Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.
Abstract Treatment with chimeric monoclonal antibody cetuximab has become the standard of care for patients with advanced colorectal cancer (CRC). Cetuximab and other EGFR-specific monoclonal antibodies predominantly inhibit cancer growth by interfering with receptor signalling. Recent analyses have shown that CRC patients with mutated KRAS and BRAF oncogenes do not profit from treatment with such antibodies. Here we have used the binding domains of cetuximab to construct a BiTE antibody that can directly engage and activate T cells by transiently connecting T cells via the CD3 subunit with EGFR-expressing target cells. The cetuximab-based BiTE antibody (C-BiTE) showed potent redirected lysis of KRAS- and BRAF-mutated human CRC lines HCT116 and HT-29, respectively, by unstimulated peripheral human T cells at half maximal concentrations of 10-40 pg/ml (ca. 0.2-0.8 picomolar). The BiTE antibody also completely prevented the outgrowth of tumors from HCT116 and HT-29 xenografts in mice at doses as low as 0.005 mg/kg when given daily i.v. for 10 days. The monoclonal antibody cetuximab was not effective in these models even at 50 mg/kg given twice weekly for 4 weeks. As a prerequisite for nonclinical safety assessment of C-BiTE in non-human primates, crossreactive binding of C-BiTE to CD3 and EGFR antigens of human and Cynomolgus monkey (Macaca fascicularis) origin was demonstrated. This binding translated into a comparable potency of redirected lysis of human EGFR-expressing cancer cells by stimulated Cynomolgus and human PBMC. Continuous i.v. infusion of 6.2 or 12.4 µg/kg C-BiTE to Cynomolgus monkeys for 3 weeks was well tolerated and led to relatively constant serum concentrations of C-BiTE between 1 and 10 ng/ml. In vitro, these serum levels would support a >90% lysis of EGFR-overexpressing CRC cells within 24 h by both Cynomolgus and human effector cells. Of note, no skin toxicity was observed during the 3-week exposure to C-BiTE. The highest doses of 31 and 154 µg/kg caused lethal damage to EGFR-expressing cells in vital organs. Inflammatory cell infiltration into the EGFR-positive tissues was observed at all dose levels but its incidence and severity showed no correlation with clinical signs. Our data suggest that C-BiTE can be safely administered to a relevant non-human primate species at dose levels similar to those of CD19-specific BiTE antibody blinatumumab, which has shown partial and complete responses in lymphoma patients. We anticipate that C-BiTE has a therapeutic window in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2429.
