Up-regulation of epidermal growth factor receptor () is a hallmark of many solid tumors， and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here， we report the isolation and functional characterization of novel camelid single-domain antibodies ( or ) directed against human EGFR. The source of these VHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost)， which is notable in that genetic immunization of large， outbred animals is generally poorly effective. The VHs targeted multiple sites on the receptor's surface with high affinity ( range: 1-40 nM)， including one epitope overlapping that of cetuximab， several e... More
Up-regulation of epidermal growth factor receptor () is a hallmark of many solid tumors， and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here， we report the isolation and functional characterization of novel camelid single-domain antibodies ( or ) directed against human EGFR. The source of these VHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost)， which is notable in that genetic immunization of large， outbred animals is generally poorly effective. The VHs targeted multiple sites on the receptor's surface with high affinity ( range: 1-40 nM)， including one epitope overlapping that of cetuximab， several epitopes conserved in the cynomolgus EGFR orthologue， and at least one epitope conserved in the mouse EGFR orthologue. Interestingly， despite their generation against human EGFR expressed from cDNA by llama cells (presumably in native conformation)， the VHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc， one of the VH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC: ∼30 nM). Thus， DNA immunization elicited high-affinity， functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.
