A new human, compact antibody was engineered by fusion of a human, antitumour ErbB2-directed scFv with a human IgG1 Fc domain. Overexpression of the ErbB2 receptor is related to tumour aggressiveness and poor prognosis. This new immunoagent meets all criteria for a potential anticancer drug: it is human, hence poorly or not immunogenic; it binds selectively and with high affinity to target cells, on which it exerts an effective and selective antiproliferative action, including both antibody-dependent and complement-dependent cytotoxicity; it effectively inhibits tumour growth in vivo. Its compact molecular size should provide for an efficient tissue penetration, yet suitable to a prolonged serum half-life.

We report the preparation and characterization of a novel, fully human antitumor immunoRNase (IR). The IR, a human RNase and fusion protein made up of a human single chain variable fragment (scFv), is directed to the ErbB-2 receptor and overexpressed in many carcinomas. The anti-ErbB-2 IR, named hERB-hRNase, retains the enzymatic activity of the wild-type enzyme (human pancreatic RNase) and specifically binds to ErbB-2-positive cells with the high affinity (K d ‫؍‬ 4.5 nM) of the parental scFv. hERB-hRNase behaves as an immunoprotoxin and on internalization by target cells becomes selectively cytotoxic in a dose-dependent manner at nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor, hERB-hRNase induced a dramatic reduction in tumor volume. hERB-hRNase is the first fully human antitumor IR produced thus far, with a high potential as a poorly immunogenic human drug devoid of nonspecific toxicity, directed against ErbB-2-positive malignancies.

ErbB2 is a prognostic factor and target of therapy for many carcinomas. In contrast with the other ErbB receptors, ErbB2 lacks a soluble direct ligand, but it is the preferred co-receptor for the ErbB family members, forming heterodimers with more potent and prolonged signalling activity than that of homodimers. We recently produced a new anti-ErbB2 antibody, Erb-hcAb, by fusion of Erbicin, a human, anti-ErbB2 scFv, selectively cytotoxic to ErbB2-positive cells, and a human Fc domain. This fully human antitumour antibody represents a compact version of an IgG1, with the cytotoxicity of the scFv moiety on target cells, combined with the ability of the Fc moiety to induce both antibody- and complement-dependent cytotoxicity. Here, we describe the main properties of Erb-hcAb, using as a reference Herceptin, an anti-ErbB2 humanized monoclonal currently employed in clinical immunotherapy. We found that both bivalent Erb-hcAb and Herceptin increase receptor phosphorylation and downregulation, whereas monovalent Erbicin does not. These results correlate with the finding that Erb-hcAb is capable of inducing apoptosis and inhibiting cell cycle progression in ErbB2-positive cells. Its powerful in vitro antitumour action matched that observed in vivo in experiments with human ErbB2-positive tumour xenografts established in athymic mice. Finally, Erb-hcAb displays a glycosylation profile virtually superimposable to that of a human IgG. These findings suggest that Erb-hcAb is a very promising new agent for the immunotherapy of carcinomas that overexpress the ErbB2 receptor.

BACKGROUND: Overexpression of ErbB2 receptor in breast cancer is associated with disease progression and poor prognosis. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer, has proven to be effective; however, a relevant problem for clinical practice is that a high fraction of breast cancer patients shows primary or acquired resistance to trastuzumab treatment. METHODS: We tested on trastuzumab-resistant cells two novel human anti-tumour immunoconjugates engineered in our laboratory by fusion of a human anti-ErbB2 scFv, termed Erbicin, with either a human RNase or the Fc region of a human IgG1. Both Erbicinderived immunoagents (EDIAs) are selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo, target an ErbB2 epitope different from that recognised by trastuzumab and do not show cardiotoxic effects. RESULTS: We report that EDIAs are active also on trastuzumab-resistant tumour cells both in vitro and in vivo, most likely because of the different epitope recognised, as EDIAs, unlike trastuzumab, were found to be able to inhibit the signalling pathway downstream of ErbB2. CONCLUSION: These results suggest that EDIAs are immunoagents that could not only fulfil the therapeutic need of patients ineligible to trastuzumab treatment due to cardiac dysfunction but also prove to be useful for breast cancer patients unresponsive to trastuzumab treatment.

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