Therapeutic window of an EpCAM/CD3-specific BiTE antibody in mice is determined by a subpopulation of EpCAM-expressing lymphocytes that is absent in humans

MuS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. A recent study has shown that muS110 has significant anti tumor activity at well-tolerated doses as low as 5 μg/kg in orthotopic breast and lung cancer models (Amann et al. in Cancer Res 68:143–151, 2008). Here, we have explored the safety profile of muS110 at higher doses. Escalation to 50 μg/kg muS110 caused in mice transient loss of body weight, and transient piloerection, hypomotility, hypothermia and diarrhoea. These clinical signs coincided with serum peaks of TNF-α, IL-6, IL-2, IFN-γ and IL-4, and an increase of surface markers for T cell activation. Because activation of T cells in response to BiTE antibodies is typically dependent on target cells, we analyzed mouse blood for the presence of EpCAM⁺ cells. Various mouse strains presented with a subpopulation of 2–3% EpCAM⁺ blood cells, mostly B and T lymphocytes, which was not detected in human blood samples. In vitro experiments in which the number of EpCAM⁺ cells in blood samples was either reduced or increased suggested that both T cell activation and cytokine release in response to muS110 was dependent on the number of target-expressing cells. In support for a role of EpCAM⁺ lymphocytes in the observed side effects, reduction of EpCAM⁺ blood cells in mice via a low-dose pre treatment with muS110 dramatically increased the tolerability of animals up to at least 500 μg/kg of the BiTE antibody. This high tolerability to muS110 occurred in the presence of non-compromised T cells. No damage to EpCAM⁺ epithelial tissues was evident from histopathological examination of animals daily injected with 100 μg/kg muS110 for 28 days. In summary, these observations suggest that side effects of muS110 in mice were largely caused by an acute T cell activation that was triggered by a subpopulation of EpCAM⁺ lymphocytes. Because humans have extremely low numbers of EpCAM⁺ cells in blood, this toxicity of an EpCAM-specific BiTE may be specific for mice.