11 months ago

2004 Scientific Report

Laboratory of Antibody

Laboratory of Antibody Technology Brian Cao, M.D. Dr. Cao obtained his M.D. from Beijing Medical University, People’s Republic of China, in 1986. On receiving a CDC fellowship award, he was a visiting scientist at the National Center for Infectious Diseases, Centers for Disease Control and Prevention (1991–1994). He next served as a postdoctoral fellow at Harvard (1994–1995) and Yale (1995–1996). From 1996 to 1999, Dr. Cao was a Scientist Associate in charge of the Monoclonal Antibody Production Laboratory at the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Dr. Cao joined VARI as a Special Program Investigator in June 1999. Staff Ping Zhao, M.S. Tessa Gieske, B.S. Laboratory Members Visiting Scientist Mei Guo, M.S. Student Yong-jun Jiao Research Interests Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional, heterodimeric polypeptide produced by mesenchymal cells that interacts with cells expressing the Met receptor. Met, the product of the c-met protooncogene, is a receptor protein tyrosine kinase of the same family as epidermal growth factor (EGF) receptors. The activation of Met by HGF/SF affects downstream signaling pathways (which include other 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 anti-cancer agents. We have generated a panel of monoclonal antibodies (mAbs) to HGF/SF. Some of these antibodies have biological neutralizing activity when used in combination, and we have characterized their antitumor effects both in vitro and in vivo. Using an in vivo xenograft model, we are particularly interested in studying glioblastoma multiforme. These tumors are the most frequent and malignant form of human brain tumors; they are highly invasive and, irrespective of their histological grade of malignancy, even low-grade tumors can be poorly demarcated and are rarely encapsulated. The 2- and 5-year survival rates for malignant glioma are less than 15% and 5%, respectively. These tumors are also considered to be endothelial-rich tumors, and prognostic relevance can be assigned to the vascular changes themselves, with poor survival correlating with increasing vascular density. HGF/SF and VEGF have been shown to be major mediators in the angiogenesis of glioblastoma multiforme, and HGF/SF is characterized through two different mechanisms: by direct-induction blood vessel formation and by induction of VEGF expression. Our previous results showed that the growth of human glioblastoma multiforme xenografts expressing HGF/SF and its receptor Met is markedly inhibited by combined neutralizing mAbs to HGF/SF; similar results have been shown with neutralizing mAb to VEGF by other labs, but none of the results have led to tumor regression. As tumors grow, they begin to produce a wide array of angiogenic molecules; if one angiogenic molecule is blocked, tumors may switch to pathways using a different molecule. We are currently developing and characterizing anti-VEGF mAbs. In collaboration with the microarray technology and cellular/molecular imaging programs at VARI, we seek to understand the relationships between key growth factors (VEGF, HGF/SF, etc.) and their receptors that stimulate tumor angiogenesis and metastasis, and to evaluate the effect of combinations of mAbs to these growth factors for clinical immunotherapeutic potential. 12

The aberrant expression of the highly tumorigenic Met receptor kinase in two-thirds of localized prostate cancers, and evidently in all osseous metastases, suggests that Met provides a strong selection for metastatic development. In a collaborative investigation, we are using our two radiolabeled anti-Met-extracellular-domain mAbs, designated Met3 and Met5, to study mouse xenograft and orthotopic models of localized and metastatic prostate cancer, with a view toward clinical nuclear imaging diagnostics and radio-immunotherapeutic applications. Moreover, we will be soon testing these two radiolabeled mAbs on dog spontaneous prostate cancers and bone metastasis models. Over the past few years, we have established the technology of a phage-display peptide library for mAb epitope mapping. A random peptide library is constructed by genetically fusing oligonucleotides coding for polypeptides of a given length to the coding sequence of a bacteriophage coat protein, resulting in display of the fused protein on the surface of the phage and its genetic element residing within. 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 phages is exposed to a plate coated with mAb. Unbound phages are washed away, and specifically-bound phages 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. We have successfully mapped epitopes of a variety of important mAbs such as anti-HGF/SF, anti- Met, and anti–anthrax lethal factor. We are now exploring this technology with protein-protein interactions—for example, mapping of the HGF/SF-Met binding site(s) in an in vitro system—and several interesting peptides have been selected from the library as having potential Met antagonistic activity. In collaboration with Nanjing Medical University of China, we have initiated the construction of a phage-display antibody fragment library. This technique involves the construction and use of human/animal, immunized/naive, Fab and scFv antibody gene repertoires by phage display. The ability to co-select antibodies and their genes allows the isolation of high-affinity, antigenspecific mAbs derived from either immunized animals or nonimmunized humans. A variety of procedures for selecting such antibodies from recombinant libraries have been described, and some useful antibodies have been produced using this approach. Over the past two years, we have closely followed the development of this technology for producing novel recombinant antibody-like molecules. We have constructed a human naive Fab library with a diversity of 2 × 10 9 , and from it we have screened out some specific mAb fragments against a tumor cell membrane protein. A new scFv library is under construction. Functioning as an antibody production facility at the Van Andel Research Institute, this lab has extensive capabilities in the generation, characterization, scaled-up production, and purification of a variety of mAbs using comprehensive, cutting-edge technologies. The technologies and services available include antigen preparation and animal immunization; peptide design and coupling to protein carriers; consultation about protein expression and purification; DNA immunization (gene-gun technology); immunization with living or fixed cells; conventional antigen/adjuvant preparation; immunization of a wide range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro immunization. Also available are conventional techniques of hybridoma production, including the generation of hybridomas from spleen cells of immunized mice and rats; hybridoma expansion and subcloning; cryopreservation of hybridomas that secrete mAbs; mAb isotyping; ELISA screening of hybridoma supernatants; consultation on other hybridoma screening techniques; production of bulk quantities of mAbs using high-density cell culture techniques; and many more. Over the past few years, this facility has generated more than 200 different mAbs, resulting in or contributing to the submission and funding of grant proposals, the filing of four patent applications, and the licensing of several of these antibodies. We have also established contract services with local biotechnological companies to generate, characterize, produce, and purify mAbs for their research and diagnostic kit development. 13

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