Heping Yan

TIP-1 translocation onto the cell plasma membrane is a molecular biomarker of tumor response to ionizing radiation

Background Tumor response to treatment has been generally assessed with anatomic and functional imaging. Recent development of in vivo molecular and cellular imaging showed promise in time-efficient assessment of the therapeutic efficacy of a prescribed regimen. Currently, the in vivo molecular imaging is limited with shortage of biomarkers and probes with sound biological relevance. We have previously shown in tumor-bearing mice that a hexapeptide (HVGGSSV) demonstrated potentials as a molecular imaging probe to distinguish the tumors responding to ionizing radiation (IR) and/or tyrosine kinase inhibitor treatment from those of non-responding tumors. Methodology/Principal Findings In this study we have studied biological basis of the HVGGSSV peptide binding within the irradiated tumors by use of tumor-bearing mice and cultured cancer cells. The results indicated that Tax interacting protein 1 (TIP-1, also known as Tax1BP3) is a molecular target that enables the selective binding of the HVGGSSV peptide within irradiated xenograft tumors. Optical imaging and immunohistochemical staining indicated that a TIP-1 specific antibody demonstrated similar biodistribution as the peptide in tumor-bearing mice. The TIP-1 antibody blocked the peptide from binding within irradiated tumors. Studies on both of human and mouse lung cancer cells showed that the intracellular TIP-1 relocated to the plasma membrane surface within the first few hours after exposure to IR and before the onset of treatment associated apoptosis and cell death. TIP-1 relocation onto the cell surface is associated with the reduced proliferation and the enhanced susceptibility to the subsequent IR treatment. Conclusions/Significance This study by use of tumor-bearing mice and cultured cancer cells suggested that imaging of the radiation-inducible TIP-1 translocation onto the cancer cell surface may predict the tumor responsiveness to radiation in a time-efficient manner and thus tailor radiotherapy of cancer.

Radiation-Guided Drug Delivery to Mouse Models of Lung Cancer

Purpose: The purpose of this study was to achieve improved cancer-specific delivery and bioavailability of radiation-sensitizing chemotherapy using radiation-guided drug delivery. Experimental Design: Phage display technology was used to isolate a recombinant peptide (HVGGSSV) that binds to a radiation-inducible receptor in irradiated tumors. This peptide was used to target nab-paclitaxel to irradiated tumors, achieving tumor-specificity and enhanced bioavailability of paclitaxel. Results: Optical imaging studies showed that HVGGSSV-guided nab-paclitaxel selectively targeted irradiated tumors and showed 1.48 ± 1.66 photons/s/cm2/sr greater radiance compared with SGVSGHV-nab-paclitaxel, and 1.49 ± 1.36 photons/s/cm2/sr greater than nab-paclitaxel alone (P < 0.05). Biodistribution studies showed >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel–treated groups as compared with either nab-paclitaxel or SGVSGHV-nab-paclitaxel at 72 hours. Both Lewis lung carcinoma and H460 lung carcinoma murine models showed significant tumor growth delay for HVGGSSV-nab-paclitaxel as compared with nab-paclitaxel, SGVSGHV-nab-paclitaxel,and saline controls. HVGGSSV-nab-paclitaxel treatment induced a significantly greater loss in vasculature in irradiated tumors compared with unirradiated tumors, nab-paclitaxel, SGVSGHV-nab-paclitaxel, and untreated controls. Conclusions: HVGGSSV-nab-paclitaxel was found to bind specifically to the tax-interacting protein-1 (TIP-1) receptor expressed in irradiated tumors, enhance bioavailability of paclitaxel, and significantly increase tumor growth delay as compared with controls in mouse models of lung cancer. Here we show that targeting nab-paclitaxel to radiation-inducible TIP-1 results in increased tumor-specific drug delivery and enhanced biological efficacy in the treatment of cancer. Clin Cancer Res; 16(20); 4968–77. ©2010 AACR.

Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers.

Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1. Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors. We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro. In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control.

Abstract 4376: Characterization and targeting of TAF15, a radiation-inducible target in multiple cancer types

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnCurrently, 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.nnNote: This abstract was not presented at the meeting.nnCitation 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

Abstract 4597: Characterization and targeting of radiation-inducible neoantigens in multiple cancer types

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnTargeted 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 cells 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.nnCitation 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