Leukemia & Lymphoma, June 2011; 52(S2): 87–90
Immunotoxins with decreased immunogenicity and improved activity
IRA PASTAN, MASANORI ONDA, JOHN WELDON, DAVID FITZGERALD, & ROBERT J. KREITMAN Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Abstract Recombinant immunotoxins, containing an Fv fragment and a bacterial toxin, frequently elicit neutralizing antibodies, nearly always against the toxin. Moxetumomab pasudotox (previously called CAT-8015 or HA22) contains an anti-CD22 Fv fused to PE38, a truncated form of Pseudomonas exotoxin, containing amino acids 253–364 and 381–613. One avenue to reducing immunogenicity is to identify B- and T-cell epitopes and remove them while retaining toxin activity. To determine B-cell epitopes on PE38, 60 monoclonal antibodies against PE38 were tested in a pairwise manner, and seven major epitope groups with 13 subgroups were identified. The locations of many of these epitopes were identified by mutating large surface-exposed residues to alanine. A mutant of moxetumomab pasudotox containing eight epitope-eliminating mutations (HA22-8X) was prepared, and greatly reduced immunogenicity in mice. In parallel, two large sections of PE38 containing lysosomal protease cleavage sites were removed, leaving only amino acids 274–284 and 394–613 of the toxin. The resulting molecule, HA22-LR,
retained cytotoxicity toward CD22þcell lines, killed primary chronic lymphocytic leukemia cells more potently than moxetumomab pasudotox, was much less toxic to mice, and had significantly improved antitumor activity toward murine xenografts. The immunogenicity and activity of recombinant immunotoxins may be optimized by combinations of these approaches.
Keywords: Recombinant immunotoxins, hairy cell leukemia Immunogenicity of recombinant immunotoxins
Recombinant immunotoxins are composed of a variable fragment (Fv) of a monoclonal antibody (MAb) fused to truncated bacterial toxin [1]. Examples include BL22 (CAT-3888), targeting CD22, and SS1P, targeting mesothelin. Each of these molecules contains PE38, a truncated form of Pseudomonas exotoxin A (PE) containing amino acids 253–364 and 381–613. Recombinant immunotoxins in clinical trials have demonstrated remarkable potency. BL22, for example, has been reported to induce complete remissions in 47–61% of patients with chemoresistant hairy cell leukemia [2,3]. Un- fortunately, neutralizing antibodies are frequently produced when recombinant immunotoxins are administered to humans or laboratory animals, and these antibodies nearly always target the bacterial toxin rather than the murine Fv. In the phase II trial
of BL22, four (11%) patients produced levels of neutralizing antibodies sufficiently high to prevent retreatment, and others made lower levels of antibodies. In solid tumors, immunogenicity is much higher; nearly all patients treated with SS1P produced significant levels of neutralizing antibodies [4,5]. One avenue for avoiding immunogenicity is to alter the toxin to mutate or remove immunogenic epitopes, which requires a mechanistic knowledge of essential toxin regions.
Intoxication of cells by Pseudomonas exotoxin
The functional domains of native PE include Ia (amino acids 1–252), which binds to cells and is not present in PE38, II (253–364), which is though to mediate translocation, III (400–613), which contains adenosine diphosphate (ADP)-ribosylating activity, and Ib (365–399), which separates domains II and III.
Correspondence: Robert J. Kreitman, Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, 37/5124b, 37 Convent Drive MSC 4255, Bethesda, MD 20892, USA. Tel: (301)496-4797. E-mail
kreitmar@mail.nih.gov
ISSN 1042-8194 print/ISSN 1029-2403 online 2011 Informa UK, Ltd. DOI: 10.3109/10428194.2011.573039
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