Isolation and characterization of camelid single-domain antibodies against HER2.

Objective: To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2.
Results: We immunized a llama with human HER2, constructed a phage-displayed V _H H library from the lymphocytes of the animal, and isolated six unique HER2-specific V _H Hs by panning. All six V _H Hs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six V _H 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 _H Hs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The V _H 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 _H Hs may be useful in the design of modular cancer immunotherapeutics.