected from the group con soluble or may be hydrophobic. Non-limiting examples of sisting of metals that have a K-edge in the X-ray energy band therapeutic agents may include immune-related agents, thy of CT. Preferred metal atoms include, but are not limited to, roid agents, respiratory products, antineoplastic agents, anti manganese, iron, gadolinium, gold, and iodine. helmintics, anti-malarials, mitotic inhibitors, hormones, tox ins, anti-protozoans, anti-tuberculars, cardiovascular 0063. The metal atoms may be metal ions in the form of products, blood products, biological response modifiers, anti +1, +2, or +3 oxidation states. For instance, non-limiting fungal agents, vitamins, peptides, anti-allergic agents, anti examples include Bat, Bi", Cs", Ca", Cr, Cr", Cr, coagulation agents, circulatory drugs, metabolic potentiators, Co?", Co", Cut, Cu2+, Cut, Ga*, Gd, Aut, Aut, Fe?", anti-virals, anti-anginals, antibiotics, anti-inflammatories, Fe", F3+, Pb2+, Mn?", Mn", Mn", Mn'", Hg", Ni?", Ni", anti-rheumatics, narcotics, cardiac glycosides, neuromuscu Ag", Sr", Sn", Sn", and Zn". The metal atoms may com lar blockers, sedatives, local anesthetics, general anesthetics, prise a metal oxide. For instance, non-limiting examples of or radioactive atoms or ions. Non-limiting examples of thera metal oxides may include iron oxide, manganese oxide, or peutic agents are described below. In a specific embodiment, gadolinium oxide. Additional examples may include magne atherapeutic agent may be a compound used in the detection tite, maghemite, or a combination thereof. diagnosis or treatment of cancer. The therapeutic agent pref 0064. In an embodiment where an antibody of the inven erably reduces or interferes with tumor growth or otherwise tion is conjugated to a non-radioactive isotope, it may be used reduces the effect of the tumor within the body or organism. in neutron capture therapy (NCT). Neutron capture therapy A therapeutic agent that reduces the symptoms produced by (NCT) is a noninvasive therapeutic modality for treating the tumor or reduces tumor growth is suitable for the present locally invasive malignant tumors. NCT is a two step proce invention. Additionally, any therapeutic agent that reduces dure: first, the Subject is injected with a tumor localizing drug the symptoms associated with tumor cell growth will work for containing a non-radioactive isotope that has a high propen purposes of the present invention. sity or cross section (O) to capture slow neutrons. The cross 0067. An antibody of the invention may be conjugated to section of the capture agent is many times greater than that of one, two, three, four, or five therapeutic agents. A linker may the other elements present in tissues such as hydrogen, oxy or may not be used to conjugate a therapeutic agent to an gen, and nitrogen. In the second step, the Subject is radiated antibody of the invention. Generally speaking, the conjuga with epithermal neutrons, which after losing energy as they tion should not interfere with the antibody binding to 14-3-3 penetrate tissue, are absorbed by the capture agent, which epsilon. In some instances, an antibody of the invention may Subsequently emits high-energy charged particles, thereby be generated with a cleavable linkage between the antibody US 2016/0194388 A1 Jul. 7, 2016 and therapeutic agent. Such a linker may allow release of the used in accordance with the presently disclosed subject mat therapeutic agent at a specific cellular location. In other ter include: thrombospondin I (Kosfeld & Frazier, 1993; instances, an antibody of the invention may be generated with Tolsma et al., 1993; Dameron et al., 1994), metallospondin an enzyme linked to it to create a prodrug. For example, proteins (Carpizo & Iruela-Arispe, 2000), class I interferons cytidine deaminase may be linked to an antibody of the inven (Albini et al., 2000), IL12 (Voest et al., 1995), protamine tion. The cytidine deaminase then cleaves the prodrug to (Ingber et al., 1990), angiostatin (O'Reilly et al., 1994), lami create a cytotoxic drug. nin (Sakamoto et al., 1991), endostatin (O'Reilly et al., 1997), 0068 Atherapeutic agent of the invention may be a toxin. and a prolactin fragment (Clapp et al., 1993). In addition, The term “toxin' means the toxic material or product of several anti-angiogenic peptides have been isolated from plants, animals, microorganisms (including, but not limited these proteins (Maione et al., 1990; Eijan et al., 1991; Wol to, bacteria, viruses, fungi, rickettsiae or protozoa), or infec tering et al., 1991). Representative proteins with both immu tious Substances, or a recombinant or synthesized molecule, nostimulatory and anti-angiogenic activities may include whatever theft origin and method of production. A toxin may IL12, interferon-Y, or a chemokine. Other therapeutic nucleic be a small molecule, peptide, or protein that is capable of acids that may be useful for cancer therapy include but are not causing disease on contact with or absorption by body tissues limited to nucleic acid sequences encoding tumor suppressor interacting with biological macromolecules such as enzymes gene products/antigens, antimetabolites, Suicide gene prod or cellular receptors. A toxin may be a “biotoxin' which is ucts, and combinations thereof. used to explicitly identify the toxin as from biological origin. 0070 A chemotherapeutic agent refers to a chemical com Biotoxins may be further classified into fungal biotoxins, or pound that is useful in the treatment of cancer. The compound short mycotoxins, microbial biotoxins, plant biotoxins, short may be a cytotoxic agent that affects rapidly dividing cells in phytotoxins and animal biotoxins. Non-limiting examples of general, or it may be a targeted therapeutic agent that affects biotoxins include: endotoxins produced by bacteria, Such as the deregulated proteins of cancer cells. A cytotoxic agent is Pseudomonas endotoxin; cyanotoxins produced by cyano any naturally-occurring, modified, or synthetic compound bacteria, Such as microcystins, nodularins, anatoxin-a, cylin that is toxic to tumor cells. Such agents are useful in the drospermopsins, lyngbyatoxin-a, saxitoxin, lipopolysaccha treatment of neoplasms, and in the treatment of other symp rides, aplysiatoxins, BMAA; dinotoxins produced by toms or diseases characterized by cell proliferation or a dinoflagellates, such as saxitoxins and gonyautoxins; necro hyperactive cell population. The chemotherapeutic agent toxins produced by, for example, the brown recluse or “fiddle may be an alkylating agent, an anti-metabolite, an anti-tumor back spider, most rattlesnakes and vipers, the puff adder, antibiotic, an anti-cytoskeletal agent, a topoisomerase inhibi Streptococcus pyogenes; neurotoxins produced by, for tor, an anti-hormonal agent, a targeted therapeutic agent, a example, the black widow spider, most Scorpions, the box photodynamic therapeutic agent, or a combination thereof. In jellyfish, elapid Snakes, the cone snail, the Blue-ringed octo an exemplary embodiment, the chemotherapeutic agent is pus, Venomous fish, frogs, palythoa coral, various different selected from the group consisting of liposomal doxorubicin types of algae, cyanobacteria and dinoflagellates, such as and nanoparticle albumin docetaxel. botulinum toxin (e.g. Botox), tetanus toxin, tetrodotoxin, 0071 Non-limiting examples of suitable alkylating agents chlorotoxin, conotoxin, anatoxin-a, bungarotoxin, caram may include altretamine, benzodopa, buSulfan, carboplatin, boxin, curare; myotoxins, found in, for example, Snake and carboquone, carmustine (BCNU), chlorambucil, chlornap lizard Venoms; and cytotoxins such as ricin, from castor hazine, cholophosphamide, chlorozotocin, cisplatin, cyclos beans, apitoxin, from honeybees, and T-2 mycotoxin, from phosphamide, dacarbazine (DTIC), estramustine, fotemus certain toxic mushrooms. In certain embodiments, a toxin is a tine, ifosfamide, improsulfan, lipoplatin, lomustine (CCNU), cytotoxin. In an embodiment, a cytotoxin is an endotoxin mafosfamide, mannosulfan, mechlorethamine, mechlore from Pseudomonas. thamine oxide hydrochloride, melphalan, meturedopa, mus 0069. A therapeutic agent of the invention may be a small tine (mechlorethamine), mitobronitol, nimustine, novem molecule therapeutic, a therapeutic antibody, a therapeutic bichin, oxaliplatin, phenesterine, piposulfan, prednimustine, nucleic acid, or a chemotherapeutic agent. Non-limiting ranimustine, Satraplatin, Semustine, temozolomide, thiotepa, examples of therapeutic antibodies may include muromo treosulfan, triaziquone, triethylenemelamine, triethylene mab, abciximab, rituximab, daclizumab, basiliximab, palivi phosphoramide (TEPA), triethylenethiophosphaoramide Zumab, infliximab, trastuzumab, etanercept, gemtuzumab, (thiotepa), trimethylolomelamine, trofosfamide, uracil mus alemtuzumab, ibritomomab, adalimumab, alefacept, omali tard and uredopa. Zumab, to situmomab, efalizumab, cetuximab, bevacizumab, 0072 Suitable anti-metabolites may include, but are not natalizumab, ranibizumab, panitumumab, eculizumab, and limited to aminopterin, ancitabine, azacitidine, 8-azaguanine, certolizumab. A representative therapeutic nucleic acid may 6-azauridine, capecitabine, carmofur (1-hexylcarbomoyl-5- encode a polypeptide having an ability to induce an immune fluorouracil), cladribine, clofarabine, cytarabine (cytosine response and/or an anti-angiogenic response in vivo. Repre arabinoside (Ara-C)), decitabine, denopterin, dideoxyuri sentative therapeutic proteins with immunostimulatory dine, doxifluridine, enocitabine, floxuridine, fludarabine, effects include but are not limited to cytokines (e.g., an inter 5-fluorouracil, gemcitabine, hydroxyurea (hydroxycarbam leukin (IL) such as IL2, IL4, IL7, IL12, interferons, granulo ide), leucoVorin (folinic acid), 6-mercaptopurine, methotrex cyte-macrophage colony-stimulating factor (GM-CSF), ate, nafoxidine, nelarabine, oblimersen, pemetrexed, pterop tumor necrosis factor alpha (TNF-C)), immunomodulatory terin, raltitrexed, tegofur, tiazofurin, thiamiprine, tioguanine cell Surface proteins (e.g., human leukocyte antigen (HLA (thioguanine), and trimetrexate. proteins), co-stimulatory molecules, and tumor-associated 0073. Non-limiting examples of suitable anti-tumor anti antigens. See Kirk & Mule, 2000; Mackensen et al., 1997: biotics may include aclacinomysin, aclarubicin, actinomy Walther & Stein, 1999; and references cited therein. Repre cins, adriamycin, aurostatin (for example, monomethyl sentative proteins with anti-angiogenic activities that can be auristatin E), authramycin, azaserine, bleomycins, cactino US 2016/0194388 A1 Jul. 7, 2016 mycin, calicheamicin, carabicin, caminomycin, carZinophi (c) Delivery Vehicle lin, chromomycins, dactinomycin, daunorubicin, detorubi cin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, I0084 Anantibody of the invention may be conjugated to a epoxomicin, esorubicin, idarubicin, marcellomycin, mitomy vehicle for cellular delivery. In these embodiments, typically cins, mithramycin, mycophenolic acid, nogalamycin, olivo an antibody of the invention, which may or may not be con mycins, peplomycin, plicamycin, potfiromycin, puromycin, jugated to a detectable label and/or therapeutic agent, is encapsulated in a suitable vehicle to either aid in the delivery quelamycin, rodorubicin, sparsomycin, Streptonigrin, Strep of the compound to target cells, to increase the stability of the toZocin, tubercidin, valrubicin, ubenimex, Zinostatin, and antibody, or to minimize potential toxicity of the antibody. As Zorubicin. will be appreciated by a skilled artisan, a variety of vehicles 0074 Non-limiting examples of suitable anti-cytoskeletal are suitable for delivering an antibody of the present inven agents may include cabazitaxel, colchicines, demecolcine, tion. Non-limiting examples of suitable structured fluid deliv docetaxel, epothilones, ixabepilone, macromycin, omacetax ery systems may include nanoparticles, liposomes, micro ine mepesuccinate, ortataxel, paclitaxel (for example, DHA emulsions, micelles, dendrimers and other phospholipid paclitaxel), taxane, tesetaxel, vinblastine, Vincristine, Vin containing systems. Methods of incorporating antibodies into desine, and vinorelbine. delivery vehicles are known in the art. Although various 0075 Suitable topoisomerase inhibitors may include, but embodiments are presented below, it will be appreciate that are not limited to, amsacrine, etoposide (VP-16), irinotecan, other methods known in the art to incorporate an antibody of mitoxantrone, RFS 2000, teniposide, and topotecan. the invention into a delivery vehicle are contemplated. 0076 Non-limiting examples of suitable anti-hormonal I0085. In one alternative embodiment, a liposome delivery agents may include aminoglutethimide, antiestrogen, aro vehicle may be utilized. Liposomes, depending upon the matase inhibiting 4(5)-imidazoles, bicalutamide, finasteride, embodiment, are suitable for delivery of the antibody of the flutamide, fluvestrant, goserelin, 4-hydroxytamoxifen, keOX invention in view of their structural and chemical properties. ifene, leuprolide, LY 1 17018, mitotane, nilutamide, onapris Generally speaking, liposomes are spherical vesicles with a tone, raloxifene, tamoxifen, toremifene, and triloStane. phospholipid bilayer membrane. The lipid bilayer of a lipo 0077. Examples of targeted therapeutic agents may Some may fuse with other bilayers (e.g., the cell membrane), include, without limit, monoclonal antibodies Such as alem thus delivering the contents of the liposome to cells. In this tuZumab, cartumaxomab, edrecolomab, epratuZumab, gem manner, the antibody of the invention may be selectively tuZumab, gemtuzumab ozogamicin, glembatumumab delivered to a cell by encapsulation in a liposome that fuses Vedotin, ibritumomab tiuxetan, reditux, rituximab, to situmo with the targeted cells membrane. mab, and trastuzumab: protein kinase inhibitors such as beva I0086 Liposomes may be comprised of a variety of differ cizumab, cetuximab, crizonib, dasatinib, erlotinib, gefitinib, ent types of phosolipids having varying hydrocarbon chain imatinib, lapatinib, mubritinib, nilotinib, panitumumab, lengths. Phospholipids generally comprise two fatty acids paZopanib, Sorafenib, Sunitinib, toceranib, and Vandetanib. linked through glycerol phosphate to one of a variety of polar 0078. Non limiting examples of angiogeneisis inhibitors groups. Suitable phospholids include phosphatidic acid (PA), may include angiostatin, bevacizumab, denileukin diftitox, phosphatidylserine (PS), phosphatidylinositol (PI), phos endostatin, everolimus, genistein, interferon alpha, interleu phatidylglycerol (PG), diphosphatidylglycerol (DPG), phos kin-2, interleukin-12, paZopanib, pegaptainib, ranibizumab, phatidylcholine (PC), and phosphatidylethanolamine (PE). rapamycin (sirolimus), temsirolimus, and thalidomide. The fatty acid chains comprising the phospholipids may 0079 Non limiting examples of growth inhibitory range from about 6 to about 26 carbonatoms in length, and the polypeptides may include bortazomib, erythropoietin, inter lipid chains may be saturated or unsaturated. Suitable fatty leukins (e.g., IL-1, IL-2, IL-3, IL-6), leukemia inhibitory acid chains include (common name presented in parentheses) factor, interferons, romidepsin, thrombopoietin, TNF-C. n-dodecanoate (laurate), n-tretradecanoate (myristate), CD30 ligand, 4-1 BB ligand, and Apo-1 ligand. n-hexadecanoate (palmitate), n-octadecanoate (Stearate), 0080. Non-limiting examples of photodynamic therapeu n-eicosanoate (arachidate), n-docosanoate (behenate), n-tet tic agents may include aminolevulinic acid, methylaminole racosanoate (lignocerate), cis-9-hexadecenoate (palmi Vulinate, retinoids (alitretinon, tamibarotene, tretinoin), and toleate), cis-9-octadecanoate (oleate), cis,cis-9,12-octade temoporfin. candienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linolenate), and all cis-5,8,11,14-eicosatetraenoate (arachi 0081. Other antineoplastic agents may include anagrelide, donate). The two fatty acid chains of a phospholipid may be arsenic trioxide, asparaginase, beXarotene, bropirimine, cele identical or different. Acceptable phospholipids include dio coxib, chemically linked Fab, efaproxiral, etoglucid, ferrugi leoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyris nol, lonidamide, masoprocol, miltefosine, mitoguaZone, tal toyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, apanel, trabectedin, and Vorinostat. palmitoyl, linolenyl PS, and the like. 0082 Also included are pharmaceutically acceptable I0087. The phospholipids may come from any natural salts, acids, or derivatives of any of the above listed agents. Source, and, as such, may comprise a mixture of phospholip The dose of the chemotherapeutic agent can and will vary ids. For example, egg yolk is rich in PC, PG, and PE, soy depending upon the agent and the type of tumor or neoplasm. beans contains PC, PE, PI, and PA, and animal brain or spinal A skilled practitioner will be able to determine the appropri cord is enriched in PS. Phospholipids may come from syn ate dose of the chemotherapeutic agent. thetic sources too. Mixtures of phospholipids having a varied 0083. Other therapeutic agents may comprise a virus or a ratio of individual phospholipids may be used. Mixtures of viral genome such as an oncolytic virus. An oncolytic virus different phospholipids may result in liposome compositions comprises a naturally occurring virus that is capable of killing having advantageous activity or stability of activity proper a cell in the target tissue (for example, by lysis) when it enters ties. The above mentioned phospholipids may be mixed, in Such a cell. optimal ratios with cationic lipids, such as N-(1-(2,3-dio US 2016/0194388 A1 Jul. 7, 2016

