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Affinity‐matured anti‐glycoprotein NMB recombinant immunotoxins targeting malignant gliomas and melanomas
Author(s) -
Kuan ChienTsun,
Wakiya Kenji,
Keir Stephen T.,
Li Jianjun,
Herndon James E.,
Pastan Ira,
Bigner Darell D.
Publication year - 2010
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.25645
Subject(s) - immunotoxin , microbiology and biotechnology , phage display , glioma , mutant , biopanning , antibody , clone (java method) , pseudomonas exotoxin , mutagenesis , peptide library , affinity maturation , biology , chemistry , cancer research , in vitro , cytotoxicity , monoclonal antibody , biochemistry , peptide sequence , immunology , gene
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.