A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity

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Blood First Edition Paper, prepublished online November 6, 2008; DOI

10.1182/blood-2008-08-173195.

A protease-resistant immunotoxin against CD22 with greatly increased activity

against CLL and diminished animal toxicity

E. Weldon, Laiman Xiang, Oleg Chertov, Inger Margulies, J. Kreitman,

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@... .

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.