leolyoxy)propyl)-N.N.N-trimethyl ammonium chloride, drops of water in oil. In an alternative embodiment, the micro 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per emulsion structure is the lamellae. It comprises consecutive chloarate, 3,3'-deheptyloxacarbocyanine iodide, 1,1'-de layers of water and oil separated by layers of surfactant. The dodecyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate, "oil of microemulsions optimally comprises phospholipids. 1,1'-dioleyl-3,3,3,3'-tetramethylindo carbocyanine methane Any of the phospholipids detailed above for liposomes are sulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium suitable for embodiments directed to microemulsions. The iodide, or 1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocya antibody of the invention may be encapsulated in a micro nine perchloarate. emulsion by any method generally known in the art. 0088 Liposomes may optionally comprise sphingolipids, 0093. In yet another embodiment, an antibody of the in which spingosine is the structural counterpart of glycerol invention may be delivered in a dendritic macromolecule, or and one of the one fatty acids of a phosphoglyceride, or a dendrimer. Generally speaking, a dendrimer is a branched cholesterol, a major component of animal cell membranes. tree-like molecule, in which each branch is an interlinked Liposomes may optionally, contain pegylated lipids, which chain of molecules that divides into two new branches (mol are lipids covalently linked to polymers of polyethylene gly ecules) after a certain length. This branching continues until col (PEG). PEGs may range in size from about 500 to about the branches (molecules) become so densely packed that the 10,000 daltons. canopy forms a globe. Generally, the properties of dendrim 0089 Liposomes may further comprise a suitable solvent. ers are determined by the functional groups at their surface. The solvent may be an organic solvent or an inorganic Sol For example, hydrophilic end groups. Such as carboxyl vent. Suitable solvents include, but are not limited to, dim groups, would typically make a water-soluble dendrimer. ethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyr Alternatively, phospholipids may be incorporated in the Sur rolidone, acetronitrile, alcohols, dimethylformamide, face of an dendrimer to facilitate absorption across the skin. tetrahydrofuran, or combinations thereof. Any of the phospholipids detailed for use in liposome 0090 Liposomes carrying the antibody of the invention embodiments are suitable for use in dendrimer embodiments. (i.e., having at least one methionine compound) may be pre Any method generally known in the art may be utilized to pared by any known method of preparing liposomes for drug make dendrimers and to encapsulate antibodies of the inven delivery, such as, for example, detailed in U.S. Pat. Nos. tion therein. For example, dendrimers may be produced by an 4,241,046, 4,394,448, 4,529,561, 4,755,388, 4,828,837, iterative sequence of reaction steps, in which each additional 4,925,661, 4,954,345, 4,957,735, 5,043,164, 5,064,655, iteration leads to a higher order dendrimer. Consequently, 5,077,211 and 5.264,618, the disclosures of which are hereby they have a regular, highly branched 3D structure, with nearly incorporated by reference in their entirety. For example, lipo uniform size and shape. Furthermore, the final size of a den Somes may be prepared by Sonicating lipids in an aqueous drimer is typically controlled by the number of iterative steps Solution, Solvent injection, lipid hydration, reverse evapora used during synthesis. A variety of dendrimer sizes are Suit tion, or freeze drying by repeated freezing and thawing. In a able for use in the invention. Generally, the size of dendrimers preferred embodiment the liposomes are formed by sonica may range from about 1 nm to about 100 nm. tion. The liposomes may be multilamellar, which have many layers like an onion, or unilamellar. The liposomes may be II. Methods of Using Anti-14-3-3 Epsilon Antibodies large or Small. Continued high-shear Sonication tends to form Smaller unilamellar lipsomes. 0094. In an aspect, the present invention provides anti-14 0091. As would be apparent to one of ordinary skill, all of 3-3 epsilon antibodies to detect 14-3-3 epsilon in vitro and/or the parameters that govern liposome formation may be var in vivo. For example, anti-14-3-3 epsilon antibodies may be ied. These parameters include, but are not limited to, tempera used to detect and measure the amount of 14-3-3 epsilon in a ture, pH, concentration of methionine compound, concentra biological sample. Alternatively, anti-14-3-3 epsilon antibod tion and composition of lipid, concentration of multivalent ies may be used to detect and measure the amount of 14-3-3 cations, rate of mixing, presence of and concentration of epsilon in a Subject. In another aspect, the present invention solvent. provides anti-14-3-3 epsilon antibodies that can be used to 0092. In another embodiment, an antibody of the inven provide tumor specific delivery, for instance, of drugs, thera tion may be delivered to a cell as a microemulsion. Micro peutic agents or imaging agents as well as enhancing the emulsions are generally clear, thermodynamically stable efficacy of radiotherapy. In still another aspect, the present Solutions comprising an aqueous solution, a surfactant, and invention provides anti-14-3-3 epsilon antibodies that can be “oil.” The “oil in this case, is the Supercritical fluid phase. used to detect a tumor in a subject. The surfactant rests at the oil-water interface. Any of a variety of surfactants are Suitable for use in microemulsion formula (a) Methods to Detect and Measure the Amount of 14-3-3 tions including those described herein or otherwise known in Epsilon in a Biological Sample the art. The aqueous microdomains suitable for use in the invention generally will have characteristic structural dimen 0095. In an aspect, the invention provides means to detect sions from about 5 nm to about 100 nm. Aggregates of this 14-3-3 epsilon in a sample. In another aspect, the invention size are poor scatterers of visible light and hence, these solu provides means to measure the amount of 14-3-3 epsilon in a tions are optically clear. As will be appreciated by a skilled sample. The method generally comprises (i) obtaining a artisan, microemulsions can and will have a multitude of sample from, and (ii) detecting and/or measuring the amount different microscopic structures including sphere, rod, or disc of 14-3-3 epsilon in the sample using an antibody that spe shaped aggregates. In one embodiment, the structure may be cifically binds 14-3-3 epsilon. Suitable antibodies are micelles, which are the simplest microemulsion structures described above in Section I. The sample may be obtained that are generally spherical or cylindrical objects. Micelles from a subject (i.e. biological sample) or may be an immor are like drops of oil in water, and reverse micelles are like talized cell line. US 2016/0194388 A1 Jul. 7, 2016

0096. As used herein, the term “biological sample” refers tacting some of the sample, or all of the sample, comprising to a sample obtained from a Subject. Any biological sample 14-3-3 epsilon with an anti-14-3-3 epsilon antibody under containing 14-3-3 epsilon is Suitable. Numerous types of conditions effective to allow for formation of a complex biological samples are known in the art. Suitable biological between the antibody and the 14-3-3 epsilon protein. Typi sample may include, but are not limited to, tissue samples or cally, the entire sample is not needed, allowing one skilled in bodily fluids. In some embodiments, the sample may be a the art to repeatedly detect and measure the amount of 14-3-3 bodily fluid comprising a cell expressing 14-3-3 epsilon on epsilon in the sample. The method may occur in Solution, or the cell surface. Non-limiting examples of suitable bodily the antibody or 14-3-3 epsilon protein comprising the sample fluids include blood, plasma, serum, lung aspirate, pleural may be immobilized on a solid Surface. Non-limiting fluid, and sputum. The fluid may be used “as is”, the cellular components may be isolated from the fluid, or a protein examples of Suitable Surfaces may include microtitre plates, fraction may be isolated from the fluid using standard tech test tubes, slides, beads, resins, and other polymers. Attach niques. In preferred embodiments, the biological sample is a ment to the Substrate may occur in a wide variety of ways, as tissue sample Such as a tissue biopsy. The tissue biopsy may will be appreciated by those in the art. For example, the be a biopsy of tumor. The biopsied tissue may be fixed, substrate and the antibody may be derivatized with chemical embedded in paraffin or plastic, and sectioned, or the biopsied functional groups for Subsequent attachment of the two. For tissue may be frozen and cryosectioned. Alternatively, the example, the substrate may be derivatized with a chemical biopsied tissue may be processed into individual cells or an functional group including, but not limited to, amino groups, explant, or processed into a homogenate, a cell extract, a carboxyl groups, oxo groups or thiol groups. Using these membranous fraction, or a protein extract. The sample may functional groups, the antibody may be attached directly also be primary and/or transformed cell cultures derived from using the functional groups or indirectly using linkers. An tissue from the subject. anti-14-3-3 epsilon antibody may also be attached to the 0097 Suitable subjects include, but are not limited to, a Substrate non-covalently. For example, a biotinylated anti-14 human, a livestock animal, a companion animal, a lab animal, 3-3 epsilon antibody may be prepared, which may bind to and a Zoological animal. A subject may or may not be known Surfaces covalently coated with Streptavidin, resulting in to have a tumor comprising a cell expressing 14-3-3 epsilon attachment. Alternatively, an antibody may be synthesized on on the cell Surface. In one embodiment, the Subject may be a the Surface using techniques such as photopolymerization rodent, e.g. a mouse, a rat, a guinea pig, etc. In another and photolithography. embodiment, the subject may be a livestock animal. Non limiting examples of Suitable livestock animals may include 0101 Contacting the sample with an antibody under effec pigs, cows, horses, goats, sheep, llamas and alpacas. In yet tive conditions for a period of time sufficient to allow forma another embodiment, the Subject may be a companion ani tion of a complex generally involves adding the anti-14-3-3 mal. Non-limiting examples of companion animals may epsilon antibody composition to the sample and incubating include pets such as dogs, cats, rabbits, and birds. In yet the mixture for a period of time long enough for the anti-14 another embodiment, the Subject may be a Zoological animal. 3-3 epsilon antibody to bind to any antigen present. After this As used herein, a "Zoological animal' refers to an animal that time, the complex will be washed and the complex may be may be found in a Zoo. Such animals may include non-human detected and the amount measured by any method well primates, large cats, wolves, and bears. In a preferred embodi known in the art. Methods of detecting and measuring an ment, the animal is a laboratory animal. Non-limiting amount of an antibody-polypeptide complex are generally examples of a laboratory animal may include rodents, based on the detection of a label or marker. The term "label, canines, felines, and non-human primates. In another pre as used herein, refers to any Substance attached to an anti ferred embodiment, the Subject is a human. body, or other substrate material, in which the substance is 0098. As will be appreciated by a skilled artisan, the detectable by a detection method. Non-limiting examples of method of collecting a biological sample can and will vary Suitable labels include luminescent molecules, chemilumi depending upon the nature of the biological sample and the nescent molecules, fluorochromes, fluorescent quenching type of analysis to be performed. Any of a variety of methods agents, colored molecules, radioisotopes, Scintillants, biotin, generally known in the art may be utilized to collect a bio avidin, stretpavidin, protein A, protein G, antibodies or frag logical sample. Generally speaking, the method preferably ments thereof, polyhistidine, Ni, Flagtags, myctags, heavy maintains the integrity of the sample such that 14-3-3 epsilon metals, and enzymes (including alkaline phosphatase, peroxi can be accurately detected and the amount measured accord dase, glucose oxidase, and luciferase). Methods of detecting ing to the invention. and measuring an amount of an antibody-polypeptide com 0099. Once a sample is obtained, it is processed in vitro to plex based on the detection of a label or marker are well detect and measure the amount of 14-3-3 epsilon using an known in the art. anti-14-3-3 epsilon antibody. All suitable methods for detect 0102. In some embodiments, an antibody-based method is ing and measuring an amount of protein using an antibody an immunoassay. Immunoassays can be run in a number of known to one of skill in the art are contemplated within the different formats. Generally speaking, immunoassays can be Scope of the invention. Methods for detecting and measuring divided into two categories—competitive immunoassays and an amount of protein using an antibody (i.e. 'antibody-based non-competitive immunoassays. In a competitive immunoas methods”) are well known in the art. Non-limiting examples say, an unlabeled analyte in a sample competes with labeled include an ELISA, a sandwich immunoassay, a radioimmu analyte to bind an antibody. Unbound analyte is washed away noassay, an immunoblot or Western blot, flow cytometry, and the bound analyte is measured. In a non-competitive immunohistochemistry, and an array. immunoassay, the antibody is labeled, not the analyte. Non 0100. In general, an antibody-based method of detecting competitive immunoassays may use one antibody (e.g. the and measuring an amount of 14-3-3 epsilon comprises con capture antibody is labeled) or more than one antibody (e.g. at US 2016/0194388 A1 Jul. 7, 2016

