Project 1: Targeting glypican-3 in hepatocellular carcinoma Liver cancer is the fifth most common malignant cancer worldwide. Hepatocellular carcinoma (HCC) accounts for approximately 75% of liver cancer cases. Cholangiocarcinoma (CCA) is the second most common primary liver tumor. Although liver cancer is uncommon in the United States, rates of liver cancer diagnosis have been increasing in recent years. While surgical resection offers a standard method for treatment of the disease, only a small portion of patients are eligible for the procedure. There is an urgent need for new treatments that can be successfully applied to a large population of patients. Our work on glypican-3 (GPC3) is to investigate the role of GPC3 in cancer pathogenesis and to identify human antibodies for cancer therapy. GPC3 is highly expressed in HCC and has been suggested as a potential target for liver cancer therapy. In FY10, we produced a recombinant GPC3 protein without the GPI anchor and found that the recombinant GPC3, functioning as a dominant-negative form, directly inhibited the growth of HCC in vitro. This observation was published in International Journal of Cancer. We hypothesize that neutralizing GPC3 oncogenic functions represents a novel therapeutic approach for treating HCC. In this manner, we successfully isolated human antibodies by phage display in FY11. The anti-GPC3 monoclonal antibodies (mAbs) may also be useful for immunoconjugates, chimeric antigen receptor-modified T cell therapy, diagnostic tumor imaging and other clinical applications. The NCI has filed two patent applications based on our GPC3 research in FY11. We hope to find an industrial partner to further develop our GPC3 mAbs in order to treat cancer patients. In FY11, the GPC3 project yielded four publications in the following journals: International Journal of Cancer, European Journal of Cancer, BioDrugs and Cancer Reports. In FY11, we were invited to present our work at 7th PEGS Protein Summit Inaugural Antibodies for Cancer Therapy Symposium and BIO2011. Project 2: Targeting mesothelin in liver cancer, mesothelioma and ovarian cancer Mesothelin is expressed at high levels in mesothelioma, as well as ovarian, pancreatic, and lung cancers. In FY10, in collaboration with Gregory Gores (Mayo Clinic) and Xin Wei Wang (NCI), we analyzed protein expression of mesothelin in liver cancer. Our results show that mesothelin is a potential novel target in cholangiocarcinoma (CCA). This work was published in the Journal of Cancer in FY11. We applied our expertise in phage display technology to identify HN1, a human mAb specific for mesothelin, by collaborating with Ira Pastan (NCI). The HN1 mAb binds cell surface-associated mesothelin with high affinity. The HN1 mAb kills cancer cells with excellent antibody-dependent cell mediated cytotoxicity (ADCC) and is promising for the treatment of mesothelin-expressing cancers. In FY11, we published this work in the International Journal of Cancer. Mesothelin is a MUC16 binding protein and the mesothelin-MUC16 interaction may lead to intra-peritoneal dissemination of tumors. To investigate this important molecular interaction, we used a truncation and alanine scanning mutagenesis approach to identify a distinct functional binding domain (named IAB) in mesothelin for MUC16 We show that IAB can block the binding of mesothelin to MUC16 and inhibit heterotypic cancer cell adhesion. This finding is important for us to understand the novel mechanisms of cancer metastasis involving mesothelin. In FY09, this work was published in the Journal of Biological Chemistry. In FY10-11, we generated the IAB-human Fc fusion protein as a novel immunoadhesin (HN125). We collaborated with Manish Patankar (University of Wisconsin-Madison) to investigate the utility of HN125 for prospective ovarian cancer therapy and publish our work in the Journal of Cancer in FY11. To evaluate the anti-tumor activity in vivo, we engineered a human mesothelioma cell line (named LMB-H226-GL) for in vivo bioluminescence tumor imaging. This is a well-suited model for mesothelioma, and may be useful for evaluating other novel agents for mesothelioma treatment in vivo. We published this work in the Journal of Cancer in FY11 and collaborated with Raffit Hassan (NCI) to use this model to study efficacy of an anti-insulin like growth factor I receptor (IGF-IR) antibody in mesothelioma. Our mesothelin project is supported in part by a Mesothelioma Applied Research Foundation grant and the Ovarian Cancer Research Fund Individual Investigator Award. We obtained two patents through the NCI on our mesothelin work related to HN1 and IAB in FY10-11. NCI provides the LMB-H226-GL mesothelioma model to the scientific community. In FY11, we were invited to present our mesothelin work in several important cancer therapy meetings such as PEGS Protein Summit, Congress of Molecular Medicine, Molecular Medicine Strategies for Clinical Oncology Drug Development symposium, Multidisciplinary Symposium in Thoracic Oncology and International Symposium on Malignant Mesothelioma. Project 3: Establish in vitro tumor spheroids to investigate antibody penetration. Most studies of anticancer drugs consider only genetic and/or cellular mechanisms at the level of the single cell. However, drug penetration is a highly important additional mechanism and requires a more complex cellular environment to study. The goal of this research is to establish in vitro spheroids in order to examine antibody penetration in solid tumors. Along with our collaborators V. Courtney Broaddus (University of California San Francisco) and Shuichi Takayama (University of Michigan), we made tumor spheroids using established cancer cell lines and primary lines isolated from cancer patients and published the method and initial observations in three journals (PLoS ONE, Analyst and Tissue Engineering) in FY11. Our spheroid research is supported by a NCI Directors Intramural Innovation Award for Principal Investigators in FY11. We were invited to give lectures at 4th Protein Discovery &Therapeutics and Protein Therapeutics Forum in FY12 to present our work on in vitro tumor spheroid model and antibody penetration.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010891-04
Application #
8349225
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2011
Total Cost
$747,016
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Li, Nan; Fu, Haiying; Hewitt, Stephen M et al. (2017) Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma. Proc Natl Acad Sci U S A 114:E6623-E6631
Zhang, Yi-Fan; Ho, Mitchell (2017) Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples. MAbs 9:419-429
Nagaya, Tadanobu; Nakamura, Yuko; Sato, Kazuhide et al. (2017) Near infrared photoimmunotherapy with avelumab, an anti-programmed death-ligand 1 (PD-L1) antibody. Oncotarget 8:8807-8817
Zhang, Yi-Fan; Ho, Mitchell (2016) Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma. Sci Rep 6:33878
Fleming, Bryan D; Ho, Mitchell (2016) Glypican-3 Targeting Immunotoxins for the Treatment of Liver Cancer. Toxins (Basel) 8:
Gao, Wei; Xu, Yongmei; Liu, Jian et al. (2016) Epitope mapping by a Wnt-blocking antibody: evidence of the Wnt binding domain in heparan sulfate. Sci Rep 6:26245
Nagaya, Tadanobu; Nakamura, Yuko; Sato, Kazuhide et al. (2016) Near infrared photoimmunotherapy with an anti-mesothelin antibody. Oncotarget 7:23361-9
Wang, Chunguang; Gao, Wei; Feng, Mingqian et al. (2016) Construction of an immunotoxin, HN3-mPE24, targeting glypican-3 for liver cancer therapy. Oncotarget :
Gao, Wei; Tang, Zhewei; Zhang, Yi-Fan et al. (2015) Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis. Nat Commun 6:6536
Gao, Wei; Kim, Heungnam; Ho, Mitchell (2015) Human Monoclonal Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Inhibits HGF-Mediated Migration and Motility of Hepatocellular Carcinoma Cells. PLoS One 10:e0137664

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