Affinity-matured anti-glycoprotein NMB recombinant immunotoxins targeting malignant gliomas and melanomas

Authors

  • Chien-Tsun Kuan,

    Corresponding author
    1. Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC
    2. Department of Pathology, Duke University Medical Center, Durham, NC
    • Department of Pathology, Duke University Medical Center, Research Drive, Box 3156, Durham, NC 27710, USA
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    • C.-T.K. and K.W. contributed equally to this work

    • Fax: +919-681-8337

  • Kenji Wakiya,

    1. Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC
    2. Department of Pathology, Duke University Medical Center, Durham, NC
    Current affiliation:
    1. Division of Neuro-Oncology, Comprehensive Cancer Center, Saitama Medical University International Medical Center, 1397-1 Yamane Hidaka-shi, Saitama 350-1298, Japan
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    • C.-T.K. and K.W. contributed equally to this work

  • Stephen T. Keir,

    1. Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC
    2. Department of Surgery, Duke University Medical Center, Durham, NC
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  • Jianjun Li,

    1. Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC
    2. Department of Pathology, Duke University Medical Center, Durham, NC
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  • James E. Herndon II,

    1. Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC
    2. Department of Biostatistics & Bioinformatics, Duke University Medical Center, Durham, NC
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  • Ira Pastan,

    1. Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD
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  • Darell D. Bigner

    1. Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC
    2. Department of Pathology, Duke University Medical Center, Durham, NC
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Abstract

Glycoprotein NMB (GPNMB), a transmembrane glycoprotein highly expressed in high-grade gliomas (HGGs), is an attractive target in cancer immunotherapy. We isolated a GPNMB-specific scFv clone, G49, from a human synthetic phage-display library. To obtain mutant single-chain variable-fragment antibodies (scFvs) with improved affinity and immunotoxins with increased activity, we subjected G49 to in vitro affinity maturation by a complementarity-determining-region (CDR) random-mutagenesis technique. Using light-chain CDR3 mutagenesis, cell-based panning by phage display, subsequent heavy-chain CDR1 mutagenesis, and flow-cytometric selection by yeast-surface display, we generated the mutant scFv clone 902V, with an overall 11-fold increase in affinity for GPNMB. Clone 902V was further randomized throughout the whole scFv by error-prone PCR, and one mutant, F6V, was selected by yeast-surface display. F6V scFv, differing from 902V by one amino-acid change in the light-chain CDR2, exhibited an affinity for GPNMB of 0.30 nM. The F6V mutant scFv clone was fused with a truncated form of Pseudomonas exotoxin A to form the immunotoxin F6V-PE38. F6V-PE38 demonstrated significant protein-synthesis-inhibition activity on GPNMB-expressing glioma and malignant melanoma cells (IC50 = 0.5 ng/ml [8 pM]), a 60-fold improvement over G49 activity, but no cytotoxicity on GPNMB-negative cells. Furthermore, F6V-PE38 exhibited significant antitumor activity against subcutaneous malignant glioma xenografts in two nude-mouse models and a melanoma neoplastic meningitis model in athymic rats. These GPNMB-specific scFv antibodies and immunotoxins hold promise as reagents in targeted therapy for HGGs and other GPNMB-expressing malignancies.

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