least one capture antibody which is unlabeled and at least one complete remission in which all evidence of the tumor or “capping or detection antibody which is labeled.) Suitable cancer disappears. Desirably, the tumor or cancer does not labels are described above. reappear or reappears after at least 5, 10, 15, or 20 years. 0103) In other embodiments, an antibody-based method is 0107 An antibody of the present invention may be indi an immunoblot or Western blot. In yet other embodiments, an rectly or directly coupled to radionuclides or chemotherapeu antibody-based method is flow cytometry. tic agents as described above in order to provide specific 0104. In different embodiments, an antibody-based delivery of radiation and chemotherapy to the site of a tumor. method is immunohistochemistry (IHC). IHC uses an anti Further, the composition of the present invention may be part body to detect and quantify antigens in intact tissue samples. of a combination therapy. Preferably, a combination therapy The tissue samples may be fresh-frozen and/or formalin would include the use of the antibody of the present invention fixed, paraffin-embedded (or plastic-embedded) tissue blocks along with a radiation therapy or chemotherapy course of prepared for study by IHC. Methods of preparing tissue block treatment. It has also been suggested that antibody composi for study by IHC, as well as methods of performing IHC are tions, such as those described herein, may increase the Sus well known in the art. ceptibility of tumor cells to the effects of chemotherapy or 0105. In alternative embodiments, an antibody-based radiation. In preferred embodiments, the composition of the method is an array. An array comprises at least one address, invention may be used to enhance the efficacy of cancer wherein at least one address of the array has disposed thereon radiotherapy. an anti-14-3-3 epsilon antibody. Arrays may comprise from 0108. In yet another aspect, the present invention provides about 1 to about several hundred thousand addresses. Several a method of detecting a tumor in a subject. The method Substrates Suitable for the construction of arrays are known in comprises exposing a target area of the Subject where the the art, and one skilled in the art will appreciate that other presence of a tumor is suspected to ionizing radiation, admin Substrates may become available as the art progresses. Suit istering to the Subject a composition to detect the presence of able substrates are also described above. In some embodi 14-3-3 epsilon in the target area, wherein the composition ments, the array comprises at least one an anti-14-3-3 epsilon comprises one or more targeting antibodies, wherein each antibody attached to the substrate is located at one or more targeting antibody specifically binds to 14-3-3 epsilon spatially defined addresses of the array. For example, an array exposed on an irradiated cell and is conjugated to a detectable may comprise at least one, at least two, at least three, at least label, and detecting the detectable label to detect the presence four, or at least five anti-14-3-3 epsilon antibodies, each anti of 14-3-3 epsilon, wherein the presence of 14-3-3 epsilon body recognizing the same or different epitope 14-3-3 epsilon indicates the presence of a tumor in the target area of the epitope, and each antibody may be may beat one, two, three, subject. In preferred embodiments, the method may be used four, five, six, seven, eight, nine, ten or more spatially defined to diagnose or image a cancer in a Subject. In some embodi addresses. ments, a method for detecting a tumor can comprise (a) exposing a suspected tumor to ionizing radiation; (b) biops (b) Tumor Specific Delivery ing a suspected tumor; (c) contacting an antibody of the 0106. In another aspect, the present invention provides a invention with the Suspected tumor in vitro; and (d) detecting method of delivering a therapeutic agent to a cell expressing the detectable label, whereby a tumor is diagnosed. 14-3-3 epsilon. Accordingly, an antibody of the present 0109 Binding may be detected using microscopy (fluo invention, as described in Section I, may be used in treating, rescent microscopy, confocal microscopy, or electron micros stabilizing and preventing cancer and associated diseases in a copy), magnetic resonance imaging (including MTI, MRS, Subject. By “treating, stabilizing, or preventing cancer is DWI and fMRI), scintigraphic imaging (SPECT (Single Pho meant causing a reduction in the size of a tumor or in the ton Emission Computed Tomography), PET (Positron Emis number of cancer cells, slowing or preventing an increase in Sion Tomography), gamma camera imaging, and rectilinear the size of a tumor or cancer cell proliferation, increasing the scanning), radiography, or ultrasound. The detectable label disease-free survival time between the disappearance of a may be detectable in situ, in vivo, ex vivo, and in vitro. tumor or other cancer and its reappearance, preventing an 0110. The antibody compositions and subject are as initial or Subsequent occurrence of a tumor or other cancer, or described above. The cancer, the radiotherapy, and the admin reducing an adverse symptom associated with a tumor or istration of the compositions are described below. other cancer. The inventors have shown that an antibody of the 0111. An antibody of the invention may be used to treat or invention activates phagocytosis of cells bound by the anti recognize a tumor derived from a neoplasm or a cancer. The body thereby reducing the amount of cancer cells expressing neoplasm may be malignant or benign, the cancer may be 14-3-3 epsilon. In a desired embodiment, the percent of tumor primary or metastatic; the neoplasm or cancer may be early or cancerous cells Surviving the treatment is at least 20, 40, stage or late stage. Non-limiting examples of neoplasms or 60, 80, or 100% lower than the initial number of tumor or cancers that may be treated include acute lymphoblastic leu cancerous cells, as measured using any standard assay (e.g., kemia, acute myeloid leukemia, adrenocortical carcinoma, caspase assays, TUNEL and DNA fragmentation assays, cell AIDS-related cancers, AIDS-related lymphoma, anal cancer, permeability assays, and Annexin V assays). Desirably, the appendix cancer, astrocytomas (childhood cerebellar or cere decrease in the number of tumor or cancerous cells induced bral), basal cell carcinoma, bile duct cancer, bladder cancer, by administration of an antibody of the invention is at least 2, bone cancer, brainstem glioma, brain tumors (cerebellar 5, 10, 20, or 50-fold greater than the decrease in the number of astrocytoma, cerebral astrocytoma/malignant glioma, non-tumor or non-cancerous cells. Desirably, the methods of ependymoma, medulloblastoma, Supratentorial primitive the present invention result in a decrease of 20, 40, 60, 80, or neuroectodermal tumors, visual pathway and hypothalamic 100% in the size of a tumor or in the number of cancerous gliomas), breast cancer, bronchialadenomas/carcinoids, Bur cells, as determined using standard methods. Desirably, at kitt lymphoma, carcinoid tumors (childhood, gastrointesti least 20, 40, 60, 80,90, or 95% of the treated subjects have a nal), carcinoma of unknown primary, central nervous system US 2016/0194388 A1 Jul. 7, 2016

lymphoma (primary), cerebellar astrocytoma, cerebral astro pelvis transitional cell cancer, urethral cancer, uterine cancer cytoma/malignant glioma, cervical cancer, childhood can (endometrial), uterine sarcoma, vaginal cancer, visual path cers, chronic lymphocytic leukemia, chronic myelogenous way and hypothalamic glioma (childhood), Vulvar cancer, leukemia, chronic myeloproliferative disorders, colon can Waldenström macroglobulinemia, and Wilms tumor (child cer, cutaneous T-cell lymphoma, desmoplastic Small round hood). In specific embodiments, the neoplasm or cancer is cell tumor, endometrial cancer, ependymoma, esophageal non-Small cell lung carcinoma. cancer, Ewings sarcoma in the Ewing family of tumors, 0112 Suitable therapeutic agents for neoplasms and can extracranial germ cell tumor (childhood), extragonadal germ cers are known in the art, and will depend upon the type and cell tumor, extrahepatic bile duct cancer, eye cancers (in stage of cancer. Suitable therapeutic agents are described in traocular melanoma, retinoblastoma), gallbladder cancer, Section I. Summaries of cancer drugs, including information gastric (stomach) cancer, gastrointestinal carcinoid tumor, regarding approved indications, may be found via the gastrointestinal Stromal tumor, germ cell tumors (childhood National Cancer Institute at the National Insitutes of Health extracranial, extragonadal, ovarian), gestational trophoblas (www.cancer.gov/cancertopics/druginfo/alphalist) and the tic tumor, gliomas (adult, childhood brain stem, childhood FDA Approved Drug Product database (www.accessdata.fda. cerebral astrocytoma, childhood visual pathway and hypotha gov/scripts/cder/drugsatfla?). In a specific embodiment, the lamic), gastric carcinoid, hairy cell leukemia, head and neck cancer is non-Small cell lung carcinoma. Suitable therapeu cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, tics agents for the treatment of non-Small cell lung carcinoma hypopharyngeal cancer, hypothalamic and visual pathway include, but are not limited to, EGFR inihibitors, VEGF glioma (childhood), intraocular melanoma, islet cell carci inhibitors, tyrosine kinase inhibitors, and chemotherapeutics. noma, Kaposi sarcoma, kidney cancer (renal cell cancer), Non-limiting examples of drugs approved for the treatment of laryngeal cancer, leukemias (acute lymphoblastic, acute NSCLC include methotrexate, Abraxane, Afatinib, Alimta, myeloid, chronic lymphocytic, chronic myelogenous, hairy Avastin, Bevacizumab, Carboplatin, Cisplatin, Crizotinib, cell), lip and oral cavity cancer, liver cancer (primary), lung Erlotinib hydrochloride, Folex, Folex PFS, gefitinib, Gilotrif, cancers (non-Small cell. Small cell), lymphomas (AIDS-re Gemcitabine hydrochloride, Gemzar, Iressa, Methotrexate, lated, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, pri Mexate, Mexate-AQ, Paclitaxel, Paclitaxel-albumin stabi mary central nervous system), macroglobulinemia (Walden lized nanoparticle formulation, Paraplat, Paraplatin, Platinol, ström), malignant fibrous histiocytoma of bone/ Tarceva, Taxol, and Xalkori. osteosarcoma, medulloblastoma (childhood), melanoma, 0113. In an aspect, the method comprises exposing a target intraocular melanoma, Merkel cell carcinoma, mesothelio area of a subject where the presence of a tumor is suspected to mas (adult malignant, childhood), metastatic squamous neck ionizing radiation. Low doses of radiation can be used for cancer with occult primary, mouth cancer, multiple endocrine selective targeting using the antibody compositions disclosed neoplasia syndrome (childhood), multiple myeloma/plasma herein. In some embodiments, the dose of radiation com cell neoplasm, mycosis fungoides, myelodysplastic Syn prises up to about 2 Gy ionizing radiation. Higher radiation dromes, myelodysplastic/myeloproliferative diseases, myel doses can also be used, especially in the case of local radiation ogenous leukemia (chronic), myeloid leukemias (adult acute, treatment as described hereinbelow. childhood acute), multiple myeloma, myeloproliferative dis 0114 Radiation can be localized to a tumor using confor orders (chronic), nasal cavity and paranasal sinus cancer, mal irradiation, brachytherapy, or stereotactic irradiation. nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin The threshold dose for inductive changes can thereby be lymphoma, non-Small cell lung cancer, oral cancer, oropha exceeded in the target tissue but avoided in Surrounding nor ryngeal cancer, osteosarcoma/malignant fibrous histiocy mal tissues. A “target tissue' as used herein refers to an toma of bone, ovarian cancer, ovarian epithelial cancer (Sur intended site for accumulation of an antibody following face epithelial-stromal tumor), ovarian germ cell tumor, administration to a subject. For example, the methods dis ovarian low malignant potential tumor, pancreatic cancer, closed herein can employ a target tissue comprising an irra pancreatic cancer (islet cell), paranasal sinus and nasal cavity diated tumor. A “control tissue' as used herein refers to a site cancer, parathyroid cancer, penile cancer, pharyngeal cancer, Suspected to Substantially lackbinding and/or accumulation pheochromocytoma, pineal astrocytoma, pineal germinoma, ofan administered antibody. For example, in accordance with pineoblastoma and Supratentorial primitive neuroectodermal the methods of the presently disclosed subject matter, a non tumors (childhood), pituitary adenoma, plasma cell neopla irradiated tumor and a non-cancerous tissue are control tis sia, pleuropulmonary blastoma, primary central nervous sys Sues. In some embodiments, doses of at least about 2 Gy tem lymphoma, prostate cancer, rectal cancer, renal cell car ionizing radiation can be used, and in some embodiments a cinoma (kidney cancer), renal pelvis and ureter transitional dose of about 10 Gy to about 20 Gy ionizing radiation can be cell cancer, retinoblastoma, rhabdomyosarcoma (childhood), used. For treatment of a Subject having two or more tumors, salivary gland cancer, sarcoma (Ewing family of tumors, local irradiation enables differential drug administration and/ Kaposi, soft tissue, uterine), Sézary syndrome, skin cancers or dose at each of the two or more tumors. Alternatively, (nonmelanoma, melanoma), skin carcinoma (Merkel cell), whole body irradiation can be used, as permitted by the low Small cell lung cancer, Small intestine cancer, Soft tissue sar doses of radiation required for targeting of antibodies dis coma, squamous cell carcinoma, squamous neck cancer with closed herein. Radiotherapy methods suitable for use in the occult primary (metastatic), stomach cancer, Supratentorial practice of the presently disclosed Subject matter can be primitive neuroectodermal tumor (childhood), T-Cell lym found in Leibel & Phillips, 1998, among other sources. phoma (cutaneous), testicular cancer, throat cancer, thymoma 0.115. In an embodiment, the radiation treatment com (childhood), thymoma and thymic carcinoma, thyroid cancer, prises administration of less than about 2 Gy ionizing radia thyroid cancer (childhood), transitional cell cancer of the tion. In another embodiment, the radiation treatment com renal pelvis and ureter, trophoblastic tumor (gestational), prises at least about 2 Gy ionizing radiation, in some enknown primary site (adult, childhood), ureter and renal embodiments about 2 Gy to about 3 Gy ionizing radiation, US 2016/0194388 A1 Jul. 7, 2016

