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Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system
Author(s) -
Asano Ryutaro,
Nakayama Makoto,
Kawaguchi Hiroko,
Kubota Tsuguo,
Nakanishi Takeshi,
Umetsu Mitsuo,
Hayashi Hiroki,
Katayose Yu,
Unno Michiaki,
Kudo Toshio,
Kumagai Izumi
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08417.x
Subject(s) - mutant , chemistry , epidermal growth factor receptor , fusion protein , cd16 , microbiology and biotechnology , humanized antibody , cancer research , antibody , receptor , biology , biochemistry , recombinant dna , immunology , gene , immune system , monoclonal antibody , cd3 , cd8
We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti‐EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR‐expressing tumor cells. After we confirmed that an anti‐EGFR × anti‐CD16 bispecific diabody (Ex16) consisting of a previously humanized anti‐EGFR variable fragment (Fv) and a mouse anti‐CD16 Fv had growth inhibitory activity, we designed a humanized anti‐CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity‐determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single‐chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti‐EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.