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. 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FG 7B Patent Application Publication Jul. 7, 2016 Sheet 11 of 20 US 2016/O194388A1 H23 3Gyx3 FG 8A Patent Application Publication Jul. 7, 2016 Sheet 12 of 20 US 2016/O194388A1 FG 8B Patent Application Publication Jul. 7, 2016 Sheet 13 of 20 US 2016/O194388A1 Lanes. 1. H23 sham 0Gy 2. H233Gyx1 3. H233Gyx3 FG 9 Patent Application Publication Jul. 7, 2016 Sheet 14 of 20 US 2016/O194388A1 ares 1. 3Gyx3 2. Scyx 3. 3Gyx i. Ogy FG 1 O Patent Application Publication Jul. 7, 2016 Sheet 15 of 20 US 2016/O194388A1 H23 cells 3Gyx3 H23 cells sham OGy FG 11A Patent Application Publication Jul. 7, 2016 Sheet 16 of 20 US 2016/O194388A1 si xixty insix strongest to 3 (Y-3 titles 3 exy xxx xi xx FG 11B Patent Application Publication Jul. 7, 2016 Sheet 17 of 20 US 2016/O194388A1 FG 12A Patent Application Publication Jul. 7, 2016 Sheet 18 of 20 US 2016/O194388A1 F.G. 12B Patent Application Publication Jul. 7, 2016 Sheet 19 of 20 US 2016/O194388A1 FG 12C Patent Application Publication Jul. 7, 2016 Sheet 20 of 20 US 2016/O194388A1 FG 12D US 2016/0194388 A1 Jul. 7, 2016 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.