and in some embodiments about 2 Gy to about 6 Gy ionizing made microbiologically acceptable. A typical composition radiation. In other embodiments, radiation treatment com for intravenous infusion could have volumes between 1-250 prises about 10 Gy to about 20 Gy ionizing radiation. mL of fluid, such as sterile Ringer's solution, and 1-100 mg 0116 Administration of a composition to a subject can be per ml, or more in anti-14-3-3 episilon antibody concentra performed by irradiating the tumor prior to, concurrent with, tion. Therapeutic agents of the discovery can be frozen or or Subsequent to administration of a composition of the inven lyophilized for storage and reconstituted in a suitable sterile tion. Accordingly, the tumor is irradiated in Some embodi carrier prior to use. Lyophilization and reconstitution may ments 0 hours to about 24 hours before administration of the lead to varying degrees of antibody activity loss (e.g. with composition, and in Some embodiments about 4 hours to conventional immune globulins, IgM antibodies tend to have about 24 hours before administration of the composition. greater activity loss than IgG antibodies). Dosages adminis 0117. In certain aspects, a pharmacologically effective tered are effective dosages and may have to be adjusted to amount of an antibody of the invention, including immuno compensate. The pH of the formulations generally pharma logically reactive fragments, may be administered to a Sub ceutical grade quality, will be selected to balance antibody ject. Administration is performed using standard effective stability (chemical and physical) and comfort to the patient techniques, including peripherally (i.e. not by administration when administered. Generally, a pH between 4 and 8 is tol into the central nervous system) or locally to the central erated. Doses will vary from individual to individual based on nervous system. Peripheral administration includes but is not size, weight, and other physiobiological characteristics of the limited to intravenous, intraperitoneal. Subcutaneous, pulmo individual receiving the Successful administration. nary, transdermal, intramuscular, intranasal, buccal, Sublin I0121 For therapeutic applications, a therapeutically gual, or Suppository administration. Local administration, effective amount of a composition of the invention is admin including directly into the central nervous system (CNS) istered to a subject. A “therapeutically effective amount is an includes but is not limited to via a lumbar, intraventricular or amount of the therapeutic composition Sufficient to produce a intraparenchymal catheter or using a Surgically implanted measurable biological tumor response (e.g., an immuno controlled release formulation. stimulatory, an anti-angiogenic response, a cytotoxic 0118 Pharmaceutical compositions for effective adminis response, or tumor regression). Actual dosage levels of active tration are deliberately designed to be appropriate for the ingredients in a therapeutic composition of the invention can selected mode of administration, and pharmaceutically be varied so as to administer an amount of the active com acceptable excipients such as compatible dispersing agents, pound(s) that is effective to achieve the desired therapeutic buffers, surfactants, preservatives, solubilizing agents, isoto response for a particular subject. The selected dosage level nicity agents, stabilizing agents and the like are used as appro will depend upon a variety of factors including the activity of priate. Remington’s Pharmaceutical Sciences, Mack Publish the therapeutic composition, formulation, the route of admin ing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest istration, combination with other drugs or treatments, tumor edition, incorporated herein by reference in its entirety, pro size and longevity, and the physical condition and prior medi vides a compendium of formulation techniques as are gener cal history of the Subject being treated. In some embodiments, ally known to practitioners. It may be particularly useful to a minimal dose is administered, and dose is escalated in the alter the solubility characteristics of the antibodies useful in absence of dose-limiting toxicity. Determination and adjust this discovery, making them more lipophilic, for example, by ment of a therapeutically effective dose, as well as evaluation encapsulating them in liposomes or by blocking polar groups. of when and how to make such adjustments, are known to 0119 Effective peripheral systemic delivery by intrave those of ordinary skill in the art of medicine. In an aspect, a nous or intraperitoneal or Subcutaneous injection is a pre typical dose contains from about 0.01 mg/kg to about 100 ferred method of administration to a living patient. Suitable mg/kg of an anti-14-3-3 epsilon antibody described herein. vehicles for Such injections are straightforward. In addition, Doses can range from about 0.05 mg/kg to about 50 mg/kg, however, administration may also be effected through the more preferably from about 0.1 mg/kg to about 25 mg/kg. mucosal membranes by means of nasal aerosols or Supposi 0.122 For diagnostic applications, a detectable amount of tories. Suitable formulations for such modes of administra a composition of the invention is administered to a subject. A tion are well known and typically include Surfactants that “detectable amount’, as used herein to refer to a diagnostic facilitate cross-membrane transfer. Such Surfactants are often composition, refers to a dose of Such a composition that the derived from steroids or are cationic lipids, such as N-1-(2, presence of the composition can be determined in vivo or in 3-dioleoyl)propyl-N,N.N-trimethyl ammonium chloride vitro. A detectable amount will vary according to a variety of (DOTMA) or various compounds such as cholesterol factors, including but not limited to chemical features of the hemisuccinate, phosphatidylglycerols and the like. drug being labeled, the detectable label, labeling methods, the 0120. The concentration of antibody informulations to be method of imaging and parameters related thereto, metabo administered is an effective amountand ranges from as low as lism of the labeled drug in the subject, the stability of the label about 0.1% by weight to as much as about 15 or about 20% by (e.g. the half-life of a radionuclide label), the time elapsed weight and will be selected primarily based on fluid volumes, following administration of the drug and/or labeled antibody Viscosities, and so forth, in accordance with the particular prior to imaging, the route of drug administration, the physi mode of administration selected if desired. A typical compo cal condition and prior medical history of the Subject, and the sition for injection to a living patient could be made up to size and longevity of the tumor or Suspected tumor. Thus, a contain 1 mL sterile buffered water of phosphate buffered detectable amount can vary and can be tailored to a particular saline and about 1-1000 mg of any one of or a combination of application. After study of the present disclosure, and in par the humanized antibody of the present discovery. In a specific ticular the Examples, it is within the skill of one in the art to embodiment, the antibody composition may have 100-300 determine Such a detectable amount. mg of antibody per administration. The formulation could be I0123. The frequency of dosing may be daily or once, sterile filtered after making the formulation, or otherwise twice, three times or more per week or per month, as needed US 2016/0194388 A1 Jul. 7, 2016 16 as to effectively treat the symptoms. The timing of adminis 0.126 Typical dosage levels can be determined and opti tration of the treatment relative to the disease itself and dura mized using standard clinical techniques and will be depen tion of treatment will be determined by the circumstances dent on the mode of administration. Surrounding the case. Treatment could begin immediately, Such as at the site of the injury as administered by emergency III. Kits medical personnel. Treatment could begin in a hospital or I0127. In another aspect, a kit is provided for use in diag clinic itself, or at a later time after discharge from the hospital nostic or therapeutic embodiments of the invention. The kit or after being seen in an outpatient clinic. Duration of treat includes an antibody of the invention, and a detectable label, ment could range from a single dose administered on a one therapeutic agent, chelating agent, and/or a linker, as time basis to a life-long course of therapeutic treatments. described in Section I. In an embodiment, each component of the kit (an antibody and a detectable label, therapeutic agent, 0124. Although the foregoing methods appear the most chelating agent, and/or linker) is separately packaged in the convenient and most appropriate and effective for adminis kit. In another preferred embodiment, the kit includes a pre tration of proteins such as antibodies, by Suitable adaptation, determined amount of the antibody and the detectable label, other effective techniques for administration, Such as intra therapeutic agent, chelating agent, and/or linker (e.g., an Ventricular administration, transdermal administration and amount Sufficient for diagnosing or treating cancer in a Sub oral administration may be employed provided properformu ject). The antibody and the detectable label, therapeutic lation is utilized herein. agent, chelating agent, and/or linker can be lyophilized to 0.125. In addition, it may be desirable to employ controlled enable long-term storage. The antibody and detectable label, release formulations using biodegradable films and matrices, therapeutic agent, chelating agent, and/or linker may be or osmotic mini-pumps, or delivery systems based on dextran sealed in a sterilized container. The kit preferably includes beads, alginate, or collagen. instructions for using the kit and its contents. TABL E B Sequence listincts

SEQ ID NO: Description SEQUENCE 1 Light chain: DNA GACATTGTGATGACACAGTCTCCATCCTCCCTGGCTATGTCAGTAGG sequence ACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTAAATA GTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGA CAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGG GGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTC TTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGT CAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCT GGAGCTGAAA

2 Heavy chain: DNA CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGC sequence CTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCT ACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGG ATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAACTACAATGAGAA GTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAG CCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTAT TACTGTGCAAGATCGGTATGGTTACGACGTGATTTTGCTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCA

3 Light chain: AA DIVMTOSPSSLAMSVGOKVTMSCKSSOSLLNSSNOKNYLAWYOOKPG sequence OSPKLLWYFASTRESGVPDRFIGSGSGTDFTL TISSVOAEDLADYFC OOHYSTPLTFGAGTKLELK 4. Heavy chain: AA OVOLOOSGAELMKPGASVKISCKATGYTFSSYWIEWWKORPGHGLEW sequence IGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMOLSSLTSEDSAVY YCARSVWLRRDFAYWGOGTLVTVSA 5 Light chain : CDR1 KSSQSLLNSSNOKNYLA 6 Light chain : CDR2 FASTRES 7 Light chain : CDR3 OOHYSTPLT 8 Heavy chain : CDR1 GYTFSSYWIE 9 Heavy chain : CDR2 EILPGSGSTNYNEKFKG 1O Heavy chain : CDR3 SWWLRRDFAY 11 Human 14-3-3 epsilon: MDDREDLVYOAKLAEQAERYDEMVESMKKVAGMDVELTVEERNLLSV isoform 1 AYKNVIGARRASWRIISSIEOKEENKGGEDKLKMIREYROMVETELK LICCDILDWLDKHLIPAANTGESKWFYYKMKGDYHRYLAEFATGNDR KEAAENSLWAYKAASDIAMTELPPTHPIRLGLALNFSWFYYEILNSP DRACRLAKAAFDDAIAELDTLSEESYKDSTLIMOLLRDNLTLWTSDM QGDGEEONKEALODWEDENO US 2016/0194388 A1 Jul. 7, 2016 17

TABLE B- continued Sequence listings

SEQ ID NO : Description SEQUENCE

12 Human 14-3-3 epsilon: MVESMKKVAGMDWELTVEERNLLSVAYKNVIGARRASWRIISSIEOK isoform SW EENKGGEDKLKMIREYROMVETELKLICCDILDVLDKHLIPAANTGE SKWFYYKMKGDYHRYLAEFATGNDRKEAAENSLWAYKAASDIAMTEL PPTHPIRLGLALNFSWFYYEILNSPDRACRLAKAAFDDAIAELDTLS EESYKDSTLIMOLLRDNLTLWTSDMQGDGEEONKEALODWEDENO 13 Epitope 1 FSWFYYEILNSPDRACRL

14 Epitope 2 HPIRLGLALNFSWFYYEI 15 Epitope 3 FSWFYYEILN

16 Heavy chain: AA MEWTWVFLFLLSVTAGVHSOVOLOOSGAELMKPGASVKISCKATGYT sequence with leader FSSYWIEWWKORPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTS SNTAYMOLSSLTSEDSAVYYCARSVWLRRDFAYWGOGTLWTVSA

17 Light chain: AA MESOTOVLMFL LLWVSGACADIVMTOSPSSLAMSVGOKVTMSCKSSO sequence with leader SLLNSSNOKNYLAWYOOKPGOSPKLLWYFASTRESGVPDRFIGSGSG TDFTLTISSVOAEDLADYFCOOHYSTPLTFGAGTKLELK

Examples cases and 159.260 deaths from lung cancer of non-small cell and Small cell combined. Among all lung cancer cases, 0128. The following examples are included to demon NSCLC is the most common type, with about 85%-90% of all strate preferred embodiments of the invention. It should be cases, and is commonly non-responsive to chemotherapy. appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques I0131 We set out to find out an antibody for use on irradi discovered by the inventors to function well in the practice of ated tumor cells. The neo-antigen discussed herein was dis the invention. Those of skill in the art should, however, in light covered and prioritized after radiation treatment of H23 cells of the present disclosure, appreciate that many changes can be by Subtractive immunization. Subtractive immunization is an approach to generate monoclonal antibodies (McAb) against made in the specific embodiments that are disclosed and still poorly immunogenic or rare antigens. Two steps are involved obtain a like or similar result without departing from the spirit in this process. The first step involves tolerization where the and scope of the invention, therefore all matter set forth or mice are immunized by normal sets of antigen after which the shown in the accompanying examples and drawings is to be mice are immune-suppressed by cyclophosphamide. After interpreted as illustrative and not in a limiting sense. immune-suppression, the mice are immunized a second time to generate antibodies against rare antigens. This study used Introduction to the Examples irradiated human lung cancer cells to create neoantigens and 0129. Ionizing radiation (IR) is a commonly employed then inoculated these neoantigens into the mice. Following treatment method for many types of human cancers. Targeted boosts with irradiated tumor cells, B cells from spleens were therapy can result in effective treatment of cancer without the harvested and hybridomas were created by fusion with systemic side effects of radiation or chemotherapy treatment myeloma cells. The hybridomas were screened with irradi alone, however remains one of the biggest challenges to ated human lung cancer cells in a high throughput Screening develop. Due to tumor heterogeneity, not all cancers respond method. After the positive clones were identified, clones of to a particular treatment regimen with similar efficacy. Thus, antibodies were purified and evaluated by binding to irradi there is an urgent need to develop targeted therapies that are ated tumor cells, immune response and characterization of the not only highly specific to cancer but also have minimal neo-antigen. toxicity towards normal tissues Surrounding tumors. IR treat 0.132. In this study, one antibody, 7D4, bound best to the ment can achieve cell killing through DNA strand breaks and irradiated H23 lung cancer cells in vitro through immunohis can illicit phenotypic changes in the cells resulting in mol tochemistry, IF, FACS and in vivo by mouse imaging. An ecules being expressed on the Surface of tumor cells. These immune response was elicited in vitro by mouse dendritic cell molecules are termed neo-antigens or radiation inducible pro (MDC) with phagocytosis of irradiated H23 tumor cells in teins. In addition, a limited number of tumor associated anti combination with 7D4 antibody treatment. The dendritic cells gens presently serve as targets for radio-immunotherapy. As engulfed the tumor cells by the receptors to the Fc domain of Such these radiation-inducible antigens can serve as potential the antibody so that the MDC could more readily bind to the targets for therapeutic antibodies, radio-immunotherapy, and antibody and opSonize the tumor. In an in vivo study, mice targeted drug delivery molecules that specifically target the were injected IV with the candidate 7D4 antibody, radiated tumor. The elimination of cancer with antibodies is based tumor cells and MDC to check for interferon gamma level upon recruitment of host effector mechanisms either through (INF-Y) through isolated lymphocytes from spleen. IFN complement activation or Fc-receptor dependent responses. gamma is a cytokine that is critical for adaptive and innate 0130 Lung cancer is a leading cause of death worldwide. immune response against viruses, tumors and indicate T cell In the United States in 2014, the NCI estimated 224,210 new activation against the tumor cells. US 2016/0194388 A1 Jul. 7, 2016

0133. The antigen bound by antibody 7D4 was character treated tumor cells compared with OGy sham H23 tumor ized and identified to be approximately 27 kDa by SDS cells. In the OGy sham H23 tumor cells IgM was absent in PAGE and Western-blot after radiation treatment of the tumor FMAT assay. Additionally, the intensity of 3Gyx3 treated cells. The antigen was proteomics sequenced and the result H23 cells was much higher compared with OGy sham H23 was shown to be a 14-3-3 epsilon protein. IHC was used to cells in the Dot-blot assay (FIG. 8B). confirm the binding on the treated H23 tumor cell. The 14-3-3 0.138. The 7D4 hybridoma clone was cultured in complete epsilon protein is an adaptor protein implicated in the regu FBS medium with all supplements. The cells were harvested, lation of a large spectrum of both general and specialized counted and then frozen in hybridoma freezing medium (90% signaling pathway. It binds to a large number of partners, FBS+10% DMSO) in 2x10/vial. The cells were shipped to usually by recognition of a phosphoserine or phosphothreo Genscript for antibody binding site sequence. The antibody nine motif. The binding generally results in the modulation of binding site amino acid sequence in variable heavy and light the activity of the binding partner. 14-3-3 epsilon coordinates chains with CDR regions are as in the following: the cross talk between the MAPK signal module and other molecular pathways/biological processes primarily including Heavy chain: Amino acids sequence (138 AA) protein metabolism and synthesis, DNA repair, and cell cycle Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 regulation. (SEQ ID NO: 16) MEWTWWFL FLILSVTAGVHSQVOLOOSGAELMKPGASVKISCKATGYTF Example 1 SSYWIEWWKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSN Generation of the 7D4 Monoclonal Antibody TAYMQLSSLTSEDSAVYYCARSWWLRRDFAYWGQGTLWTVSA 0134. Irradiation of cancer elicits a stress response with Light chain: Amino acids sequence (133 AA) transport of stored proteins to the Surface of cancer cells. Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 These inducible Surface proteins can be exploited as targets (SEO ID NO : 17) for antibody based immunotherapy to destroy cancer cells. In MESQTOVLMFLLLWVSGACADIVMTOSPSSLAMSVGQKVTMSCKSSQS order to develop monoclonal antibodies (McAb) targeted to radiation-inducible neoantigens, Subtractive immunization LLNSSNOKNYLAWYOOKPGOSPKLLWYFASTRESGVPDRFIGSGSGTD was employed to produce antibodies against Surface proteins FTLTISSWQAEDLADYFCQQHYSTPLTFGAGTKLELK on irradiated lung cancer tumor model H23. TABLE 1.

