Submitted August 11, 2008
Accepted October 28, 2008
A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity
John E. Weldon, Laiman Xiang, Oleg Chertov, Inger Margulies, Robert J. Kreitman, David J. FitzGerald, and Ira Pastan*
National Institute of General Medical Sciences, National Institutes of Health, Bethesda, MD, United States
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
Protein Chemistry Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD, United States
* Corresponding author; email: pastani{at}mail.nih.gov.
Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anti-cancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps, but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biological activity. HA22-LR killed CLL cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced anti-tumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.
