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Two novel human antitumor immunoconjugates, engineered by fusion of a single-chain antibody fragment against human ErbB2 receptor, termed Erbicin, with either a human RNase or the Fc region of a human IgG(1) , are selectively cytotoxic for ErbB2-positive cancer cells in vitro and in vivo. These Erbicin-derived immunoagents (EDIAs) do not show the most negative properties of Herceptin, the only humanized mAb against ErbB2 used in the therapy of breast carcinoma: cardiotoxicity and the inability to act on resistant tumors. These differences are probably attributable to the different ErbB2 epitopes recognized by EDIAs and Herceptin, respectively, as we have previously reported that they induce different signaling mechanisms that control tumor and cardiac cell viability. Thus, to accurately identify the novel epitope recognized by EDIAs, three independent and complementary methodologies were used. They gave coherent results, which are reported here: EDIAs bind to a different ErbB2 epitope than Herceptin and the other human/humanized antibodies against ErbB2 reported so far. The epitope has been successfully located in region 122-195 of extracellular domain I. These findings could lead to the identification of novel epitopes on ErbB2 that could be used as potential therapeutic targets to mitigate anti-ErbB2-associated cardiotoxicity and eventually overcome resistance., (2011 The Authors Journal compilation; 2011 FEBS.)

Overexpression of the ErbB2 receptor is associated with the progression of breast cancer, and is a sign of a poor prognosis. Herceptin, a humanized antibody directed to the ErbB2 receptor, has been proven to be effective in the immunotherapy of breast cancer. However, it can result in cardiotoxicity, and a large fraction of breast cancer patients are resistant to Herceptin treatment. We have engineered three novel, fully human, anti-ErbB2 immunoagents: Erbicin, a human single-chain antibody fragment; ERB-hRNase, a human immunoRNase composed of Erbicin fused to a human RNase; ERB-hcAb, a human 'compact' antibody in which two Erbicin molecules are fused to the Fc fragment of a human IgG1. Both ERB-hRNase and ERB-hcAb strongly inhibit the growth of ErbB2-positive cells in vivo. The interactions of the Erbicin-derived immunoagents and Herceptin with the extracellular domain of ErbB2 (ErbB2-ECD) were investigated for the first time by three different methods. Erbicin-derived immunoagents bind soluble extracellular domain with a lower affinity than that measured for the native antigen on tumour cells. Herceptin, by contrast, shows a higher affinity for soluble ErbB2-ECD. Accordingly, ErbB2-ECD abolished the in vitro antitumour activity of Herceptin, with no effect on that of Erbicin-derived immunoagents. These results suggest that the fraction of immunoagent neutralized by free extracellular domain shed into the bloodstream is much higher for Herceptin than for Erbicin-derived immunoagents, which therefore may be used at lower therapeutic doses than those employed for Herceptin.