(205f) A Suite of Anti-Fibroblast Activation Protein-Alpha Antibodies for Targeting the Tumor Microenvironment

An expanding body of work suggests that the tumor stroma plays an integral role in the initiation and development of epithelial cancers. Within the stroma, cancer-associated fibroblasts (CAFs) are involved in several functions that form key aspects of tumor progression, including immune suppression, metabolism, and invasion and metastasis. Despite the demonstrated importance of CAFs within the tumor microenvironment, targeting CAFs in a therapeutic context remains a challenge. The work presented here details the development of a suite of antibodies targeting multiple epitopes of fibroblast activation protein-alpha (FAP), a membrane-bound protease selectively overexpressed on CAFs in numerous epithelial cancers. The combination of a minimalist antibody fragment library and yeast surface display allowed for the isolation of more than 50 unique antibodies recognizing the human or murine forms of FAP, with close to 30 exhibiting cross-reactivity. The cross-reactive subset of antibody fragments was secreted in an antibody-like scFv-Fc format from yeast for further characterization of binding function. These antibody-like structures, usable without purification, led to the identification of binding proteins that recognize the membrane-bound form of FAP and further categorization of antibodies into those recognizing two or more distinct epitopes of the protein. Several of these fragments have been affinity matured and converted to full-length IgGs, with the conventional antibody formats retaining the binding function and distinct epitope recognition observed with scFv-Fcs. These antibodies are expected to provide biological insights into the roles of CAFs and therapeutic approaches to cancer treatment. Furthermore, the general antibody isolation and characterization approaches used in this work are accessible to many single-investigator laboratories and should be applicable to the development of antibodies against numerous therapeutic targets.