Abstract Many tumors compensate with a resistance mechanism once a signaling pathway is inhibited, as evidenced in patients treated with small molecule inhibitors of EGFR which frequently show increased expression of c-Met or HGF. Since both EGFR and c-Met signal through the same survival and anti-apoptotic pathways, inhibition of the pair of receptors could improve overall efficacy. This was demonstrated clinically, where the combination of onartuzumab and erlotinib showed promising Phase II trials in lung cancer patients. Centyrins are small proteins (~10 kDa) of an emerging class of proteins termed alternative scaffolds. They are engineered to bind to target molecules with high specificity and affinity, but are structurally simpler molecules than antibodies in that that they are single chain, unglycosylated proteins that lack disulfide bonds. In addition, monomeric Centyrins can be linked together such that one molecule can bind to and inhibit multiple targets. Anti-EGFR and anti-c-Met Centyrins were selected for their ability to inhibit ligand-induced phosphorylation. Molecules with a wide range of affinities were identified and bispecific molecules were generated. Phosphorylation of EGFR, c-Met, and ERK were monitored in cells treated with individual monomeric Centyrins, a mixture of monomeric Centyrins, or linked bispecific Centyrins. One bispecific EGFR/c-Met Centyrin provided a 134-fold increase in potency in inhibition of phosphorylation of c-Met compared to the mixture of the two Centyrins. The increase in potency observed in cell signaling translated to enhanced anti-proliferative activity with a potency >100-fold compared to that of the mixture of monospecific Centyrins. The bispecific EGFR/c-Met Centyrins were produced as fusion proteins linked to an albumin binding domain in order to reduce kidney filtration and evaluated in SCID-beige mice implanted with tumor cells engineered to express human HGF. Interestingly, all of the Centyrins tested significantly reduced the size of the tumors compared to the control and the degree of tumor growth inhibition correlated with the affinity to both EGFR and c-Met. In a second tumor model, complete tumor regressions were observed in all mice treated with the bispecific EGFR/c-Met Centyrin. The Centyrin platform could offer benefits over currently available treatment options. Our data demonstrate that in addition to inhibiting two targets simultaneously, a single bispecific molecule allows for significant avidity at the cellular level. This could translate into a lower dosing regimen that allows for improved efficacy through avidity and decreased toxicity. We anticipate that avidity will allow for improved specificity for tumor compared to normal tissue. In addition, the small size of Centyrins may provide an advantage for tumor targeting and accumulation. Citation Format: Donna Klein, Steve Jacobs, Moores Sheri, Mark Anderson, Ricardo Attar, Alexander Barnakov, Kerry Brosnan, Barbara Bushey, Kristen Chevalier, Diana Chin, Carla Cornejo, Mike Diem, Linus Hyun, Elise Kuhar, Francis McCabe, Kristen Picha, Tracy Spinka-Doms, Edward Swift, Karyn O'Neil. Bispecific Centyrin simultaneously targeting EGFR and c-Met demonstrates improved activity compared to the mixture of single agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-312. doi:10.1158/1538-7445.AM2013-LB-312