Mouse immunization schedule. Mouse strain: BALB, C or AJ

Cell line H23 H23 H23 - H460 - HISO H23 - H460 - HS2O IR treatment Time interval 0, 6, 24h 2h 2 h, cell mixture 0, 6, 24h, cell mixture 2 x 10°/IP 2 x 106/IP 2 x 10/IP 2 x 10/IP

0135 0Gy or 3Gyx3 irradiated H23; H460 and H520 cells TABLE 2 were used as antigen for Subtractive immunization. Cyclo phosphamide was used as an immuno-Suppressing reagent. Days of The mice with high immune response to irradiated cancer Tumor cell IRTreatment Immunization Positive 1000 cells cells were chosen for cell fusion. The hybridomas were Samples of BALB/C mouse “L” screened using both cells by FMAT and Dot-blot assays. H23 3 Gy/2 h 40 5 0.136 Specifically, H23 lung tumor cells were cultured in 56 145 384 well plates and were either sham irradiated or irradiated 70 26S as described in Table 1. There were 1x10" cells per well. PBS H23 3Gy x 3/0, 6, 24h 40 10 56 200 diluted mouse sera was added to the 384 microtiter plate 70 235 wells. The cells and the sera were then incubated at 37°C. in H23 O Gy 40 10 a CO incubator for 2 hours. The plates were then treated with 56 115 goatanti-mouse IgGFc conjugated to Alexafluor 647 dye and 70 120 goat anti-mouse IgM conjugated to Alexafluor 680 dye. The Sample of AJ mouse “O'” cells were then incubated overnight and were read by FMAT H23 3 Gy/2 h 40 40 system (Model 8100). The results of immunized mice serum 56 250 against H23 tumor cells showed that positive cells increased 70 260 at day 56 and were highest at day 70 compared to day 40, H23 3Gy x 3/0, 6, 24h 40 25 which was the first boost with irradiated tumor cells (Table 2). 56 210 70 245 0.137 After 15 days of cell fusion, the hybridoma super H23 O Gy 40 50 natants in culture plates were harvested and screened with 56 125 FMAT and Dot-blot assays. The results indicated that hybri 70 135 doma Supernatants contained mouse IgG (green) and IgM (blue) (FIG.8A). Clone 7D4 had high binding to H233Gyx3 US 2016/0194388 A1 Jul. 7, 2016

Example 2 isoform 1 (FIG. 1), and with light to medium absorbance (moderate binding) to a peptide comprising amino acids 145 Antigen Identification and Purification 162 (HPIRLGLALNFSVFYYEI:SEQID NO:14) of 14-3-3 epsilon SV (FIG.2). There is 10 amino acid shared homology 0139 H23 cells were grown and irradiated as indicated (FSVFYYEILN; SEQID NO:15) in both recognized amino and cell lysates were prepared as the Source of antigen. The acid sequences. The data also indicated that purified anti same amount of cell lysate was loaded on each lane. The protein was separated by SDS-PAGE and transferred to a NC 14-3-3 epsilon 7D4 antibody is a mouse IgG1 sub-class. membrane. Western-blot assay was used for analysis of the Example 4 inducible protein in cell lysates. Three bands can be observed located around 27 kDa. The intensities of bands increased Anti-14-3-3 Epsilon Antibody Displays Specificity from 0Gy to 3Gyx1 (FIG. 9). for Irradiated Cancer Cells 0140 IR or sham 0Gy treated H23 cell lysates were pre pared and specific protein was captured by a 7D4 affinity 0145 H23 human lung cancer cells were irradiated with column. Eluted protein from the column was dialyzed against 3Gyx3. Cells were treated with 4% paraformaldehyde and PBS pH 7.2 and concentrated. SDS-PAGE and Western-blot rehydrated. Cells were then treated with blocking buffer fol assay was then performed using the 7D4 McAb. FIG. 10 lowed by the addition of the 7D4 mouse monoclonal antibody shows that the intensity of bands recognized by 7D4 McAb. to 14-3-3 epsilon or addition of a commercial rabbit anti The 3Gyx3 treated H23 cells had a higher intensity band human 14-3-3 epsilon antibody purchased from Bio-Legend compared with other treatments. The band around 27Kda was Company. Cells were then washed and incubated with the cut out and sent to Mass-Spectrometry Research Center for secondary antibody conjugated to HRP Cells were then proteomics analysis of the protein. It was reported that the stained with DAB and counterstained with Hematoxylin. affinity purified proteinband from is a 14-3-3 protein epsilon. 0146 Shown in FIG. 3A is the immunocytochemistry of It is composed of 255 amino acids and is coded by a the commercial antibody binding to human lung cancer cell GN=YWHAE, AC=P62258 OS=Homosapiens (human). The H23 following irradiation. Upon incubation with the com full protein sequence is as following: MDDREDLVYO mercial antibody, intensity of staining was higher in the AKLAEQAERY DEMVESMKKV AGMDVELTVE ERN 3Gyx3 treatment group as compared to the 3Gyx 1 treatment LLSVAYKNVIGARRASW RIISSIEQKEENKGGEDKLK group. However, background staining in the OGy treatment MIREYRQMVE TELKLICCDI LDVLDKHLIP AANT group was observed. Shown in FIG. 3B is the immunocy GESKVF YYKMKGDYHRYLAEFATGND RKEAAEN tochemistry of 7D4 antibody binding to human lung cancer SLV AYKAASDIAM TELPPTHPIR LGLALNFSVF cell H23 following irradiation in comparison with the com YYEILNSPDRACRLAKAAFD DAIAELDTLSEESYKD mercial antibody. These data show that the 7D4 mouse mono STLI MQLLRDNLTL WTSDMQGDGE EQNKEALQDV clonal antibody binds to lung cancer cells following treatment EDENQ (SEQ ID NO:11). with radiation with minimal binding to cells that were not 0141. In summary, Western immunoblot showed a specific treated with radiation (lower panel). The commercial anti band with molecular weight of about 27 kDa using this anti body to 14-3-3 was used as a positive control and shown in the body. The intensity of this band increased with the dosage of center panels. This also showed that the anti-14-3-3 epsilon irradiation on H23 and highest intensity occurred in 3Gyx3. antibody binds following irradiation, however, there is also Proteomics analysis revealed that the 27 kDa molecule is non-specific binding in the absence of irradiation. The nega 14-3-3 epsilon. tive control was secondary antibody that showed no increased binding to H23 cells without irradiation. Example 3 0147 To determine normal tissue cross reactivity, anti body binding to normal tissues was evaluated. Antibody bind 14-3-3 Protein Epsilon Epitope Mapping ing to colon, liver, lung, pancreas, kidney and tongue was examined (FIG. 4A, FIG. 4B). These immunohistochemistry 0142. The human 14-3-3 epsilon epitope and human experiments showed that the 7D4 mouse monoclonal anti 14-3-3 epsilon isoform SV epitope recognized by antibody body to 14-3-3 epsilon showed no binding to colon, liver, 7D4 was determined by epitope mapping using peptides from pancreas, kidney or tongue. There was minimal binding to the whole human 14-3-3 protein epsilon sequence (Sigma Ald lung. In comparison, the isotype control on secondary anti rich). These peptides span the full length of human 14-3-3 bodies showed no binding to any organ. epsilon isoform 1 and 14-3-3 epsilon isoform SV. Each pep 0148 H23 cells were treated with radiation, 3Gyx3 or 0Gy tide contains 18 amino acids from N-terminal to C-terminal. sham. Cells were then treated with blocking buffer followed Two amino acids overlap in each peptide, for a total of 31 by the addition of the 7D4 anti-14-3-3 epsilon monoclonal peptides. antibody. Cells were then washed and stained with the sec 0143. Two batches of purified anti 14-3-3 epsilon mono ondary antibody conjugated to the fluorescent marker Alexa clonal antibody 7D4 were used for testing all 31 peptides by Fluor 488. Commercial rabbit anti-human 14-3-3 epsilon ELISA. Three concentrations of each peptide had been used antibody was used as positive control. Negative controls for coating ELISA plate at 30, 15, 10 ug/mL, 50 ul/each well included 7D4 without secondary antibody and normal mouse were reacted against diluted purified antibodies at same con IgG with secondary antibody. Shown in FIG. 5 is the percent centration in all assay. Similar results were obtained for each age of cells staining positive with the anti-14-3-3 antibodies peptide concentration coated on the ELISA plate. as measured by flow cytometry (FACS). Cells treated with 0144. The epitope mapping experiments showed that the 3Gyx3 showed a 20 fold increase in the percentage of cells monoclonal antibody reacted with high absorbance (strong staining positive with the 7D4 antibody following irradiation. binding) to a peptide comprising amino acids 177-194 (FSV In comparison, only 4% of cells showed positive staining with FYYEILNSPDRACRL: SEQ ID NO:13) of 14-3-3 epsilon no irradiation. The commercial antibody showed less radia US 2016/0194388 A1 Jul. 7, 2016 20 tion induced binding as compared to the 7D4 monoclonal. ogy, Ltd.). The ELISPOT shows that lymphocytes from mice Control antibodies included isotype control and secondary injected with irradiated H23 tumor cells cultured with mouse antibody following irradiation which showed no increase in DCs and 7D4 monoclonal antibody to 14-3-3 showed the antibody binding. greatest amount of IFN-y production (FIG. 7A). FIG. 7B 0149. To further evaluate specificity of 7D4 for irradiated shows the quantification of interferon production. The num tumor cells, radiation treated tumor cells were harvested, ber of cells producing interferon-gamma were counted. Cells mounted on slides, and then fixed with 4% paraformalde treated with 3Gyx3 followed by the 7D4 mouse monoclonal hyde. The slides were stained with purified 7D4 monoclonal to 14-3-3 and the mouse dendritic cells showed the greatest antibody followed by a secondary goat anti-mouse IgG Fc number of T cell activation (Group 1). In comparison, cells conjugated to HRP Substrate DAB was used for IHC or Alexa treated with radiation and antibody alone without dendritic fluro 594 conjugated antibody was used for IF. IHC results cells showed a modest T cell activation (Group 2). Likewise, indicated that purified 7D4 antibody reacted with H233Gyx3 cells treated with radiation and isotype control antibody irradiated cells as high intensity brown color was observed showed modest T cell activation (Group 3). Negative controls (FIG. 11A). Minimum or negative signals were observed on showed minimal T cell activation (Group 4 and Group 5). sham 0Gy H23 cells. The same results were observed in the IF These findings indicate that the 7D4 anti-14-3-3 epsilon anti assay as indicated by red staining in FIG. 11B. Antibody 7D4 body can activate phagocytosis and human lung cancer anti strongly reacted with 3Gyx3 H23 cells while only minor gen presentation to T cells. staining on OGy H23 cells was observed. The controls were 0153. In summary, ADCP and ELISPOT indicated that the negative. 3Gyx 1 H23 tumor cells were positive but the inten antibody binds to irradiated H23 cells co-cultured with acti sity was less than 3Gyx3 H23 tumor cells (FIG. 11B). vated MDC, the phagocytic events increased with increasing 0150 Taken together, these results show that monoclonal irradiation dosage while OGy or control antibody was absent. antibody 7D4 showed high specificity and affinity to irradi Moreover, the IFN-Y levels on ELISPOT increased with the ated H23 cells. The intensity of binding on tumor cells irradiation dosages and highest level at 3Gyx3 group. This increased in response to irradiation while OGy or control indicated that immune responses can be triggered by the antibody on irradiated H23 was negative on IHC and IF antibody through ADCP and increasing of IFN-gamma from assayS. lymphocytes. Example 5 Example 6 Anti-14-3-3 Epsilon Antibody Activates Antibody Binds to H23 Human Tumor in Mouse Phagocytosis by Dendritic Cells and Monocytic Model Imaging Assays Cells 0154) 50 g of AF750 fluorescent dye conjugated 7D4 0151 Mouse dendritic cells and H23 lung cancer cells antibody or 50 ug of control normal mouse IgG conjugated in were co-cultured in vitro and measured qualitatively and 100 ul PBS were injected intravenously into irradiated or quantitatively for an immune response. Phagocytosis was sham 0Gy tumor bearing mice. Images were recorded every measured following 7D4 antibody incubation with irradiation 24 hours in a Kodak Imager. The NIR imaging showed that treated H23 tumor cells. FIG. 6A shows that the 7D4 antibody antibody signals were obtained at time points of 24, 48 and 96 activates phagocytosis by dendritic cells and monocytic cells hours and lasted for 10 days in 3Gyx3 treated (left hindlimbs) (red Staining). Extended pseudopodia and adhesions were H23 bearing mice. Control sham 0Gy treated side (right hind observed in the 3Gyx3 treated H23 tumor cell. H23 cells were limbs) had much less binding signals compared with left side treated with 3Gyx3 and incubated with the 7D4 monoclonal (in group A). The peak time was 96 hours after antibody antibody to 14-3-3 epsilon. Incubation with the antibody injection in 3Gyx3 treated H23. The signal in 5Gyx 1 tumor following irradiation activates phagocytosis of the antibody bearing mice can be seen at the same time points but the coated cancer cells. In comparison, the isotype control shows intensities were less than 3Gyx3 treated mice in both hind no increase in phagocytosis (FIG. 6B) nor does the absence of limbs (in group B). Controls with OGysham treated plus 7D4 irradiated cells (FIG. 6C). The number of dendritic cells that antibody or 5Gyx 1 with controls normal mouse IgG indicated activate phagocytosis were then quantified by use of flow minor or no binding (FIG. 12). cytometry. FIG. 6D shows the quantification of phagocytosis. 0.155. In summary, near infrared images of mice showed H23 cells were labeled with the fluorescent marker Dil. Dil is that the fluorophore-conjugated antibody bound to irradiated contained in the cell membrane and is incorporated into tumors in the 3Gyx3 group for 240 hours while the 3Gyx1 phagocytic cells. The number of dendritic cells that engulf DiI group showed a lower level of binding. In contrast the OGy from H23 cells was then quantified. FIG. 6D shows that H23 group and control antibody group showed minimal to no cells treated with 3Gyx3 activated phagocytosis in 32 cells. In tumor binding. comparison, lower dose irradiation activated phagocytosis in 18 cells. Cells treated with no radiation activated phagocyto sis in 14 cells. The negative control was the isotype control Discussion for the Examples IgG which activated phagocytosis in 7 dendritic cells. 0156 Ionizing irradiation (IR) is employed to treat many 0152 Next, irradiated H23 tumor cells were co-cultured types of human cancers in addition to Surgery and chemo 1:1 with activated mDCs and injected into C57 mice. Two therapy. Due to DNA damage during treatment, the pheno booster injections were performed at one month intervals, and type of the cells changes, resulting in translocation of protein then spleens were removed. The spleens were homogenized to the surface of tumor cells. These molecules are called IR and lymphocytes were isolated by gradient centrifugation and inducible molecules or neo-antigens and are expressed in assayed using an anti-mouse IFN-Y antibody. Spot counts small amounts that are difficult to isolate and purify for anti were tallied via the ImmunoSpot scanner (Cellular Technol body production. US 2016/0194388 A1 Jul. 7, 2016

