Recombinant immunotoxins (RITs) are fusions of an Fv-based targeting moiety and a toxin. Pseudomonas exotoxin A (PE) has been used to make several immunotoxins that have been evaluated in clinical trials. Immunogenicity of the bacterial toxin and off-target toxicity have limited the efficacy of these immunotoxins. To address these issues, we have previously made RITs in which the Fv is connected to domain III (PE24) by a furin cleavage site (FCS), thereby removing unneeded sequences of domain II. However, the PE24 containing RITs do not contain the naturally occurring disulfide bond around the furin cleavage sequence, because it was removed when domain II was deleted. This could potentially allow PE24 containing immunotoxins to be cleaved and inactivated before internalization by cell surface furin or other proteases in the blood stream or tumor microenvironment. Here, we describe five new RITs in which a disulfide bond is engineered to protect the FCS. The most active of these, SS1-Fab-DS3-PE24, shows a longer serum half-life than an RIT without the disulfide bond and has the same anti-tumor activity, despite being less cytotoxic in vitro. These results have significance for the production of de-immunized, low toxicity, PE24-based immunotoxins with a longer serum half-life.

Mesothelin-targeted immunotoxin RG7787 has synergistic anti-tumor activity when combined with taxanes

LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomonas exotoxin A (PE) payload. PE kills cells by inhibiting synthesis of new proteins. We found that treatment of pancreatic cancer cells with LMB-100 for 24–48 h did not change total protein level despite inducing protein synthesis inhibition (PSI). Further, increased levels of ubiquitinated proteins were detected, indicating that cells may have limited ability to compensate for PSI by reducing protein degradation. Together, these data suggest that PE depletes concentrations of a minority of cellular proteins. We used reverse phase protein array and Luminex assay to characterize this subset. LMB-100 decreased the abundance of 24 of 32 cancer-related proteins (including Bcl-x, Her2, Her3 and MUC16) without compensatory increases in other analytes. Further, cancer cells failed to maintain extracellular concentrations of cancer cell secreted growth factors (CCSGFs), including Vascular Endothelial Growth Factor (VEGF) following treatment with cytostatic LMB-100 doses both in culture and in mouse tumors. Decreased VEGF concentration did not change tumor vasculature density, however, LMB-100 caused tissue-specific changes in concentrations of secreted factors made by non-cancer cells. In summary, our data indicate that PSI caused by cytostatic LMB-100 doses preferentially depletes short-lived proteins such as oncogenic signaling molecules and CCSGFs.