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2006 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report 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 director of MATC. Mapping an antibody-binding epitope to a protein-protein or antigen binding site using a phage-display random peptide library is a powerful technology for studying protein-protein interactions and the kinetics of antibody interactions, as well as in screening for small peptides that have potential therapeutic applications. A random peptide library is constructed by genetically fusing oligonucleotides coding for polypeptides to the DNA coding for a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the virion. Phage display has been used to create a physical linkage between a vast library of random peptide sequences and the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules such as antibodies. A library of phage is exposed to a plate coated with mAb. Unbound phage are washed away and then specifically bound phage are eluted by lowering the pH. The eluted pool of phage is amplified, and the process is repeated for two more rounds. Individual clones are isolated, screened by ELISA, and sequenced. 12 With this technology, we have been able to screen out and identify a small peptide that specifically binds to Met, the receptor of hepatocyte growth factor/scatter factor (HGF/SF). Activation of Met by HGF/SF affects downstream signaling pathways (including additional protein kinases) responsible for cellular differentiation, motility, proliferation, organogenesis, angiogenesis, and apoptosis. Aberrant expression of the Met-HGF/SF receptor-ligand complex—resulting either from mutations in the complex or in conjunction with mutations in other oncogenes—is associated with an invasive/metastatic phenotype in most solid human tumors. Met-HGF/SF and downstream kinases are therefore attractive targets for new agents aimed at clinical diagnosis, prognosis, and treatment of cancer. We have also successfully epitope-mapped several neutralizing anti-HGF/SF mAbs. The information about the antibody-binding site obtained from these experiments should lead us to new strategies and new reagents for cancer intervention. In collaboration with Nanjing Medical University, China, we have initiated a project to construct a phage-display antibody fragment library, which will involve the construction and use of human/animal immunized/naïve Fab and scFv antibody gene repertoires. The ability to co-select antibodies and their genes enables the isolation of high-affinity, antigen-specific mAbs derived from either immunized animals or non-immunized humans. Over the past two years, we have closely followed the development of this technology for producing novel recombinant antibody-like molecules. We constructed a human naïve Fab 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. Moreover, by modifying and improving biopanning strategies, we have selected Fab fragments that recognize both the Met and EGFR extracellular domains in native confirmation with reasonable affinity and, importantly, with an internalization property that makes these Fabs attractive as conjugate reagents for immuno-chemotherapy or immuno-radiation therapy against cancer. External Collaborators We have established strong collaborations internationally and locally with universities, institutions, and companies. Some examples are the Key Laboratory of Antibody Technology, Nanjing Medical University, China; the Medical Research Council (MRC), Cambridge, England; the Protein Research Department, Mubarak Scientific City, Egypt; Neogen Corporation, Lansing, Michigan; and Assay Designs, Inc., Ann Arbor, Michigan.

VARI | 2006 13 Recent Publications From left: Grabinski, Nelson, Cao, Wang, Zhang, Zhao Tsarfaty, G., G.Y. Stein, S. Moshitch-Moshkovitz, D.W. Kaufman, B. Cao, J. Resau, G.F. Vande Woude, and I. Tsarfaty. In press. HGF/SF increases tumor blood volume: a novel tool for in vivo functional molecular imaging of Met. Neoplasia. Hay, Rick V., Brian Cao, R. Scot Skinner, Yanli Su, Ping Zhao, Margaret F. Gustafson, Chao-Nan Qian, Bin T. Teh, Beatrice S. Knudsen, James H. Resau, Shuren Shen, David J. Waters, Milton D. Gross, and George F. Vande Woude. 2005. Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek). Clinical Cancer Research 11(19): 7064s–7069s. Jiao, Yongjun, Ping Zhao, Jin Zhu, Tessa Grabinski, Zhengqing Feng, Xiaohong Guan, R. Scot Skinner, Milton D. Gross, Rick V. Hay, Hiroshi Tachibana, and Brian Cao. 2005. Construction of human naïve Fab library and characterization of anti-Met Fab fragment generated from the library. Molecular Biotechnology 31(1): 41–54. Ren, Yi, Brian Cao, Simon Law, Yi Xie, Ping Yin Lee, Leo Cheung, Yongxong Chen, Xin Huang, Hiu Man Chan, Ping Zhao, John Luk, George Vande Woude, and John Wong. 2005. Hepatocyte growth factor promotes cancer cell migration and angiogenic factors expression: a prognostic marker of human esophageal squamous cell carcinomas. Clinical Cancer Research 11(17): 6190–6197.

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