0157. In this study it was shown that subtractive immuni Gemini Bio-Products, CA; PEG 1500 (Roche, cat. Zation after radiation of the human lung cancer cells resulted #10817300); Dil dye (Biotium Inc). in the creation of multiple antibodies to these neo-antigens. In (0165 Reagents: total, 96 original positive clones and 54 sub-cloned hybrido 0166 Murine Granulocyte macrophage-colony stimulat mas were picked up by FMAT and Dot-Blot assays. One of ing factor (GM-CSF, ProSpec, Israel); Recombinant murine the antibodies, 7D4, showed the best binding and immune tumor necrosis factor C. (TNFC.) (Pierce, Ill.); Recombinant response using irradiation treated H23 cells. The 7D4 anti mouse CD40 Ligand/TRAP (Biovision Research Products, body showed binding to H23 in vitro and in vivo by IHC, IF Mountain View, Calif.); Lympholyte-M (Cedarlane Labora and mouse NIR imaging. An immune response was generated tories LTD. Ontario, Canada); IFN-Y detection kit (BD Bio and qualitatively shown in a phagocytosis experiment and Sciences, San Diego, Calif); Goat anti mouse IgGFc Y Spe quantitatively measured for immune response in ELIPSOT cific—HRP (Jackson Immuno Research); Goat anti mouse for IFN-gamma levels. The antigen was characterized by IgGFc-Alexafluor 647 conjugate (Molecular Probes); Goat Western-blot with the 7D4 antibody at which point the anti anti mouse IgM Alexa Fluor 680 conjugate (Molecular gen was sequenced to show that it was 14-3-3 epsilon. The Probes); Rabbit anti 14-3-3 epsilon antibody (BioLegend purified antigen was reconfirmed by IHC when the 7D4 anti Company); M-pre Mammalian protein extraction reagent body bound to the irradiated H23 cell. (Pierce, Ill.); Alexa Fluor 750 (Molecular Probes). 0158. The antigen characterized in the H23 irradiated cell line with the 7D4 antibody treatment was 14-3-3 epsilon. Subtractive Immunization 14-3-3 is a family of highly homologous proteins encoded by separate genes. There are seven protein known mammalian 0.167 Antigens: 14-3-3 isoforms, named with Greek letters f, e, Y, m, 6, t. 0168 Human Lung Cancer cell lines: (A) NCI—H23, The MW ranged 26-29 KDa. The 14-3-3 family of proteins Non Small cell lung cancer, adenocarcinoma; (B) NCI— plays a key regulatory role in wide range of vital regulatory H460, Large cell lung cancer, carcinoma; and (C) NCI— processes including signal transduction, checkpoint control, H520, Squamous cell carcinoma. apoptosis, cell cycle progressing, DNA replication and nutri 0169 Treatment of Lung Cancer Cells with Irradiation for ent-sensing pathways. 14-3-3 proteins are highly conserved Subtractive Immunization: and ubiquitously expressed. It has been shown to interact with (0170 The cultured tumor cells in Petri-dishes were CDC25 phosphatases, RAF1 and IRS1 proteins, suggesting exposed to X-ray (80kVp X-ray generator (Pantak North its role in diverse biochemical activities related to signal Branford, Conn., USA) with doses of 3Gyx1 or 3Gyx3 with transduction, such as cell division and regulation of insulin 6 hours intervals or sham 0Gy when the cells became 75% sensitivity'''. As such, future studies will to look at tumor confluent. The irradiation schema is shown in Table 1. After shrinkage models after antibody treatment, the function of treatment, culture medium in the dishes was removed, the this antigen and its role after radiation treatment, and the use cells were washed three times with warmed PBS and then of the 7D4 antibody for drug delivery. detached with pre-chilled 4°C. PBS. Three kinds of untreated tumor cells were used for primary injection and treated tumor Methods for the Examples cells were used for the secondary and boost immunization of the mice in the following four groups: (1) H23: 3Gyx3 irra Animals, Cell Lines and Reagents diated at 0, 6, 24 hours over 24 hour period; (2) H23: 3Gyx1 0159 Animals: irradiated cells and incubated 2 hours after treatment; (3) (0160 Four to five week old female BALB/C or AJ and H23+H460+H520:3Gyx1 irradiated individually, incubated C57BL/6 mice were purchased from Harlan Laboratories Inc. for 2 hour after treatment and then mixed together for immu and maintained in Animal Facility of Office of Animal Care nization; and (4) H23+H460+H520: 3Gyx3 irradiated indi Welfare Assurance in School of Medicine in Vanderbilt Uni vidually at 0, 6, 24 hours and then mixed together for immu versity. All animal studies were performed in accordance with nization. the guidelines of the IACUC and with protocols approved by (0171 Mice Immunization Schedule: Institutional Animal Care and Use Committee in Vanderbilt 0172 Balb/C and AJ mice were pre-bled, sera separated University. and stored in -80° C. for self negative controls. The immu (0161 Cell Lines: nization was as following: On day 0, 2x10' non-irradiated 0162 Three human lung cancer cells (H23, adenocarci human lung cancer cells were intraperitoneal (IP) injected. noma, non-Small cell lung cancer, H460, carcinoma, large On day two and day four, cyclophosphamide was IP injected cell lung cancer; and H520, squamous cell carcinoma) were at 200 mg/kg to Suppress antibody production to non-irradi purchased from ATCC (American Type Culture Collection, ated lung cancer cells. On day 18, irradiated human lung Manassas City, Va., USA). The tumor cells were maintained cancer cells were injected for subtractive immunization (See in RPMI 1640 medium with the supplements of 2 mM Table 1). On day 40 and 56, the mice were boosted with L-glutamine, penicillin plus streptomycin, 10% fetal bovine irradiated human lung cancer cells. On day 70, the mice were serum, after which it was split every three days. SP2/O hyper-immunized with irradiated cancer cells. All the mice myeloma cells were kindly provided by Dr. Clint E. Carter in were bled on day 0, 18, 40, 56 and day 70 and the sera were the Department of Biological Sciences at Vanderbilt Univer collected for antibody titer screening by a Dot-blot and sity, Nashville, USA. FMAT analysis. Three days prior to cell fusion, the high (0163 Chemicals: antibody titer B cell donor candidate mouse was boosted with 0164 HAT (hypoxanthine, Thymidine and AZaserine); irradiated tumor cells again. Four mice/group/strain of mouse Cyclophosphamide: DAB subtract PHA-L and PMA mito were used in the immunization. At day 0, all mice were gens: Proteinase inhibitor cocktail (Sigma); Fetal Bovine injected with non-irradiated tumor cells. At day 2 and day 4, serum was pre-selected for cell fusion and purchased from the mice were injected cyclophosphamide and then mice US 2016/0194388 A1 Jul. 7, 2016 22 primary or boost immunization with IR tumor cells at days cell pellet was gently re-suspended in a HAT medium and 18, 40, 56 and 70, cell numbers were as above. dropped into the culture plates. The culture plates were trans ferred in a 37° C. CO incubator to select culture the hybri Immunized Mice Sera Screening domas. After 7 days of culture, HAT medium was removed, fresh HAT medium was replaced and grown for another 7-10 (0173 Dot Blot Assay: days in a CO incubator for hybridoma screening. 0.174 Treatments of human lung tumor cells were (0179 Hybridoma Screen: described in Table 1. IR or sham treated tumor cells were 0180 Fifteen days after the cell fusion, antibodies in the collected and resuspended in PBS. The cells were coated on a supernatant were tested by FMAT and Dot-Blot. The positive nitrocellulose membrane with 4x10° cells per 7.5x11.5 cm clones were plated into 24 well plates with the HAT medium membrane. The membranes were air dried, washed three so that the hybridoma could grow and then replaced with times with PBS containing 0.1% Tween 20, and air dried complete FBS medium; the supernatants were collected and again. The pre-diluted immunized mice Sera starting from assayed once again by FMAT and Dot-Blot to confirm, then 1/1000 and then 1:2 dilution until 1764,000 stored in 384 well positive clones were ready for Sub-cloning. plates was replicated onto the appropriate membrane by a 0181 Hybridoma Sub-Cloning, Positive Clone Freeze, replicator with 384 pins and then incubated for 2 hours at Antibody Production and Purification: room temperature (RT) in a humid chamber. After washing 0182 Positive hybridomas were sub-cloned into 384 well three times with the same buffer as above, membranes were plates using limited dilution. The Supernatants were tested blocked with 5% non-fat dry milk (NFDM)+0.5% BSA in the again by Dot-Blot and FMAT. Sub-cloned positive clones buffer for at least 45 minutes and washed again. The mem were transferred into 24 well plates for freezing and expand branes were incubated with 1:4,000 diluted goat anti mouse ing culture in serum free (SF) medium. Antibody was purified IgGFc gamma chain specific antibody that was conjugated to from accumulated SF supernatants with Protein G and Pro the HRP enzyme for two hours. The membranes were then tein A columns. The purified IgG was dialyzed against 4 liter washed 3x5 minutes; ECL was applied for 2 minutes. The PBS at 4°C. with two changes. The concentration was tested films were developed to pick up the positive screen where it by spectrophotomatry and IgG sub-class was distinguished was viewed on dot blot films on a light box. using an isotyping kit (Southern Biotech, Birmingham Ala.). (0175 FMAT-HTS (Fluorometric Microvolume Assay Technology, High Throughput Screen System): 0183 IHC (Immunohistochemistry) or IF (Immunofluo rescent) Stain: 0176 The lung tumor cells were cultured in 384 well 0184 The H23 cells were cultured in RPMI 1640 medium plates with 1x10" cells per well and were either sham irradi supplemented with 10% FBS plus Penicillin, streptomycin in ated or radiated at the same dosage as described in Table 1. 10x10 cm Petri dish. The cells were irradiated with either 3 The diluted sera were added to the designated wells and then Gy/2h, 3 Gyx3/0, 6, 24 hrs or sham 0Gy treatment when 75% incubated at 37°C. in a CO incubator for 2 hours. The cells confluent. The culture medium was discarded; the tumor cells were then reacted with goat anti-mouse IgGFc conjugated to were rinsed by warmed PBS three times, and then detached the Alexafluor 647 dye or goat anti-mouse IgM conjugated to with pre-chilled PBS. The cells were counted and centrifuged the Alexafluor 680 dye and incubated overnight. The plates at 1,200 rpm for 5 minute and the Supernatant was discarded. were read by FMAT machine (Applied Biosystems, Model The cells were resuspended in fresh PBS and 1x10 cells were 8100) the next day for mouse IgG and IgM levels in immu mounted on pre-clean Fisher Brand Superfrost microscope nized mice Sera. slides. The cells were air dried, fixed with 4% buffered paraformaldehyde for 10 minutes, and then air dried again, Cell Fusion and Hybridoma Screening and stored at -20°C. Immunohistochemistry (IHC) or immu 0177 Cell Fusion: nofluorescence (IF) was performed by using the purified 0.178 Mice chosen from BALB/C and AJ immunized monoclonal antibodies. The slides were re-hydrated and groups with higr titres of antibody were sacrificed and the washed three times with PBS, and then endogenous hydrogen spleen was removed aseptically and stored in separate dishes peroxidase (HRP) was blocked (if a hydrogen peroxidase with plain RPMI 1640 medium. The spleen was dissociated conjugated secondary antibody was used). The slides were into single cells by a homogenizer and collected into a 50 ml blocked with 5% BSA+5% normal goat serum in PBS for sterile centrifuge tube. The spleen cells were washed three 30–45 minutes. After blocking, the diluted purified mono times with pre-warmed plain RPMI 1640 medium for cell clonal antibodies were added onto the slides, and then incu fusion. Pre-cultured Sp2/O myeloma cells were collected in a bated for 2 hours in a humid chamber at room temperature. sterile 50 ml centrifuge tube and washed three times with After washing 3x with PBS, a secondary goatantimouse IgG pre-warmed plain RPMI 1640 medium also. After centrifu Fc conjugated to HRP or conjugated to Alexa Fluor 594 was gation of spleen and the Sp2/O separately, the medium was added onto the smears, then incubated for 1.5 hour at room removed and cells were re-suspended in 5 ml of the same temperature. The slides were washed three times with PBS medium individually. The cells were counted and combined then DAB was applied onto the cell smears for 2-5 minutes at a ratio of spleen cell: Sp2/O cell of 2:1 or 5:1, then centri (HRP 2nd Ab). The reaction was stopped by rinsing with fuged. The Supernatant was removed, and mixed cells were d-water at which point it was counterstained with Hemotoxy washed once again and then the medium was removed com line. The slides were de-hydrated and mounted with cover pletely, the pellet was gently resuspended and loosened by slips. finger flicking. 1 ml of PEG 1500 was added slowly to perform 0185. Flow Cytomery Assay (FACS): cell fusion within 1 to 3 minutes. Afterwards, plain RPMI 0186 H23 lung cancer cells were cultured and irradiated 1640 medium was added to stop the reaction. The cell sus as above. 1x10 cells were incubated with primary purified pension was incubated at 37° C. for 5 minutes, and then McAb and then washed three times with PBS by centrifuga centrifuged at low speed, the Supernatant was removed. The tion. A goat anti mouse IgG Fc Y chain specific secondary US 2016/0194388 A1 Jul. 7, 2016 antibody conjugated with FITC was added to an individual 0.195 Prepare Lymphocyte Suspension: tube and incubated for one hour. The samples were washed 0196. The spleen was homogenized into single cells in three times then analyzed by Flow cytomety assay (MACS plain RPMI 1640 medium and the cell concentration was Quant Analyzed, Miltenyl Biotec). adjusted to a maximum of 1.0x107 nucleated cells/ml. 5 ml of the cell suspension was added to a 15 ml centrifuge tube Dendritic Cell Phagocytosis Through Fc Receptors where a large Pasteur pipette was put at the bottom of the tube. In Vitro Assay (ADCP) Lympholyte-M was slowly added to the Pasteur pipette and 0187 Preparing Mouse Dendritic Cells: layered under the cell Suspension. The Suspension was cen 0188 Immature mouse dendritic cell (MDC) line JAWSII trifuged at 1,000-1,500 g for 20 minutes at room temperature. was obtained from American Type Culture Collection After centrifugation, the lymphocyte layer at the interface (ATCC, Manassas Va., USA.) The immature mouse dendritic which was harvested carefully and transferred to a new cen cells were grown in a 100x20 mm dish with RPMI 1640 trifuge tube. The cells were washed 3 times with plain RPMI medium with 10% IgG depleted fetal bovine serum plus 1640 medium at 800 g for 10 minute to pellet the lympho antibiotics and 5 ng/ml of GM-CSF. Once the MDCs were cytes. The lymphocytes were re-suspended in plain RPMI 50-60% confluent, the MDCs were harvested by detaching 1640 and counted, then diluted in plain RPMI 1640 to a with 0.05% trypsin-EDTA. The cells were washed with designed concentration for use in the next step. medium, then resuspended in medium and dispensed into a (0197) ELISPOT to Measure of INF-y Level: small culture dish (35x10 mm) with sterile 22x22 mm cover (0198 ELISPOT was employed for measuring of IFN-y slips. The MDCs were grown overnight in a 37° C. CO levels generated in immunized C57BL/6 mice in vivo. The incubator. After overnight incubation, the JAWSII cells were IFN-Y detection kit was used for the test and the protocol was activated and matured in vitro with 20 ng/ml of TNF-C. and followed as per the company datasheet. Briefly, the ELISPOT 200 ng/ml of CD40 ligand/TRAP. The dendritic cells were plate was coated with 100 ul of diluted IFN-Y capture anti cultured in a CO incubator for 48 hours until confluent. body in the concentration of 5 g/ml in PBS and incubated at 0189 Preparing the H23 Human Lung Tumor Cells: 4°C. overnight. The plate was washed with blocking buffer (0190. The H23 cells were cultured in IgG depleted FBS and blocked with 200 ul of this buffer for 2 hours. The lym medium until the cells became confluent at 70%. The H23 phocytes were added to designed wells in the amount of cells were irradiated with X-ray treatments or sham 0Gy 10x10, 0.5x10, 0.25x10, 0.1x10, 0.05x10° and 0.025x radiated as above. The culture medium was removed and 10° in 100 ul volume followed by discarding blocking solu rinsed with warmed PBS three times. The cells were detached tion. The cells were activated by adding 100 ul of diluted with cold PBS and counted. The tumor cells were incubated mitogen mixture in 10% FBS RPMI 1640 to the plate (PHA with purified monoclonal antibody in PBS in 10 ug/ml for 1-2 L., 5 g/ml, PMA, 5 ng/ml). The ELISPOT plate was incu hours and washed with warmed PBS three times. The cells bated in the CO incubator for 24 hours. 200 ul of the cell were spun down at 1000 RPM for 5 minutes, then resus Suspension in wells was aspirated and plate was washed three pended in fresh PBS and stained with DiI dye at 2 ug/ml in times by soaking wells for 3-5 minutes for each washing. 100 PBS for 30 minutes in the 37° C. CO, incubator. Finally, the ul of biotinylated anti mouse IFN-Y detection antibody was cells were washed with warmed PBS 3 times to remove free added to each well and then incubated for 2 hours at room dye. temperature. The plate was emptied and washed three times with supplied buffer and then diluted streptavidin HRP con (0191 Co-Culture of Mouse Dendritic with H23 Rumor jugate was added to each well, and incubated for one hour at Cells for Phagocytosis: room temperature. The plate was washed 4 times with wash (0192. The antibody reacted and Dil stained H23 cells were ing buffer I and then twice with washing buffer II. Substrate added to the matured MDC dishes in a 1:1 ratio. These cells DAB was added to each well. Spot development was moni were co-cultured overnight in the CO incubator at 37°C. The tored for 30 minutes. The reaction was stopped by washing next day, the cultured medium was removed and the cells the wells with DI water and the plate was air-dried overnight were rinsed 3 times with warmed PBS. The cells were fixed at room temperature in the dark. The ELISPOT was read and with 10% formalin for 8 minutes. The cells were thenwashed analyzed by an ELISPOT scanner (ImmunoSpot, Cellular, with PBS 3 times again and stained with DAPI at 10 ug/ml for Technology Ltd.) 10 minutes followed by washing with PBS 3 times. The slides were mounted on coverslip with 80% glycerol for ADCP (0199 Antibody Targeted Human Tumor H23 Mouse events viewing. Model Imaging: 0200. The H23 human lung cancer cells were cultured in ELISPOT Assay 10x10 cm dishes in RPMI 1640 medium with 10% FBS plus supplements. The cells were detached by using cold PBS and 0193 C57BL6/J Mice Immunization: collected in 15 ml centrifuge tubes when tumor cells sub 0194 The H23 and MDC mixture was prepared as above. confluent. The tumor cells were injected in athymic Nu/Nu Antibody coated either IR or sham 0Gy treated H23 tumor mice at 1x10°/hind limb. The AF750 fluorescent dye was cells and controls were included in this assay. 2x10 cells in conjugated to purified 7D4 antibody. The conjugation method 0.1 ml PBS were injected into C57BL/6 mice by intraperito was following the protocol from Molecular probes. Briefly, neal (IP) injection. There were 3 mice/group for primary dissolving the lyophilized powder in DMSO to form 10 immunization. The mice were boosted twice after the first mg/ml and then aliquot in Small amount, and stored at -20C. immunization by the same treatment and cell number, with Whence antibody conjugation performance, 1M sodium the last boost being performed three days prior to termination. bicarbonate (pH 9.0) was added into 2C6F3 antibody to form Three days after the boost, the mouse spleen was harvested 0.1 M sodium bicarbonate in antibody solution (pH8.3) and aseptically into individual tubes containing RPMI 1640 then added AF750 to antibody solution. The ratio is Antibody: medium for lymphocyte isolation. AF750=10:1 by amount and mixed well. The reaction rotated US 2016/0194388 A1 Jul. 7, 2016 24 over two hours wrapped by an aluminum foil. The free dye 7D4 produced in house. Normal mouse IgG or secondary was cleaned by a using a 3K size exclusion centrifugation antibody only was used for the controls. column (Pall Corporation). Conjugated antibody was har (0209 Specific Anti 14-3-3 Epsilon Antibody Epitope vested. Concentration of labeled antibody and degree of Mapping and Sub-Class Identification: labeling were tested according to the method from the com 0210 Human 14-3-3 Protein epsilon and its isoform SV pany. Finally, the labeled antibody was adjusted to 50 lug/100 whole amino acid sequences were obtained through UniPro ul PBS then injected through tail vein once the tumor size tKB/Swiss-Prot. The Taxonomic identifier 9606 (NCBI) reached to 0.5-0.8 cm, then irradiated with 3Gyx3 and 5Gyx1 organism is Homosapiens (Human), Gene name is YWHAE. and sham 0Gy. The mice were anesthetized for imaging every Peptide designed from whole sequences of 14-3-3 protein 24 hours by using an in vivo multi-spectral NIR system epsilon and its isoform 14-3-3 protein epsilon SV. The (Kodak imager) (Optical Radiology Laboratory). designed peptides contain 18AA from N terminal to C termi 0201 Antigen Identification Targeted by Monoclonal nal and had 2AA overlapped in each peptide. A total of 31 Antibody: peptides synthesized by Sigma. The synthesized peptide was (0202 Cultured H23 cell was irradiated at 3Gyx3, 3Gyx1 prepared in DMSO and then further diluted in PBS for coating and sham OGy. The cells were washed three times with an ELISA plate. 30 ug/ml and 10 g/ml of peptides in PBS warmed PBS at 37° C. and 10 ml of cold PBS was added to the were coated on ELISA plates, 50 uL/well. The plates incu dishes to detach for 5 minutes, the tumor cells were harvested bated overnight at 4°C., were washed with 0.1% Tween20 into 50 ml centrifuge tubes. The cells were centrifuged at PBS and blocked with 3% BSA in same buffer for 1 hour. 50 1400 rpm for 8 minutes. The cell pellets were lysed by the ul dilution of purified anti 14-3-3 protein Epsilon antibody at freeze/thaw method 5 times in liquid nitrogen and 37° C. the concentration of 5 g/ml was added to each well, and water bath with lysis buffer by supplement proteinase inhibi incubated for 2 hours in a humid chamber in RT. A goat tor cocktail. The cell lysate was centrifuged at 5000 rpm for anti-mouse IgG Fc-gamma chain specific HRP conjugated 30 minutes and the Supernatant was harvested as the source of secondary antibody was diluted at 1:4000 and added to each cell lysate antigens. Protein concentration was determined well followed by washing of the plates 5 times. After washing and Western-blot was used for analysis of X-ray induced again, substrate ABTS was added to each well to monitor antigen recognized by the purified monoclonal antibodies as color change by reading at 405 nm in an ELISA reader (Bio above. 10 micro-gram of cell lysate was loaded on the desig Tek Instrument Inc.). A mouse Ig isotyping kit was purchased nated lanes and the proteins were separated by 10% gel then from Southern Biotech (Birmingham Ala.) included IgG1. transferred to a nitrocellulose (NC) membrane at 4°C. over 2a, 2b, IgG3, IgM and kappa lambda chain for purified anti night at low current. The NC membranes were washed three body isotype test. times in 0.1% Tween 20 in PBS and then blocked with 5% 0211 Antibody Binding Site AA Sequence: NFDM plus 0.5% BSA in the same buffer. The purified mono 0212 Hybridoma clone 7D4 was cultured in complete clonal antibody was added to the membrane at the concentra FBS medium with all supplements. The cells were harvested, tion of 5ug/ml in 5% NFDM then incubated for one hour at counted and then frozen in 10% DMSO-90% FBS hybri RT. The NC membranes were then washed three times again doma frozen medium, 2x10/vial. The vials were shipped to and incubated with secondary antibody goat anti mouse IgG Genscript (Piscataway, N.J. USA) for antibody binding site Fc conjugated to HRP at 1:4000 in 5% NFDM in 0.1% Tween Sequence. 20 PBS for another 2 hours. Finally, ECL was added to the membranes after completed washing. The photographic films REFERENCES were developed for analysis protein bands. 0213 1. Ghazal Hariri, Ying Zhang; Zhaozhong Han; 0203 Specific Antigen Affinity Purification by IP and Allie Fu: Martin Brechbiel: Noor Tantawy: Todd Peterson, Identification: Hallahan, Dennis. Radiation-guided P-selectin targeted 0204 The purified monoclonal antibody 7D4 was conju tumor imaging in a lung tumor model. Annals of Biomedi gated to cyanogens bromide-activated Sepharose 4B cal Engineering. 36(5): 821-830, 2008 (Sigma). X-ray treated H23 cell lysates were prepared as 0214) 2. Wong JY C, Chu DZ, Yamauchi DM, Williams above and applied to antibody conjugated beads individually L E. Liu A, Wilczynski S, Wu AM, Shively J. E. Doroshow (pre-conjugated to each column). Based off the cell lysate, J. H. Raubitschek A A. A phase I radioimmunotherapy trial various treatment groups of OGy, 3Gyx1, 3Gyx3 were mixed evaluating 90yttrium-labeled anti-carcinoembryonic anti for conjugation at 4° C. for overnight. The columns were gen (CEA) chimeric T84.66 in patients with metastatic packed and washed with PBS and then eluted with a 0.1 M CEA-producing malignancies. Clin Cancer Res. 2000 Oct. Na-citric elution buffer pH2.8 to collect the specific antigens 6(10):3855-63. captured by purified antibody. Western-blotassays were used 0215 3. Richman CM, Denardo SJ, O'Donnell RT. Yuan to confirm the purified antigens from affinity column. A, Shen S, Goldstein DS, Tuscano JM, WunT, Chew HK, 0205 Antibody Identified Protein Sequence: Lara PN. Kukis DL, Natarajan A. Meares CF, Lamborn K 0206. The specific protein bands detected through West R. DeNardo G. L. High-dose radioimmunotherapy com ern-blot by purified antibody were excised in a SDS-PAGE bined with fixed, low-dose paclitaxel in metastatic prostate gel and sent to Vanderbilt University Mass-Spectrometry and breast cancer by using a MUC-1 monoclonal antibody, Research Center for proteomics analysis of the protein. m170, linked to indium-111/yttrium-90 via a cathepsin 0207. Purified Protein Identification and Localization: cleavable linker with cyclosporine to prevent human anti 0208. IHC was used to confirm the binding on the treated mouse antibody. Clin Cancer Res. 2005 Aug. 15; 11 (16): tumor cells. Briefly, a commercial rabbit anti 14-3-3 epsilon 5920-7. antibody was used for this assay. X-ray treated H23 cells at 0216 4. Hallahan DE, et al. Control of pharmacokinetics 3Gyx3, 3Gyx1 or sham 0Gy cell smear on slides were pre and pharmacodynamics of drug delivery by use of radia pared and performed as IHC above to compare with purified tion. Am J. Clin Oncol 2001 b: 24:473-480 US 2016/0194388 A1 Jul. 7, 2016

