title: Affinity-matured anti-glycoprotein NMB recombinant immunotoxins targeting malignant gliomas and melanomas.
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 (IC(50) = 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.
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, NC 27710, USA. kuan@duke.edu
authors.forename: Chien-Tsun
authors.initials: CT
authors.lastname: Kuan
authors.forename: Kenji
authors.initials: K
authors.lastname: Wakiya
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Keir
authors.forename: Jianjun
authors.initials: J
authors.lastname: Li
authors.forename: James E
authors.initials: JE
authors.lastname: Herndon
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
authors.forename: Darell D
authors.initials: DD
authors.lastname: Bigner
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D005910:Glioma / Q000473:pathology*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D008545:Melanoma / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
chemical_list: C503042:GPNMB protein, human; D000922:Immunotoxins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins
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