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: 5.52). 01/2007; 177(12):8822-34. DOI: 10.4049/jimmunol.177.12.8822
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: CD133 expression enriches for tumor-initiating cells and is a negative prognostic factor in numerous cancers. We previously developed an immunotoxin against CD133 by fusing a gene fragment encoding the scFv portion of an anti-CD133 antibody to a gene fragment encoding deimmunized PE38KDEL. The resulting fusion protein, dCD133KDEL, demonstrated potent antitumor activity following intratumoral delivery into head neck cell carcinoma xenografts. However, the efficacy against other tumors and the tolerability of systemic administration remained unclear. The purpose of this study was to evaluate the tolerability and efficacy of dCD133KDEL in a systemic human breast carcinoma model. Time course viability studies showed that dCD133KDEL selectively inhibited MDA-MB-231 ductal breast carcinoma cells that contained a minority CD133+ subpopulation, implicating CD133+ cells as a source for self-renewal within this cell line. Furthermore, systemic administration of dCD133KDEL caused regression or inhibition of tumor growth in mice bearing an intrasplenic MDA-MB-231 tumor challenge as a model for metastatic disease. In the same model, combined therapy with dCD133KDEL and another immunotoxin designed to target the bulk tumor mass was the most effective therapy, supporting the idea that such combination therapies might better address tumor heterogeneity. dCD133KDEL shows promise as a therapeutic agent and as a biologic tool to study cancer stem cells.
    Drug Delivery and Translational Research. 3(2).
  • [Show abstract] [Hide abstract]
    ABSTRACT: OBJECTIVES: While most women with ovarian cancer will achieve complete remission after treatment, the majority will relapse within two years, highlighting the need for novel therapies. Cancer stem cells (CSC) have been identified in ovarian cancer and most other carcinomas as a small population of cells that can self-renew. CSC are more chemoresistant and radio-resistant than the bulk tumor cells; it is likely that CSC are responsible for relapse, the major problem in cancer treatment. CD133 has emerged as one of the most promising markers for CSC in ovarian cancer. The hypothesis driving this study is that despite their low numbers in ovarian cancer tumors, CSC can be eradicated using CD133 targeted therapy and tumor growth can be inhibited. METHODS: Ovarian cancer cell lines were evaluated using flow cytometry for expression of CD133. In vitro viability studies with an anti-CD133 targeted toxin were performed on one of the cell lines, NIH:OVCAR5. The drug was tested in vivo using a stably transfected luciferase-expressing NIH:OVCAR5 subline in nude mice, so that tumor growth could be monitored by digital imaging in real time. RESULTS: Ovarian cancer cell lines showed 5.6% to 16.0% CD133 expression. dCD133KDEL inhibited the in vitro growth of NIH:OVCAR5 cells. Despite low numbers of CD133-expressing cells in the tumor population, intraperitoneal drug therapy caused a selective decrease in tumor progression in intraperitoneal NIH:OVCAR5-luc tumors. CONCLUSIONS: Directly targeting CSC that are a major cause of drug resistant tumor relapse with an anti-CD133 targeted toxin shows promise for ovarian cancer therapy.
    Gynecologic Oncology 05/2013; · 3.93 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Immunotoxins are a group of protein-based therapeutics, basically comprising two functional moieties: one is the antibody or antibody Fv fragment that allows the immunotoxin to bind specifically to target cells; another is the plant or bacterial toxin that kills the cells upon internalization. Immunotoxins have several unique features which are superior to conventional chemotherapeutics, including high specificity, extraordinary potency, and no known drug resistance. Development of immunotoxins evolves with time and technology, but significant progress has been achieved in the past 20 years after introduction of recombinant DNA technique and generation of the first single-chain variable fragment of monoclonal antibodies. Since then, more than 1,000 recombinant immunotoxins have been generated against cancer. However, most success in immunotoxin therapy has been achieved against hematological malignancies, several issues persist to be significant barriers for effective therapy of human solid tumors. Further development of immunotoxins will largely focus on the improvement of penetration capability to solid tumor mass and elimination of immunogenicity occurred when given repeatedly to patients. Promising strategies may include construction of recombinant antibody fragments with higher binding affinity and stability, elimination of immunodominant T- and B-cell epitopes of toxins, modification of immunotoxins with macromolecules like poly(ethylene glycol) and liposomes, and generation of immunotoxins with humanized antibody fragments and human endogenous cytotoxic enzymes. In this paper, we briefly reviewed the evolution of immunotoxin development and then discussed the challenges of immunotoxin therapy for human solid tumors and the potential strategies we may seek to overcome the challenges.
    Journal of basic and clinical medicine. 01/2013; 2(2):1-6.

Full-text (2 Sources)

Available from
Jul 28, 2014