Engineered Anti-Mesothelin and Anti-CD25 Immunotoxins

Clinical-grade anti-mesothelin SS1P and anti-CD25 LMB-2 [anti-Tac(Fv)-PE38] were manufactured and provided by Advanced BioScience Laboratories, Inc. (Kensington, MD). huSS1(Fab)-LR-GGS-LO10-PE24 (RG7787) was supplied by Roche Innovation Center Penzberg, Germany under a Cooperative Research and Development Agreement (#2791). Anti-mesothelin SS1(dsFv)-LR-GGS-PE24 (SS1-LR-GGS) and the anti-CD71 immunotoxin HB21(Fv)-PE40 were produced in our laboratory, according to a standard protocol [23 (link)]. Both SS1-LR-GGS and RG7787 are re-engineered low-immunogenic versions of SS1P that consist of a PE24 fragment. Specific modifications include removing the bulk of PE domain II, leaving a furin cleavage site, and adding a Gly–Gly–Ser (GGS)-based peptide linker after the furin cleavage site. RG7787 is further optimized for clinical use by replacing the mouse anti-mesothelin Fv (SS1) with a humanized Fab (huSS1), to increase size and therefore circulatory half-life, and by introducing seven mutations (R505A, R427A, R490A, R467A, D463A, R456A, and R538A) in the catalytic domain III of PE to silence B-cell epitopes [19 (link)]. 5-azacytidine (AZA) (Sigma) is a DNA methyltransferase inhibitor, and was dissolved in RPMI-1640 medium.