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_HH library from the lymphocytes of the animal, and isolated six unique HER2-specific V_HHs by panning. All six V_HHs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six V_HHs 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_HHs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The V_HHs 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_HHs may be useful in the design of modular cancer immunotherapeutics.