title: A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity.
abstract: Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer 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 biologic activity. HA22-LR killed chronic lymphocytic leukemia 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 antitumor 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.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4264, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Weldon
authors.forename: Laiman
authors.initials: L
authors.lastname: Xiang
authors.forename: Oleg
authors.initials: O
authors.lastname: Chertov
authors.forename: Inger
authors.initials: I
authors.lastname: Margulies
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: David J
authors.initials: DJ
authors.lastname: FitzGerald
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases / Q000009:adverse effects / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000009:adverse effects / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000009:adverse effects / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D002986:Clinical Trials as Topic; D011992:Endosomes / Q000378:metabolism; D005098:Exotoxins / Q000009:adverse effects / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000009:adverse effects / Q000235:genetics / Q000494:pharmacology*; D000922:Immunotoxins / Q000009:adverse effects / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009154:Mutation; D051918:Sialic Acid Binding Ig-like Lectin 2; D037521:Virulence Factors / Q000009:adverse effects / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D051918:Sialic Acid Binding Ig-like Lectin 2; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C497867:CD22 protein, human; C497868:Cd22 protein, mouse