Figure 6.
Tuning the valency of monospecific and bispecific CSANs controls non-targeted T cell activity. A, Reducing the valency of αCD3 monomers in the dimerized CSAN is done by increasing the molar ratios of either the αB7-H3 monomers or non-targeted DHFR2 monomers and decreasing the αCD3 monomers in the oligomerized CSAN. Shown is the average valency of the reduced αCD3 monomer CSANs. B, Target cells, unactivated PBMCs, and “valency-tuned” CSANs were co-cultured as previously described in a monolayer. ONS 2303 cell viability was monitored over a 72 hour period using an Incucyte SX5 at a fixed CSAN concentration (400nM) and PBMCs added at a 20:1 E:T ratio (Significance is indicated as **P<0.01, ***P<0.001 by two-tailed unpaired t-test between hours 14–72, n = 3 wells per time point ± SEM). C, Following the 72 hour viability study, the media from the co-culture was analyzed for IFNγ and IL-2 using a sandwich ELISA. (Significance is indicated as *P<0.05, **P<0.01 by two-tailed unpaired t-test, n = 3 wells per time point ± SD). Incucyte and ELISA data was obtained using one PBMC donor.