Discovery and Characterization of Human Recombinant anti-uPAR antibodies
- Author(s): Duriseti, Sai
- Advisor(s): Craik, Charles S
- et al.
The urokinase plasminogen activator receptor (uPAR) is involved in the proliferation and migration of cells from diverse lineages. As a heavily glycosylated, glycosyl phosphotidyl inositol (GPI) linked protein of approximately 300 amino acids, its ubiquitousness in many aggressive cancer types-triple negative breast cancer (TNBC) in particular-makes it an attractive target for uPAR-directed therapies. uPAR participates in many protein/ protein interactions, which leads to pericellular proteolysis and "outside-in" signaling that is mediated by specific integrins. Here, the Craik Lab's human antigen-binding fragment (Fab) phage display library was panned for anti-uPAR antibodies. In total, 22 unique anti-uPAR Fabs were found. Two of these, 2G10 and 3C6, were found to antagonize the the uPAR/uPA and uPAR/β1 integrin interactions. While antagonism of these interactions did not effect cytotoxic responses, the antagonism did prevent uPAR over-expressing H1299 cells from migrating through a layer of cross-linked extracellular matrix. Further investigation revealed coarse-level binding sites of 2G10 and 3C6, as well as a unique feature of 3C6's ability to antagonize the uPAR/β1 integrin interaction by binding uPAR in an extended conformation. While surface plasmon resonance (SPR) revealed high binding affinities of 2G10 and 3C6 for uPAR, apparent affinities of 2G10 and 3C6 for the uPAR over-expressing MDA-MB-231 cells were different, and suggested that 3C6 Fab has a very poor affinity for uPAR. Regardless, both antibodies preferentially accumulated in MDA-MB-231-derived tumors, and demonstrated favorable pharmacokinetics and high tumor uptake. As a promising result, 2G10 IgG labeled with 177Lu demonstrated potent anti-tumor activity. The results of my research provide both reagents and a framework for the development of a multi-modal clinical probe and therapeutic for cancers, and other disorders, where uPAR over-expression plays a role.