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Recombinant immunotoxins (RITs) are genetically engineered proteins designed to kill cancer cells. The RIT HA22 contains the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A (PE38). As PE38 is a bacterial protein, patients frequently produce antibodies that neutralize its activity, preventing retreatment. We have earlier shown in mice that PE38 contains 7 major B-cell epitopes located in domains II and III of the protein. Here we present a new mutant RIT, HA22-LR-6X, in which we removed most B-cell epitopes by deleting domain II and mutating 6 residues in domain III. HA22-LR-6X is cytotoxic to several lymphoma cell lines, has very low nonspecific toxicity, and retains potent antitumor activity in mice with CA46 lymphomas. To assess its immunogenicity, we immunized 3 MHC-divergent strains of mice with 5 μg doses of HA22-LR-6X, and found that HA22-LR-6X elicited significantly lower antibody responses than HA22 or other mutant RITs with fewer epitopes removed. Furthermore, large (50 μg) doses of HA22-LR-6X induced markedly lower antibody responses than 5 μg of HA22, indicating that high doses can be administered with low immunogenicity. Our experiments show that we have correctly identified and removed B-cell epitopes from PE38, producing a highly active immunotoxin with low immunogenicity and low animal toxicity. Future studies will determine if these properties carry over to humans with cancer.