Utilization of the Conserved Antibody Nucleotide Binding Site (NBS): From Antibody Purification and Advanced Medical Diagnostics to Next Generation Therapeutics

There are two approaches often taken to advance cancer research and improve patient outcomes: i) developing more sensitive medical diagnostics to detect specific disease biomarkers earlier and ii) through the development of more advanced drug delivery technologies to more effectively, and selectively target and kill cancerous cells. This dissertation will demonstrate how an underutilized, highly conserved binding domain located on the antibody variable fragment, known as the nucleotide binding site (NBS), can be implemented for antibody purification, advanced medical diagnostic development and for next generation antibody based therapeutics. Through overlaying all available antibody crystal structures it has been shown that the NBS is conserved across nearly all species and all antibody isotypes. An in silico screening of small molecules was carried out to select for a high binding nucleotide analog to selectively target the NBS leading to the identification of indole-3-butyric acid (IBA), with a monovalent Kd = 1-10 micromolar, based on various IgG and IgE antibodies tested. By targeting the antibody NBS with surface immobilized IBA a unique small molecule antibody purification method has been developed. A stable covalent bond can also be formed through a photo-chemical reaction in a UV energy dependent manner between IBA and the antibody NBS to provide for a unique, homogenous, site-specific conjugation of a target molecule selectively to the antibody light chains. Using this method we have developed an oriented antibody immobilization technique to produce immunosensors that maintain an exceedingly high level of antibody activity, including Fc recognition, allowing for greatly improved biomarker detection. This method of crosslinking also has implications in the development of next generation pharmaceutical antibodies by providing a site-specific insertion of functional moieties such as: chemotherapeutics, cell penetrating peptides, targeting sequences for bispecific antibody preparation, imaging molecules, and immobilization of antibodies to various nanoparticle drug delivery platforms, without negatively impacting antibody-antigen binding interactions. Utilization of this highly conserved binding pocket allows for simple, site-specific, covalent antibody modification with numerous applications in both clinical and diagnostic laboratory settings for rapid antibody modification and surface immobilization.