Targeting the Tight Junction: Immunotherapy of Colon Cancer

A33 is a cell surface glycoprotein of colon epithelium with a long clinical history as a target in antibody-based cancer therapy. Despite being present in normal colon, radiolabeled antibodies against A33 are selectively retained by tumors at long time points. Accordingly, we have studied the trafficking and kinetic properties of the antigen to determine its promise in multi-step, pretargeted immunotherapy. In vitro, the localization, mobility, and persistence of the antigen were investigated, and this work has demonstrated that the antigen is both highly immobile and extremely persistent, properties which may contribute to the prolonged retention of the clinically administered antibodies, and their uncommon ability to penetrate solid tumors. Secondly, because poor tissue penetration is a significant obstacle to the development of successful antibody drugs for immunotherapy of solid tumors, we assess the contribution of antigen density and turnover rate by evaluating the distance to which antibodies penetrate spheroids when these properties are systematically varied. The results agree well with the quantitative modeling predictions, and demonstrate that dosing distal regions of tumors is best achieved by selecting slowly internalized targets that are not expressed above the level necessary for recruiting a toxic dose of therapeutic. Lastly, we describe the in vitro characteristics and report the promising in vivo biodistribution of a multi-step tumor targeting therapy utilizing a novel bispecific antibody which recognizes both the A33 antigen and a small molecule radiometal chelate. Following these studies, several protein engineering techniques are presented. First, a new method of conducting de novo protein engineering utilizing highly avid magnetic beads is described, in which extremely weak interactions can be captured from large library populations. Secondly, an in vitro assay which utilizes these highly avid magnetic beads is used to score the clinical immunogenicity of therapeutic protein drugs is presented. Finally, the use of sortase A as a means to generate fusion proteins posttranslationally is described. Taken together, this additional work demonstrates a productive intersection of basic research and protein engineering methods. Thesis Advisor: K. Dane Wittrup Title: C.P. Dubbs Professor of Chemical Engineering & Biological Engineering Table of