Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed “reverse biopanning,” which can then be selected for further development based on their peptide receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in the cell surface in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide-TIP-1 binding. Pseudo-binding energies were calculated to be −360.645 kcal/mol, −487.239 kcal/mol, and −595.328 kcal/mol for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide in vitro via electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor.
Targeted therapy remains one of the biggest challenges in the effective treatment of cancer. Due to their heterogeneity, not all tumors respond to a particular treatment regimen with similar efficacy. Thus, there is an urgent need to develop targeted therapies that are not only highly specific to cancer subtypes but also have minimal toxicity towards normal tissue. Currently, development of anti-cancer antibodies is limited to antigens that are either overexpressed or antigens that are only expressed in a few patients. This limits both the number of available targets and their selectivity for tumors. Using phage display technology, we have identified proteins that are induced by clinical doses of ionizing radiation (IR): tax-interacting protein 1 (TIP-1) and TATA box-associated factor 15 (TAF-15). Following IR treatment, cell surface expression of both TIP-1 and TAF-15 is enhanced in a number of human cancers, including brain, lung, breast and pancreatic cancer. The underlying premise is that IR induces a stress response resulting from DNA strand breaks and activation of ATM repair pathways. After IR stress, these antigens translocate to the surface where they can be targeted for therapeutic purposes. These radiation-inducible neoantigens present a new synergistic model for the treatment of cancer using IR. In this study, we characterized both radiation-inducible targets in vitro and in vivo. Western blotting, immunohistochemistry and flow cytometry data all demonstrated increased cell surface expression of both TIP-1 and TAF-15 after irradiation. We also developed a monoclonal antibody and a single chain antibody against TIP-1 and are currently in the process of developing antibodies against TAF-15. ELISA and near infra-red whole mouse imaging results demonstrate high affinity and high specificity of our lead anti-TIP-1 antibody and anti-TAF15 peptide in lung, brain, breast and pancreatic cancer. Dosimetry studies using immunofluorescence and Western blot analysis show a dose-dependent increase in expression of TIP-1 and TAF15, suggesting that these proteins may play a key role in the cancer cell's response to radiation stress. We are currently in the process of characterizing the mechanisms of how these proteins contribute to radioresistance and cancer survival processes such as proliferation, apoptosis and metastasis. Citation Format: Lincoln Muhoro, Heping Yan, Jeremy Hunn, Dinesh Thotala, Daniel Ferraro, Dennis Hallahan. Characterization and targeting of radiation-inducible neoantigens in multiple cancer types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4597. doi:10.1158/1538-7445.AM2014-4597
EpCAM is a transmembrane glycoprotein that functions as a homophilic cell adhesion molecule. EpCAM is expressed in normal epithelium and at even higher levels in epithelial cancers, including breast, pancreatic and lung cancers. Since its discovery in the 1980s, EpCAM has been targeted in several different epithelial cancers. Even though numerous novel therapeutic strategies targeting EpCAM have been investigated, the biological role and functional significance of EpCAM expression in cancer remains unclear. EpCAM expression appears to be associated with prognosis in several epithelial cancers -in some cancers it is associated with a favorable prognosis (renal cell, rectal) and in others it is associated with a poor prognosis (lung, pancreatic, breast). Thus, the function of EpCAM appears to be context-dependent in epithelial cancers. To understand the impact of EpCAM in human cancer, we specifically ablated EpCAM expression in over 40 cancer cell lines, and assessed the impact on invasion. We identified the A431 epidermoid cancer cell line as a cell line that overexpresses EpCAM, but is exquisitely sensitive to manipulation of EpCAM expression. A431 is known to overexpress EGFR and serves as a model cell line for EGFR signaling. Specific ablation of EpCAM significantly enhanced the migration and invasion of the A431 cell line. Specific ablation of EpCAM was also associated with increased EGFR activity, as demonstrated by phospho-immunoblot. Immunoprecipitation studies demonstrate that EpCAM binds directly to EGFR. Using specific EpCAM mutant constructs, we showed that the N-terminal domain of EpCAM is required for binding and suppression of EGFR activity. Using a soft agar colony growth assay, we were able to confirm that N-terminal EpCAM domain mutants (NTD288) inhibited colony growth in 3T3-transfected cells relative to the wildtype. In addition, a transformation assay showed that the same N-terminal mutant was able to inhibit transformation of 3T3 cells. To assess the effects of EpCAM ablation on in vivo tumor growth, we specifically ablated EpCAM and then performed tumor challenge experiments using immunocompromised mice. Specific ablation of EpCAM increased tumor growth relative to control. These data suggest that EpCAM may modulate EGFR signaling. Further studies are ongoing to define the mechanisms by which EpCAM inhibits EGFR activity. Citation Format: Lincoln Muhoro, Timothy Fleming, Narendra Sankpal, William Gillanders. Role of EpCAM in EGFR signaling and tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5065.
