Isolation and characterization of camelid single-domain antibodies against HER2

To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2. We immunized a llama with human HER2, constructed a phage-displayed V H library from the lymphocytes of the animal, and isolated six unique HER2-specific V Hs by panning. All six V Hs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six V Hs recognized recombinant human HER2 ectodomain with monovalent affinities ranging from 1 to 51 nM, had comparable affinities for cynomolgus monkey HER2, and bound HER2 SKOV3 cells by flow cytometry. Three of the V Hs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The V Hs recognized three major epitopes on HER2 (including one conserved across the human, simian and murine orthologues), all of which were distinct from that of trastuzumab. These V Hs may be useful in the design of modular cancer immunotherapeutics.