2009 Scientific Report

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Van Andel Research Institute | Scientific Report Research Interests Functioning as an antibody production core facility at VARI, our lab develops state-of-the-art services and technology platforms for monoclonal antibody (mAb) production and characterization. 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. 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. We have also taken part over the past year in the following research projects. • A single neutralizing mAb against the HGF/SF alpha domain. Hepatocyte growth factor/scatter factor (HGF/ SF) is a multifunctional heterodimeric polypeptide produced by mesenchymal cells; it is an effector of cells expressing the Met tyrosine kinase receptor. We previously generated a cocktail of three or four neutralizing mAbs against HGF/SF that significantly inhibited the HGF-Met signaling pathway in Met-expressing cells. In a glioblastoma multiforme xenograft model, our cocktail showed potent inhibition of tumor growth. Amgen and others have reported a single anti-HGF/SF b-subunit mAb that is able to inhibit biological activities of HGF/SF; it is in early clinical trials. We hypothesized that two mAbs that react with different subunits (a and b) of HGF/SF in combination would have stronger anti-tumor activity than any single antibody. Using a unique immunization protocol, we have generated a mAb against the HGF/SF a subunit (designated HGF8) that has neutralizing activity. Our current results show that HGF8 is able to block HGF/SF-induced scattering of MDCK cells, and in collaboration with the Vande Woude lab, we have shown that HGF8 also significantly inhibits the Met-HGF/SF signaling pathway in vitro using uPA and cell proliferation assays. The in vivo antitumor activity of HGF8 is now under investigation in a brain tumor xenograft model using HGF/SF transgenic mice established by the Vande Woude lab. • 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 the selection of individual patients being considered for treatment with MET antagonistic drugs. 12
VARI | 2009 • Generation of monoclonal antibodies against pancreatic cancer biomarkers. In March 2008, the Lustgarten Foundation officially launched the Pancreatic Cancer Biomarker Development Initiative. Identifying key pancreatic cancer biomarkers and producing antibodies against them is the first step toward developing a blood test for this disease. A consortium of investigators representing four leading cancer research organizations— including the Canary Foundation, Dana-Farber Cancer Institute, University of California, San Francisco, and Van Andel Research Institute—will study a total of 60 candidate biomarkers. We have been assigned 15 biomarkers and funding for 18 months. The project is to develop monoclonal antibodies against those biomarkers, including paired mAbs for sandwich ELISA development, mAbs specifically for western blotting and immunohistochemical study, etc. All biomarkers need to be expressed and purified by the lab. This project also requires collaboration with other labs and core facilities. For example, we will collaborate with Brian Haab’s VARI lab to identify paired mAbs for sandwich ELISA development using antibody array technology. We will also use James Resau’s VARI histology/pathology core and tissue microarray technology to characterize the mAbs that work best for immunohistochemical staining. Recent Publications Knudsen, Beatrice S., Ping Zhao, James Resau, Sandra Cottingham, Ermanno Gherardi, Eric Xu, Bree Berghuis, Jennifer Daugherty, Tessa Grabinski, Jose Toro, et al. 2009. A novel multipurpose monoclonal antibody for evaluating human c-Met expression in preclinical and clinical settings. Applied Immunohistochemistry and Molecular Morphology 17(1): 56–67. Nguyen, Melissa L., Sherry R. Crowe, Sridevi Kurella, Simon Teryzan, Brian Cao, Jimmy D. Ballard, Judith A. James, and A. Darise Farris. 2009. Sequential B cell epitopes of Bacillus anthracis lethal factor bind lethal toxin–neutralizing antibodies. Infection and Immunity 77(1): 162–169. Wang, Xin, and Brian B. Cao. 2009. Screening of specific internalization Fab fragments from human naïve phage library by combinational bio-panning. In Therapeutic Antibodies: Methods and Protocols, Antony S. Dimitrov, ed. Methods in Molecular Biology series, Vol. 525. New York: Humana Press, pp. 161–174. Chen, Jindong, Kunihiko Futami, David Petillo, Jun Peng, PengFei Wang, Jared Knol, Yan Li, Sok Kean Khoo, Dan Huang, Chao-Nan Qian, et al. 2008. Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia. PLoS One 3(10): e3581. Xie, Qian, Ryan Thompson, Kim Hardy, Lisa DeCamp, Bree Berghuis, Robert Sigler, Beatrice Knudsen, Sandra Cottingham, Ping Zhao, Karl Dykema, et al. 2008. A highly invasive human glioblastoma pre-clinical model for testing therapeutics. Journal of Translational Medicine 6: 77. From left: Ding, Lin, Wang, Cao, Grabinski, Zhu, Nelson, Zhao 13
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