(4ea) Engineering the Next Generation of Therapeutic Proteins

Recombinant protein-based reagents, diagnostics, and therapeutics are beginning to come of age. However, the development of proteins tailored for specific applications, especially therapeutic, remains an ongoing challenge. Cell surface display technologies provide an especially powerful set of tools for protein engineers to utilize in the development of new therapeutic molecules. The work presented here introduces and exploits improvements in cell surface display technologies in order to develop proteins with therapeutic properties and novel functionalities.

In many cancers that present as solid tumors, 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 known to contribute to immune suppression, metabolism, invasion, and metastasis. Despite the demonstrated importance of CAFs in the tumor microenvironment, targeting CAFs within the tumor microenvironment remains a challenge. To facilitate interference with these cells, we have developed 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. Screening a billion-member synthetic antibody library displayed on the surface of yeast yielded more than 50 unique antibodies recognizing FAP, of which 28 were crossreactive for both the human and murine forms of the antigen. We developed secondary screens to verify that candidate binding proteins could recognize the membrane-bound form of the antigen and identify candidates recognizing distinct epitopes of the antigen. These secondary screens utilized soluble, yeast-produced antibody-like structures in the efficient identification of a handful of candidate proteins with promising properties. These candidates were subjected to affinity maturation, converted to full-length IgGs, and produced with reasonable yields in mammalian cells. The full-length IgGs exhibit excellent binding properties, recognize multiple distinct epitopes of FAP, and modulate the surface levels of FAP on activated fibroblasts in culture. Further in vitro characterization and in vivo studies are underway. 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.

Protein engineering using proteins containing the conventional 20 amino acids has yielded a startling array of proteins with druglike properties. However, the chemical diversity present in the conventional set of amino acid side chains is quite limited and has motivated efforts to introduce new functionalities into proteins using noncanonical amino acids (ncAAs) and other approaches. Although therapeutic applications of proteins containing functionalities beyond those encoded in the common 20 amino acids appear promising, proteins with these added functionalities will likely require further improvement using library-based screening methods. To address this issue, we have developed an E. coli cell surface display platform for evolving antibody fragments containing analogs of the amino acid methionine. In our initial efforts, screening proteins containing various methionine analogs, including “clickable” analogs, led to the emergence of highly functional variants that exhibit unique properties modulated in part by ncAA side chains. These properties include azide-containing fragments with binding kinetics improved 2-fold relative to their methionine-containing counterparts and fragments exhibiting chemical reactivity in strain-promoted azide-alkyne cycloadditions. Characterization of the azide-containing fragments indicates that these fragments are quite functional, with both soluble and cell surface-displayed fragments retaining their ability to bind antigen after chemical modification. The development of a ncAA-compatible cell surface display platform and the isolation of clickable, functional fragments using the platform raises intriguing possibilities for future therapeutic screening efforts, including the modulation of molecular recognition events with ncAA side chains and screening libraries of chemically modified proteins for functional variants.