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2007 Scientific Report

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VARI |

VARI | 2007 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 diagnostic applications for clinical medicine. ELISA and radioimmunoassay systems are antibody-based. Analysis of cells and tissues in pathology laboratories includes the use of antibodies on tissue sections and in flow cytometry analyses. Further, antibodies are making rapid inroads into medical therapeutics, driven by technological evolution from chimeric and humanized to fully human antibodies. The therapeutic antibody market has the potential to reach billion by 2010. Our Antibody Technology laboratory has developed several technologies over the last few years: 1) state-of-the-art monoclonal antibody (mAb) production and characterization, followed by scaled-up production and purification; 2) antibody-binding-site epitope mapping using a phage-display peptide library; 3) a human-antibody-fragment phage-display library and screening of specific fragments from the library; and 4) characterization of these human antibody fragments and conjugation with chemotherapeutics to generate immuno-chemotherapeutic reagents for preclinical studies. In collaboration with Nanjing Medical University, China, we constructed our own human naïve Fab fragment phage-display library, with a diversity of 2 × 10 9 , in late 2004. In 2005, we screened out several Fab fragments from the library that specifically recognize HGF/SF, Met, and EGFR. By modifying and improving biopanning strategies, we have selected Fab fragments that recognize the Met and EGFR extracellular domains in native conformation with reasonable affinity and, importantly, with the internalization property that makes these Fabs attractive as conjugate reagents for immuno-chemotherapy or immuno-radiation therapy against cancer. In the past year, we have conjugated anti-EGFR human Fab to paclitaxel (Taxol) as an immuno-chemotherapy agent and investigated its in vitro anti-tumor efficacy on A431 epidermoid carcinoma cells using cell proliferation inhibition and apoptosis assays. The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner; more than 70% inhibition was observed at 52 pM. Furthermore, almost 100% of the cells underwent apoptosis after treatment with Fab-Taxol at 26 pM for 48 hours. The in vitro anti-tumor efficacy is four- to fivefold more potent than Taxol alone. We are modifying the Taxol conjugation conditions and working with other drug conjugations to investigate their in vivo anti-tumor efficacy in xenograft and orthotopic animal models. 11

Van Andel Research Institute | Scientific Report Functioning as an antibody production core facility, this lab has extensive capabilities. Our technologies and services include antigen preparation and animal immunization; peptide design and coupling to protein carriers; DNA immunization (gene-gun technology); immunization with living or fixed cells; conventional antigen/adjuvant preparation; immunizing a wide range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro immunization. Our work also includes the generation of hybridomas from spleen cells of immunized mice, rats, and rabbits; hybridoma expansion and subcloning; cryopreservation of hybridomas secreting mAbs; isotyping of mAbs; ELISA screening of hybridoma supernatants; mAb characterization by immunoprecipitation, Western blot, immunohistochemistry, immunofluorescence staining, FACS, and in vitro bioassays; generation of bi-specific mAbs by secondary fusion; conjugation of mAbs to enzymes, biotin/streptavidin, or fluorescent reporters; and development of detection methods/kits such as sandwich ELISA. We also contract services to biotechnology companies, producing and purifying mAbs for their research and for diagnostic kit development. The Michigan 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 CTA in March 2005. Our goals are to provide state-of-the-art antibody technologies and services to research scientists; to generate, characterize, produce, and purify a wide variety of monoclonal antibodies; to make human antibody fragments and humanize murine 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. 12 From left: Gu, Zhang, Xu, Zhao, Nelson, Grabinski, Cao Recent Publications Wang, X., J. Zhu, P. Zhao, Y. Jiao, N. Xu, T. Grabinski, C. Liu, C.K. Miranti, T. Fu, and B. Cao. In press. In vitro efficacy of immunochemotherapy with anti-EGFR human Fab-Taxol conjugate on A431 epidermoid carcinoma cells. Cancer Biology & Therapy. Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276. Tsarfaty, Galia, Gideon Y. Stein, Sharon Moshitch-Moshkovitz, Dafna W. Kaufman, Brian Cao, James H. Resau, George F. Vande Woude, and Ilan Tsarfaty. 2006. HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging of Met. Neoplasia 8(5): 344–352.

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