Figure 6.

Optimization for manufacturability, efficacy and predicted immunogenicity. (a) CTL cytotoxicity assay (T84 cells, 48 hr, 5:1 E:T) demonstrates potent killing activity of PF-07062119 as compared to the parental GUCY2C-0247. (b) PF-07062119 demonstrates substantially reduced self-association potential and polyreactivity compared with GUCY2C-0247, as measured by AC-SINS and DNA binding assays. (c) Thermal stability in the T-BsAb demonstrated an increase in T_m1 of over 8°C compared to the parental molecule. (d) Clipped species in the parental anti-CD3 antibody H2B4 indicated by arrows (blue curve) were successfully removed in H2B5v6 (red curve) as demonstrated by cGE. (e) Long term stability analysis demonstrated good stability and no increase in HMMS for over 6 weeks at 4°C or at 25°C. (f) PF-07062119 also demonstrated low viscosity at concentrations over 100 mg/mL. Limit of 20 cP indicated by dashed red line. (g) Crystal structure of PF-07062119 (side view) shows the anti-GUCY2C domain in yellow and orange, the anti-CD3 in light and dark green, the linker sequences in blue, the stabilizing mutations in red, the immunogenicity-reducing mutations in pink, the deamidation mutation in bright green, and the disulfide constrained C-terminal domain (at bottom) also in blue. (h) Top view of tightly packed diabody structure. (i) Zoomed in view of GUCY2C VH-VL interface highlighting key stabilizing mutations with same coloring pattern of domains and mutations. Mutations at positions L94 (Ala) and H60 (His) were observed in most stability optimized clones. These mutations appear to help stabilize the VH-VL interface