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2008 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests Antibodies are primary tools of biomedical science. In basic research, the characterization and analysis of almost any molecule involves the production of specific monoclonal or polyclonal antibodies that react with it. Antibodies are also widely used in clinical diagnostic applications. Further, antibodies are making rapid inroads into clinical treatment of a variety of diseases, driven by technological evolution from chimeric and humanized to fully human antibodies. Functioning as an antibody production core facility at VARI, our lab’s primary responsibility is to develop state-of-the-art services and technology platforms for monoclonal antibody (mAb) production and characterization. Our technologies and services include antigen preparation and animal immunization; peptide design and coupling to protein carriers; immunization with living or fixed cells; conventional antigen/adjuvant preparation; and immunizing a wide range of antibody-producing models (including mice, rats, rabbits, and transgenic or knock-out mice). Our work also includes the generation of hybridomas from spleen cells of immunized mice and rats; hybridoma expansion and subcloning; cryopreservation of hybridomas; mAb isotyping; ELISA screening of hybridoma supernatants; mAb characterization by immunoprecipitation, immunohistochemistry, immunofluorescence staining, Western blot, FACS, and in vitro bioassays; conjugation of mAbs to enzymes, biotin/streptavidin, or fluorescent reporters; and development of detection kits such as sandwich ELISA. We contract our services to biotechnology companies, producing and purifying mAbs for their research and for diagnostic kit development. Over the last year, this core has finished 14 antibody development projects for researchers and industrial users in Michigan and nationwide. Michigan’s Core Technology Alliance (CTA), funded by the state government, was created in 2001. The Antibody Technology Core at VARI and the Hybridoma Core at the University of Michigan in Ann Arbor joined together to form the Michigan Antibody Technology Core (MATC) and became the seventh core of the CTA in March 2005. The goals of MATC are to provide state-ofthe-art antibody technologies and services to research scientists; to generate, characterize, produce, and purify a wide variety of mAbs for clinical diagnostic/therapeutic applications; and to advance biomedical research and development. The Antibody Technology lab at VARI serves as the core’s hub, and Dr. Brian Cao is the director of MATC. We also carry out research and collaboration projects, which use both murine mAbs and human antibody fragments generated in our lab, aimed at developing cancer diagnostic and therapeutic applications. • Epitope mapping and characterization of a Met-binding peptide using phage-display peptide libraries. This project is to screen for a specific Met-binding peptide from a random-peptide phage-display library that could be used as an in vivo imaging agent (and possibly as a therapeutic carrier) when labeled with radioisotopes or conjugated with chemotherapeutics. A subtractive bio-panning approach on intact cells was used. A Met-binding peptide was obtained that recognizes the Met extracellular domain under native conditions and internalizes upon binding to the Met receptor. In vivo imaging showed that the radiolabeled peptide in a mouse xenograft model had tumor-associated activity. We are modifying this peptide to increase its binding affinity, and we are screening new Met-binding peptides having higher affinity for future clinical applications. • Development of highly specific anti-Met mouse mAbs with potential application for clinical immunohistochemical diagnosis. In collaboration with Beatrice Knudsen’s lab at the Fred Hutchinson Cancer Research Center, we have developed a monoclonal antibody, designated MET4, with the goal of accurately and reproducibly measuring MET in formalin-fixed paraffin-embedded (FFPE) tissues. MET4 was selected as the best probe from a pool of MET-avid monoclonal antibodies, based on its specific staining pattern in FFPE preparations of normal human prostate tissues. The reliability of MET4 immunohistochemistry was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis, which demonstrated a high avidity of MET4 for formalin-treated MET. These properties encourage further development of MET4 as a multipurpose molecular diagnostic reagent to help guide selection of individual patients being considered for treatment with METantagonistic drugs. 10

VARI | 2008 • Characterization of anti-EGFR and anti-Met human Fab fragments and conjugation with chemotherapeutics to generate reagents for preclinical studies. In collaboration with the Ministry of Health’s Key Laboratory of Antibody Technology in Nanjing Medical University, we screened several Fab fragments (from a naïve human Fab phage library constructed in our lab in late 2004) that specifically recognize Met and EGFR. By modifying and improving bio-panning strategies, we have selected Fab fragments that recognize the Met and EGFR extracellular domains in native confirmation with reasonable affinity. These fragments have internalization properties making them attractive as conjugate reagents for immuno-chemotherapy or immuno-radiation therapy against cancer. We have conjugated anti-EGFR human Fab to paclitaxel as an immuno-chemotherapy reagent and investigated its in vitro anti-tumor efficacy using cell proliferation and apoptosis assays. We will further explore its in vivo anti-tumor efficacy in xenograft or orthotopic animal models, and we will label this Fab fragment with radioisotopes to evaluate its potential as an immuno-radiation reagent for in vivo imaging diagnosis and immuno-radiation therapy. From left: Zhao, Sun, Nelson, Ding, Grabinski, Cao Recent Publications Wang, Xin, Jin Zhu, Ping Zhao, Yongjun Jiao, Ning Xu, Tessa Grabinski, Chao Liu, Cindy K. Miranti, Tao Fu, and Brian B. Cao. 2007. In vitro efficacy of immuno-chemotherapy with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy 6(6): 980–987. Zhao, Ping, Tessa Grabinski, Chongfeng Gao, R. Scot Skinner, Troy Giambernardi, Yanli Su, Eric Hudson, James Resau, Milton Gross, George F. Vande Woude, Rick Hay, and Brian Cao. 2007. Identification of a Met-binding peptide from a phage display library. Clinical Cancer Research 13(20): 6049–6055. 11

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