0217. 5. Clynes RA, Towers TL, Presta LG, Ravetch JV. delivery to radiation-induced neoantigens in tumor Inhibitory Fc receptors modulate in vivo cytoxicity against microvasculature. J Control Release. 2001 Jul. 6: 74(1-3): tumor targets. Nat Med. 2000 April; 6(4):443-6. 183-91 0218 6. Raymond L. Mernaugh, Heping Yan, Dong Chen, 0226, 14. Oriente F. Andreozzi F. Romano C. Perruolo G, Jennifer Edl, Gregory Hanley, Ambra Pozzi, Roy Zent. Perfetti A, Fiory F, Miele C, Beguinot F. Formisano P. Production and Characterization of Mouse Ureteric Bud Protein kinase C-alpha regulates insulin action and degra cell-specificrat hybridoma antibody utilizing subtractive dation by interacting with insulin receptor Substrate-1 and 14-3-3 epsilon. J Biol Chem. 2005 Dec. 9; 280(49):40642 immunization and high-throughput Screening. Jornal of 9. Epub 2005 Oct. 10. Immunological Methods 306 (2005) 115-127. 0227 15. Telles E. Hosing AS, Kundu ST, Venkatraman P. 0219. 7. Ning Y. Wang Y. Li Y. Hong Y, Peng D, Liu Y. Dalal SN. A novel pocket in 14-3-3 epsilon is required to Wang J, Hao W. Tian X, Wu F, Dong W, Wang L, Wu Q, Liu mediate specific complex formation with cdc25C and to X, Gao J. He F, Qian X, Sun Q H. Li M. An alternative inhibit cell cycle progression upon activation of checkpoint strategy for high throughput generation and characteriza pathways. Exp Cell Res. 2009 May 1; 315 (8): 1448-57. tion of monoclonal antibodies against human plasma pro Epub 2009 Jan. 31. teins using fractionated native proteins as immunogens. 0228 16. Lee E. K. Lee YS, Lee H, Choi CY. Park S. H. Proteomics. 2006 January; 6(2):438-48. 14-3-3 epsilon protein increases matrix metalloprotein 0220) 8. Clynes R. Takechi Y. Moroi Y. Houghton A, ase-2 gene expression via p38 MAPK signaling in NIH3T3 Ravetch J.V.Fc receptors are required in passive and active fibroblast cells. Exp Mol Med. 2009 Jul. 31; 41(7):453 immunity to melanoma. Proc Natl Acad Sci USA. 1998 561. Jan. 20:95(2):652-655 0229. 17. Heidi major Sleister, A Gururaj Rao. Subtractive 0221) 9. Desjarlais J. R. Lazar GA, Zhukovsky EA, Chu S immunization: A tool for the generation of discriminatory Y. Optimizing engagement of the immune system by anti antibodies to proteins of similar sequence. Journal of tumor antibodies: an engineer's perspective. Drug Discov Immunological Methods. 261 (1-2) 213-220, March 2002. Today. 2007 November; 12(21-22):898-910. 0230 18. Raymond L. Mernaugh, Heping Yan, Dong 0222 10. Zuo S, Xue Y. Tang S. Yao J, Du R, Yang P. Chen Chen, Jennifer Edl, Gregory Hanley, Ambra Pozzi, Roy X. 14-3-3 epsilon dynamically interacts with key compo Zent Production and Characterization of mouse ureteric nents of mitogen-activated protein kinase signal module bud cell-specific rat hybridoma antibodies utilizing sub for selective modulation of the TNF-alpha-induced time tractive immunization and high-throughput screening. course-dependent NF-kappaB activity. J Proteome Res. Journal of Immunological Methods. 306 115-127, 2005. 2010 Jul. 2:9(7):3465-78. 0231. 19. Mariana Ferreira Leal, Danielle Queiroz, Cal 0223) 11. DivgiCR, Bander NH, Scott AM, O'Donoghue cagno, Samia Demachki, Paulo Pimentel Assumpção, JA, Sgouros G. Welt S, Finn RD, Morrissey F, Capitelli P. Roger Chammas, Rommel Rodríguez Burbano, and Williams.JM, Deland D, Nakhre A, Oosterwijk E, Gulec S. Marília de Arruda Cardoso Smith Clinical implication of Graham MC, Larson S M, Old L. J. Phase I/II radioimmu 14-3-3 epsilon expression in gastric cancer. World J. Gas notherapy trial with iodine-131-labeled monoclonal anti troenterology. 18(13)1531-1537, 2012. body G250 in metastatic renal cell carcinoma. Clin Cancer 0232 20. Qi W1, Liu X, Qiao D, Martinez J. D. Isoform Res. 1998 November, 4(11):2729-39. specific expression of 14-3-3 proteins in human lung can 0224 12. Cai, Weibo, Yun Wu, Kai Chen, Qizhen Cao, certissues. Int. J. cancer. 113(3) 359-363, 2005. David A. Tice, and Xiaoyuan Chen. In-Vito and In-vivo 0233. 21. Zhihui Wang, Jahn M. Nesland, Zhenhe Suo, characterization of 64Cu-labeled AbegrinTM, a humanized Claes G. Trope, Ruth Holm. The Prognostic Value of monoclonal antibody against integrin AVB3. Cancer Res 14-3-3 Isoforms in Vulvar Squamous Cell Carcinoma 2006; 66: (19).9673. Cases: 14-3-3? and e Are Independent Prognostic Factors 0225 13. Hallahan DE, Geng L. Cmelak A.J. Chakravar for These Tumors. PloS ONE Volume 6 Issue 9, September thy Aft Martin W. Scarfone C, Gonzalez A. Targeting drug 2011.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 17

