(649b) Engineering Anti-EGFR Fibronectin Nanorings for Cancer Immunotherapy

Engineered protein scaffolds have gained interest in recent years to overcome the challenges faced with antibody-based drugs. Several scaffolds have shown promise in both preclinical models and clinical trials. One of these promising scaffolds is the tenth type III domain of human fibronectin, which consists of seven β-strands connected with flexible loops. Analogous to the complementarity determining regions of antibodies, the amino acid sequences of these loop regions can be engineered to provide target-specific binders with high affinity and specificity. One such target, epidermal growth factor receptor (EGFR) is a well-studied cancer biomarker and a promising target for cancer therapeutics. While some groups have already evolved high affinity EGFR-binding fibronectins using different methods, their use in cancer immunotherapy applications has not yet been explored.

 Our lab has developed Chemically Self-Assembling Nanorings (CSANs) as a non-genetic approach for cell-directed immunotherapies. Multimeric CSANs are formed when a bis-methotrexate dimerizer induces the oligomerization of dihydrofolate reductase-dihydrofolate reductase (DHFR²) fusion proteins. An advantage of the CSAN platform is that it can be easily expanded through fusion of additional protein domains or by chemical modifications to the dimerizer.

By fusing an EGFR-targeting fibronectin to the DHFR² subunits, we formed fibronectin CSANs with both high affinity and high avidity for EGFR-overexpressing cancer cells. After investigating the biological outcomes of the fibronectin nanorings on cancer cells, we developed the bispecific nanorings that will target both the cancer cells and the T cells. We have formed bispecific-CSANs by oligomerizing the fibronectin-DHFR² subunits alongside anti-CD3 scFv-DHFR² subunits that target T cells. Furthermore, T cells functionalized with EGFR-CD3 bispecific CSANs are able to selectively recognize and eradicate EGFR-overexpressing tumor cells. A unique advantage of this bispecific CSAN system is the ability to disassemble the CSANs in vitro and in vivo by introducing the FDA-approved antibiotic, trimethoprim, thus providing control over the T cell-directing activity of the CSANs. As the findings here suggest, fibronectin CSANs provide unique features for enhanced cancer immunotherapy.