Currently, development of anti-cancer therapies is limited to antigens that are either overexpressed or that are specific to cancer. This limits both the number of available targets and their tumor selectivity. Using phage display technology, we identified a number of proteins in multiple cancer types that are induced by sub-lethal doses of ionizing radiation (IR), including TATA-box-binding protein associated factor 15 (TAF-15), a key transcriptional regulator. TAF15 presents a novel synergistic molecular target in cancer for both therapeutic and imaging purposes. In the first part of this study, we characterized TAF15 as a radiation-inducible molecular target both in vitro and in vivo using various analytical approaches. Surface expression analysis of TAF15 via flow cytometry showed that TAF15 is not only expressed on the surface of brain, lung and breast cancer, but is also induced by IR reproducibly on the surface of breast cancer and endothelial cells, and not in normal cells. Using a heterotopic tumor model in athymic nude mice, near-infrared (NIR) imaging analysis results showed that TAF15 can be successfully targeted in several different irradiated human tumor xenografts using both an anti-TAF15 antibody (Ab) and our in-house anti-TAF15 peptide. More importantly, we demonstrated that TAF15 can also be targeted in an orthotopic primary tumor model; NIR results showed high affinity binding of an anti-TAF15 Ab to irradiated primary breast tumors established in the mammary fat pads of nude mice as compared to isotype-matched controls. Furthermore, we have successfully genetically modified the knob domain of an adenovirus (Ad) type 5 fiber protein to contain our lead anti-TAF15 peptide and showed that this novel Ad-TAF15 targeting peptide binds strongly to irradiated breast tumor xenografts in a recent pilot study using bioluminescence imaging (BLI), a result that has major therapeutic and tumor imaging implications. We are currently evaluating the microscopic biodistribution of TAF15 in the primary tumor microenvironment using immunofluorescence and whether cell surface binding of TAF15 can activate an immune response, either via antibody-dependent cell mediated cytotoxicity (ADCC) or antibody dependent cell mediated phagocytosis (ADCP). In the second part of this study, we initiated anti-TAF15 monoclonal antibody (mAb) production against 3 different TAF15 epitopes using hybridoma technology. After screening for binding specificity to TAF15 binding via Western and dot blot analysis, we identified several lead anti-TAF15 antibody clones for further sub-cloning. We are currently evaluating the binding affinity to TAF15, in vivo efficacy and cancer cell specificity of these lead clones. Future studies include humanization of these candidate mAbs and evaluation of their cancer specificity and anti-cancer cytotoxicity with the goal of introducing the most efficacious and selective anti-TAF15 mAbs into clinical trials. Note: This abstract was not presented at the meeting. Citation Format: Lincoln Muhoro, Heping Yan, Sergey Kaliberov, Jerry Jaboin, David Curiel, Dennis Hallahan. Characterization and targeting of TAF15, a radiation-inducible target in multiple cancer types. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4376. doi:10.1158/1538-7445.AM2015-4376
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