Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 14; pp. 5742 - 5747
• Main Authors: Onda, Masanori, Beers, Richard, Xiang, Laiman, Lee, Byungkook, Weldon, John E, Kreitman, Robert J, Pastan, Ira
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 05.04.2011; National Acad Sciences
• Subjects: ADP Ribose Transferases - genetics; ADP Ribose Transferases - metabolism; Amino acid composition; Amino Acid Sequence; Amino acids; Animals; Antibodies; Antibodies, Monoclonal; antibody formation; Antibody response; Antigenicity; antineoplastic activity; Antitumor activity; B lymphocyte epitopes; B-lymphocytes; Bacteria; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Biological Sciences; Cell Line, Tumor; Cell lines; Cells; Chronic lymphatic leukemia; Cytotoxicity; Drug resistance; Enzyme-Linked Immunosorbent Assay; Epitopes; Epitopes, B-Lymphocyte - genetics; exotoxin A; Exotoxins; Exotoxins - genetics; Exotoxins - metabolism; humans; Immune response; Immunization, Passive - methods; Immunogenicity; Immunotoxins; Inhibitory concentration 50; Intravenous administration; intravenous injection; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell - drug therapy; Leukemia, Lymphocytic, Chronic, B-Cell - immunology; Lymphocytes; Lymphocytes B; lymphocytic leukemia; Malignancy; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutation; patients; Point mutation; Protein Engineering - methods; Proteins; Pseudomonas; Pseudomonas aeruginosa Exotoxin A; Reactivity; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - therapeutic use; Remission; Rodents; Statistics, Nonparametric; Toxins; Virulence Factors - genetics; Virulence Factors - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1102746108

Many nonhuman proteins have useful pharmacological activities, but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22, CAT8015, moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38, a 38-kDa portion of Pseudomonas exotoxin A, has produced many complete remissions in drug-resistant hairy-cell leukemia when several cycles of the agent can be given, but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas exotoxin. Using this information, we eliminated these epitopes to produce an immunotoxin (HA22-LR-8M) that is fully cytotoxic against malignant B-cell lines, has high cytotoxic activity against cells directly isolated from patients with chronic lymphocytic leukemia, and has excellent antitumor activity in mice. HA22-LR-8M does not induce antibody formation in mice when given repeatedly by intravenous injection and does not induce a secondary antibody response when given to mice previously exposed to HA22. HA22-LR-8M also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to HA22. The properties of HA22-LR-8M make it an excellent candidate for further clinical development.