The broad, long-term objective is to produce an effective diagnostic and therapeutic humanized antibody against adenocarcinomas. Humanized antibodies (huAbs) are rapidly emerging as a novel treatment for various diseases. Several clinical trials are presently underway that have resulted from advances in the ability to discover putative cancer markers or physiologically relevant targets. The proposed target, labyrinthin, was discovered and cloned using a mouse monoclonal antibody (MCA 44-3A6) that reacted with adenocarcinoma tumors arising in human lungs. Labyrinthin has subsequently been identified in over 600 adenocarcinoma cell lines, tissues and specimens, and further characterized as a membrane associated protein with extracellular epitopes that are accessible to antibodies. Functional relevance has been implicated by recent data in which labyrinthin overexpression in normal cells promotes a cancer phenotype, whereas adenocarcinoma cells appear normal after decreased expression via antisense transfection. Labyrinthin has 94% amino acid identity with a recently discovered protein, junctate, that regulates calcium in the endoplasmic reticulum of normal cells. Because labyrinthin lacks junctate's ER targeting signal it may contribute to the elevated intracellular calcium phenomenon in cancer cells. The data collectively support the hypothesis that labyrinthin is a diagnostic marker and therapeutic target for adenocarcinomas without respect to origin. Computer analyses of the extracellular epitopes indeed signify several diagnostic and/or therapeutic immunogenic sites, and have provided a basis to construct labyrinthin-based peptides for a vaccine that is presently in PhaseI/II clinical trials. The present aims are to design humanized antibodies as an alternative (or complementary) approach. Specifically, we will: 1) Develop prototypic anti-labyrinthin huAb fragments. The MCA 44-3A6 sequence will be computer analyzed by a proprietary method that models a high affinity huAb. The resulting computer-based design is used to develop a phage display library that will be selected by phage ELISA against immobilized labyrinthin-based antigens that correspond to extracellular domains. 2) Evaluate the huAbs affinity and specificity. The binding kinetics of those individual clones from Aim 1 that recognize labyrinthin-based epitopes will be analyzed by surface plasmon resonance array (Biacore). The final huAB candidates' ability to bind native labyrinthin in normal vs. adenocarcinoma cells will be detected by immunocytochemistry. Successful development of an anti-labyrinthin huAb would provide the framework for:1) a useful tool for diagnostics; 2) support of labyrinthin as a widespread therapeutic target via further development as a diagnostic antibody.
