[Proceedings of the 11th NCI · EORTC · AACR Symposium]
Copyright © 2000 Stichting NCI-EORTC Symposium on New Drugs in Cancer Therapy
Published by the AACR.
176 Identification of targets for immunotherapeutic antibodies by mRNA expression profiling clinical prostate tumor samples. Hevezi P.1, Gish K.1, Henshall S.2, Sutherland R.2, Holloway A.3, Venter D.3, Mack D.1. 1 Eos Biotechnology, South San Francisco, CA, USA, 2 The Garvan Institute, Sydney, Australia 3 Peter MacCallum Cancer Institute, Melbourne, Australia.
Fully humanized monclonal antibodies have the promise to be specific and potent antitumor agents. We have utilized a proprietry Affymetrix GeneChip® microarray to select genes that are specifically expressed in tumors, a subset of which encode plasma membrane or secreted proteins and potential targets for monoclonal antibody immunotherapy. This custom array contains probes to interogate >35,000 unique genes and EST clusters on a single array. Total RNA is isolated from fresh frozen tumor biopsy samples (40 independent samples per tumor type), or, normal body tissues (26 tissue and organ types) and is labeled and hybridized to our proprietary array for expression profiling. Following normalization, data is queried for "tumor specific" genes (genes whose expression is elevated >3 fold in tumors and low or absent in normal tissues). Of the >35,000 genes and EST clusters represented on the propriety GeneChip® array, typically 30 - 40% are detected in a given tissue. Of these, approximately 200 exhibit the desired tumor specific expression pattern (target genes). Target EST sequences are extended in silico and/or in vitro to identify the open reading frame and allow prediction of subcellular localization of the gene product. In parallel experiments, expression specificity, at the tumor and transformed cell level, are confirmed via in situ hybridization (ISH) of target mRNA to tissue arrays. Target genes that are predicted to encode membrane or secreted proteins and exhibit tumor specific expression by ISH are expressed in tissue culture to confirm subcellular localization, provide material to generate antibodies and enable functional validation of the gene product as a target of immunotherapuetic antibodies. To exemplify our approach, a subset of targets identified in our prostate cancer program will be presented including previously identified prostate specific markers (PSA, PMSA, Kallikrin-2) and examples of novel disease-specific cell surface targets. |