<21 Os SEQ ID NO 1 &211s LENGTH: 339 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs SEQUENCE: 1

gacattgttga tigacacagtic to catcCtcc ctggctatgt cagtaggaca galaggt cact 60

atgagctgca agtc.cagtica gagcc tittta aatagtagca atcaaaagaa ct atttggcc 12O

tgg taccagc agaaaccagg acagt ct colt aaacttctgg tatactittgc atccac tagg 18O

gaatctgggg to cctgatcg Ctt cataggc agtggatctg ggacagattt cact Cttacc 24 O US 2016/0194388 A1 Jul. 7, 2016 26

- Continued atcagcagtg to aggctga agacctggca gattact tct gtcagcaa.ca ttatagc act 3OO cc.gct cacgt toggtgctgg gaccalagctg gagctgaaa 339

<210s, SEQ ID NO 2 &211s LENGTH: 357 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 2 Caggttcagc tigcagcagtic tigagctgag Ctgatgaagc Ctgggggct C agtgaagata 6 O t cctgcaagg Ctactggcta Cacattcagt agctactgga tagagtgggit aaa.gcagagg 12 O

Cctgga catg gccttgagtggattggagag attttacctg. galagtgg tag tactalactac 18O aatgagaagt toaagggcaa gogo cacattc actgcagata catcc to caa cacagcc tac 24 O atgcaactica gcagoctgac atctgaggac totgc.cgt.ct attactgtgc aagat.cggta 3OO tggittacgac gtgattittgc titactggggc Caagggactic tigt cactgt Citctgca 357

<210s, SEQ ID NO 3 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 3 Asp Ile Val Met Thr Glin Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1. 5 1O 15 Gln Llys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Lieu. Asn Ser 2O 25 3O Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45 Ser Pro Llys Lieu. Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O Ile Ser Ser Val Glin Ala Glu Asp Lieu Ala Asp Tyr Phe Cys Glin Glin 85 90 95 His Tyr Ser Thr Pro Leu. Thir Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 1OO 105 11 O Lys

<210s, SEQ ID NO 4 &211s LENGTH: 119 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 4 Glin Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Met Llys Pro Gly Ala 1. 5 1O 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr 2O 25 3O US 2016/0194388 A1 Jul. 7, 2016 27

- Continued Trp Ile Glu Trp Wall Lys Glin Arg Pro Gly His Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe SO 55 6 O Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 7s 8O Met Glin Leu Ser Ser Lieu. Thir Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Trp Lieu. Arg Arg Asp Phe Ala Tyr Trp Gly Glin Gly 1OO 105 11 O

Thir Lieu Val Thir Wal Ser Ala 115

<210s, SEQ ID NO 5 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYTHENSIZED

<4 OOs, SEQUENCE: 5 Llys Ser Ser Glin Ser Lieu. Lieu. Asn. Ser Ser Asn. Glin Lys Asn Tyr Lieu. 1. 5 1O 15

Ala

<210s, SEQ ID NO 6 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 6 Phe Ala Ser Thr Arg Glu Ser 1. 5

<210s, SEQ ID NO 7 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OO > SEQUENCE: 7 Gln Gln His Tyr Ser Thr Pro Leu. Thr 1. 5

<210s, SEQ ID NO 8 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 8 Gly Tyr Thr Phe Ser Ser Tyr Trp Ile Glu 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 17 212. TYPE: PRT US 2016/0194388 A1 Jul. 7, 2016 28

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 9 Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 10 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 10 Ser Val Trp Lieu. Arg Arg Asp Phe Ala Tyr 1. 5 1O

<210s, SEQ ID NO 11 &211s LENGTH: 255 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 11 Met Asp Asp Arg Glu Asp Lieu Val Tyr Glin Ala Lys Lieu Ala Glu Glin 1. 5 1O 15 Ala Glu Arg Tyr Asp Glu Met Val Glu Ser Met Llys Llys Val Ala Gly 2O 25 3O Met Asp Val Glu Lieu. Thr Val Glu Glu Arg Asn Lieu Lleu Ser Val Ala 35 4 O 45 Tyr Lys Asn Val Ile Gly Ala Arg Arg Ala Ser Trp Arg Ile Ile Ser SO 55 6 O Ser Ile Glu Gln Lys Glu Glu Asn Lys Gly Gly Glu Asp Llys Lieu Lys 65 70 7s 8O Met Ile Arg Glu Tyr Arg Gln Met Val Glu Thr Glu Lieu Lys Lieu. Ile 85 90 95 Cys Cys Asp Ile Lieu. Asp Val Lieu. Asp Llys His Lieu. Ile Pro Ala Ala 1OO 105 11 O Asn Thr Gly Glu Ser Lys Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr 115 12 O 125 His Arg Tyr Lieu Ala Glu Phe Ala Thr Gly Asn Asp Arg Lys Glu Ala 13 O 135 14 O Ala Glu Asn. Ser Lieu Val Ala Tyr Lys Ala Ala Ser Asp Ile Ala Met 145 150 155 160 Thr Glu Lieu Pro Pro Thr His Pro Ile Arg Lieu. Gly Lieu Ala Lieu. Asn 1.65 17O 17s

Phe Ser Val Phe Tyr Tyr Glu Ile Lieu. Asn Ser Pro Asp Arg Ala Cys 18O 185 19 O

Arg Lieu Ala Lys Ala Ala Phe Asp Asp Ala Ile Ala Glu Lieu. Asp Thr 195 2OO 2O5

Lieu. Ser Glu Glu Ser Tyr Lys Asp Ser Thr Lieu. Ile Met Glin Lieu. Lieu. 21 O 215 22O

Arg Asp Asn Lieu. Thir Lieu. Trp Thir Ser Asp Met Glin Gly Asp Gly Glu 225 23 O 235 24 O US 2016/0194388 A1 Jul. 7, 2016 29

- Continued

Glu Glin Asn Lys Glu Ala Lieu. Glin Asp Val Glu Asp Glu Asin Glin 245 250 255

<210s, SEQ ID NO 12 &211s LENGTH: 233 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 12 Met Val Glu Ser Met Lys Llys Val Ala Gly Met Asp Val Glu Lieu. Thr 1. 5 1O 15 Val Glu Glu Arg Asn Lieu Lleu Ser Val Ala Tyr Lys Asn Val Ile Gly 2O 25 3O Ala Arg Arg Ala Ser Trp Arg Ile Ile Ser Ser Ile Glu Glin Lys Glu 35 4 O 45 Glu Asn Lys Gly Gly Glu Asp Llys Lieu Lys Met Ile Arg Glu Tyr Arg SO 55 6 O Glin Met Val Glu Thr Glu Lieu Lys Lieu. Ile Cys Cys Asp Ile Lieu. Asp 65 70 7s 8O Val Lieu. Asp Llys His Lieu. Ile Pro Ala Ala Asn Thr Gly Glu Ser Lys 85 90 95 Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr His Arg Tyr Lieu Ala Glu 1OO 105 11 O Phe Ala Thr Gly ASn Asp Arg Lys Glu Ala Ala Glu ASn Ser Lieu Val 115 12 O 125 Ala Tyr Lys Ala Ala Ser Asp Ile Ala Met Thr Glu Lieu Pro Pro Thr 13 O 135 14 O His Pro Ile Arg Lieu. Gly Lieu Ala Lieu. Asn Phe Ser Val Phe Tyr Tyr 145 150 155 160 Glu Ile Lieu. Asn. Ser Pro Asp Arg Ala Cys Arg Lieu Ala Lys Ala Ala 1.65 17O 17s Phe Asp Asp Ala Ile Ala Glu Lieu. Asp Thir Lieu. Ser Glu Glu Ser Tyr 18O 185 19 O Lys Asp Ser Thir Lieu. Ile Met Glin Lieu. Lieu. Arg Asp Asn Lieu. Thir Lieu. 195 2OO 2O5 Trp. Thir Ser Asp Met Glin Gly Asp Gly Glu Glu Glin Asn Lys Glu Ala 21 O 215 22O Lieu. Glin Asp Val Glu Asp Glu Asin Glin 225 23 O

<210s, SEQ ID NO 13 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 13 Phe Ser Val Phe Tyr Tyr Glu Ile Lieu. Asn Ser Pro Asp Arg Ala Cys 1. 5 1O 15

Arg Lieu.

<210s, SEQ ID NO 14 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2016/0194388 A1 Jul. 7, 2016 30

- Continued

<4 OOs, SEQUENCE: 14 His Pro Ile Arg Lieu. Gly Lieu Ala Lieu. Asn Phe Ser Val Phe Tyr Tyr 1. 5 1O 15

Glu Ile

<210s, SEQ ID NO 15 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 15 Phe Ser Val Phe Tyr Tyr Glu Ile Lieu. Asn 1. 5 1O

<210s, SEQ ID NO 16 &211s LENGTH: 138 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 16 Met Glu Trp Thir Trp Val Phe Leu Phe Lieu. Leu Ser Val Thr Ala Gly 1. 5 1O 15 Val His Ser Glin Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Met Lys 2O 25 3O Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe 35 4 O 45 Ser Ser Tyr Trp Ile Glu Trp Val Lys Glin Arg Pro Gly His Gly Lieu. SO 55 6 O Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn 65 70 7s 8O Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Lieu. Thir Ser Glu Asp Ser Ala Val 1OO 105 11 O Tyr Tyr Cys Ala Arg Ser Val Trp Lieu. Arg Arg Asp Phe Ala Tyr Trp 115 12 O 125 Gly Glin Gly Thr Lieu Val Thr Val Ser Ala 13 O 135

<210s, SEQ ID NO 17 &211s LENGTH: 133 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: SYNTHESIZED

<4 OOs, SEQUENCE: 17 Met Glu Ser Glin Thr Glin Val Lieu Met Phe Leu Lleu Lleu Trp Val Ser 1. 5 1O 15

Gly Ala Cys Ala Asp Ile Val Met Thr Glin Ser Pro Ser Ser Leu Ala 2O 25 3O

Met Ser Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser Ser Glin Ser 35 4 O 45

Lieu. Lieu. Asn. Ser Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin SO 55 6 O US 2016/0194388 A1 Jul. 7, 2016

- Continued

Llys Pro Gly Glin Ser Pro Llys Lieu. Leu Val Tyr Phe Ala Ser Thr Arg 65 70 7s Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Lieu. Thir Ile Ser Ser Val Glin Ala Glu Asp Lieu Ala Asp Tyr 1OO 105 11 O Phe Cys Glin Gln His Tyr Ser Thr Pro Leu. Thr Phe Gly Ala Gly Thr 115 12 O 125 Llys Lieu. Glu Lieu Lys 13 O

1. An isolated antibody, wherein the antibody specifically a) exposing a target area of the Subject where the presence binds 14-3-3 epsilon and comprises a heavy chain CDR3 of a tumor is suspected to ionizing radiation; comprising the amino acid sequence of SEQID NO:10 with b) administering to the Subject a composition to detect the Zero to two amino acid Substitutions and/or a light chain presence of 14-3-3 epsilon in the target area, wherein the CDR3 comprising the amino acid sequence of SEQID NO:7 composition comprises one or more targeting antibod with Zero to two amino acid Substitutions. ies, wherein each targeting antibody specifically binds to 2. The isolated antibody of claim 1, wherein the antibody 14-3-3 epsilon exposed on an irradiated cell and is con jugated to a detectable label; and further comprises at least one amino acid sequence selected c) detecting the detectable label to detect the presence of from the group consisting of SEQID NO:3, SEQ ID NO:4, 14-3-3 epsilon, wherein the presence of 14-3-3 epsilon SEQID NO:5, SEQID NO:6, SEQID NO:8, and SEQ ID indicates the presence of a tumor in the target area of the NO: 9 Subject. 3. The isolated antibody of claim 1, wherein the antibody is 12. The method of claim 11, wherein the exposing com encoded by a nucleic acid sequence comprising a nucleic acid prises exposing the tumor to at least about 2 Gy ionizing sequence selected from the group consisting of SEQID NO:1 radiation. and SEQID NO:2. 13. The method of claim 11, wherein the administering 4. The isolated antibody of claim 1, further comprising the comprises administering the antibody 0 hours to about 24 amino acid sequence of SEQID NO:8 with Zero to two amino hours following radiation exposure. acid substitutions and the amino acid sequence of SEQ ID 14. The method of claim 11, wherein the detectable label is NO:9 with zero to two amino acid substitutions. a radionuclide. 5. The isolated antibody of claim 1, further comprising the 15. The method of claim 14, wherein the detecting com amino acid sequence of SEQID NO:5 with Zero to two amino prises detecting the radionuclide label using positron emis acid substitutions and the amino acid sequence of SEQ ID sion tomography, single photon emission computed tomog NO:6 with zero to two amino acid substitutions. raphy, gamma camera imaging, or rectilinear Scanning. 6. The isolated antibody of claim 1, wherein the antibody 16. The method of claim 11, wherein the tumor is a lung specifically binds to an epitope selected from the group con carcinoma. sisting of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID 17. A method of enhancing radiotherapy in a subject, the NO:15. method comprising administering to the Subject a pharmaco 7. The isolated antibody of claim 1, wherein the antibody is logically effective amount of an isolated anti-14-3-3 epsilon selected from the group consisting of a single-chain antibody, antibody, wherein the isolated anti-14-3-3 epsilon antibody an antibody fragment, a chimeric antibody, or a humanized comprises a heavy chain CDR3 comprising the amino acid antibody. sequence of SEQ ID NO:10 with Zero to two amino acid 8. The isolated antibody of claim 1, wherein the antibody is substitutions and/or a light chain CDR3 comprising the conjugated directly or indirectly to a payload selected from amino acid sequence of SEQID NO:7 with zero to two amino the group consisting of a detectable label, a therapeutic agent, acid Substitutions and is conjugated to a therapeutic agent, or a combination thereof. Such that radiotherapy is enhanced. 9. The isolated antibody of claim 1, wherein the antibody is 18. The method of claim 17, wherein the subject has cancer. conjugated directly or indirectly to a nanoparticle or a lipo 19. The method of claim 18, wherein the cancer is lung SO. carcinoma. 10. The isolated antibody of claim 8, wherein the detect 20. The method of claim 17, wherein the administering able label and/or therapeutic agent is a radionuclide. comprises administering the antibody Subsequent to radiation 11. A method of detecting a tumor in a subject, the method exposure. comprising: