Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892, USA.
The Journal of Immunology (Impact Factor: 4.92). 01/2007; 177(12):8822-34. DOI: 10.4049/jimmunol.177.12.8822
Source: PubMed


Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Download full-text


Available from: BK Lee, Jul 28, 2014
    • "This effort is driven by experiments designed to identify epitopes that react with serum from patients involved in clinical trials. For example, antibodies isolated from the serum of patients treated with PE38 immunoconjugates identified seven B cell epitope groups within PE38 (Roscoe et al., 1997, Onda et al., 2006). Elimination or mutation of these large hydrophilic residues to small nonpolar residues decreased immunogenicity of the immunotoxin (Onda et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Over the past 20 years monoclonal antibodies (mAbs) have evolved to become a major class of therapeutics for the treatment of a variety of indications, including cancer. The evolution of mAbs into front-line cancer therapies required significant advances in the strategies used to both isolate and optimize these agents. Here we discuss development of the steps that facilitated this evolution and the criteria that drive current development of next-generation mAb-based cancer therapies. KeywordsImmunogenicity-Humanized-Phage-display-Effector-Immunoconjugate
    No preview · Chapter · May 2011
  • Source
    • "Finally, a tail vein injection of up to 4 mg/kg in mice did not result in any signs of toxicity (results not shown). Overall, these findings suggest that the repeated injections of SLT-1 A subunit variants such as SLT-1AIYSNKLM elicit modest humoral responses in mice and compare well with responses observed in patients treated with other targeted toxin therapies [21,22]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1A IYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells. SLT-1A IYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1A IYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AI YSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1A IYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1A IYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1A IYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice. These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1A IYSNKLM can specifically kill human melanoma cells in vitro and in vivo.
    Full-text · Article · Feb 2010 · Molecular Cancer
  • Source
    • "agents for the treatment of mesothelioma and glioma revealed that 73 and 88% of the patients respectively developed neutralising IgG antibodies, with one study showing the majority of the antibodies were directed to the toxin fragment of the molecule (Hassan et al, 2007; Vogelbaum et al, 2007). To address this issue, Onda and Pastan used serum samples from patients receiving a BL22, a PE38- containing anti-leukemia agent, to map out regions of the molecule that elicited the strongest antibody response (Onda et al, 2006). Upon identifying seven major reactive epitopes, they proved that mutations could be made to these regions without compromising tumour cell killing (Onda et al, 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy. To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut. In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (P<0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14-16 days post tumour innoculation. These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.
    Full-text · Article · Sep 2009 · British Journal of Cancer
Show more