Abstract 2976: Engineering next-generation anti-CD25 immunotoxins with improved cytotoxic activity and low immunogenicity

Objective: CD25 is expressed on many T cell malignancies, including adult T cell leukemia, and is a target for cancer therapy. Immunotoxins against CD25+ cells containing a portion of Pseudomonas exotoxin A (PE38) show extremely high cytotoxic activity. Our lab has previously created a de-immunized PE immunotoxin in which toxin domain II was removed. Removal of domain II confers two major advantages: i) the immunogenic sequences found in domain II are removed, generating a less immunogenic toxin; ii) the non-specific toxicity of domain II-truncated toxins is much lower, allowing much higher doses of immunotoxin to be given safely (7-10-fold higher safe dosage). However, removal of domain II, which does not affect or enhances the cytotoxic activity of immunotoxins targeting the B cell marker CD22 or the mesothelioma marker mesothelin, lowers the activity of anti-CD25 immunotoxins 30-fold. Because lowering toxin immunogenicity and animal toxicity are critical for the success of immunotoxins in a clinical setting, we have engineered domain II truncated immunotoxins with improved activity against CD25+ cells using a novel adaptation of an established screening system. Methods: Eighteen domain II truncation mutants were designed based upon the crystal structure of the toxin. To bypass the resource intensive protein refolding of standard immunotoxins, these domain II mutants were expressed as fusions with the ZZ protein, which is a small (13.6kD) derivative of protein A. Fusion to ZZ allowed easy production of these ZZ-domain II mutant fusion proteins in the E. coli periplasm and fast purification using IgG coated beads. The purified ZZ-domain II mutant toxins were then bound to anti-CD25 IgG by mixing (the ZZ-domain binds human Fc) and tested for cytotoxic activity on the CD25+ T-cell leukemia HUT-102 cell line. The most active domain II mutants were made into standard recombinant immunotoxins and retested. Results: Of the 18 tested ZZ-domain II mutants, 6 were completely inactive, 8 were active at near parental levels, and 4 were as active as the parental toxin. The 4 high-activity and 2 structurally-interesting, medium-activity mutants were then produced as standard recombinant immunotoxins and retested for cytotoxic activity. Two of the 4 high-activity mutants showed a 2-4-fold increase in activity compared to parental domain II truncated immunotoxin. While still being 8-fold less active than immunotoxins containing domain II (PE38), these high-activity muta