Abstract 5554: A bispecific T cell engager targeting CEACAM5/6 exhibits strong anti-tumor efficacy in pre-clinical studies

Background: The carcinoembryonic antigen-related adhesion molecules (CEACAM), containing seven members, are involved in many biological processes including cell adhesion, proliferation, differentiation, and tumor suppression. Some variants of CEACAMs such as CEACAM5 and CEACAM6 have long been recognized to be overexpressed in many cancers and highly associated with cancer development. Immunotherapies targeting CEACAM have achieved some clinical benefit, e.g. Tusamitamab Ravtansine (anti-CEACAM5 ADC), that has shown decent disease control in NSCLC. Recently, with the success of CAR-T and T cell engager (TCE) therapies against blood cancers, TCEs against solid tumors have also been developed. For instance, Cibisatamab, targeting CEACAM5 and CD3, is currently under clinical investigation in CRC patients. Here, we report the discovery of a bispecific antibody (biAb) anti-CEACAM5/6 x CD3 that mediates direct T cell killing against CEACAM5- and/or CEACAM6-expressing cancer cells. Methods: Anti-CEACAM5/6 x CD3 TCE consists of two CEACAM5/6 binding Fabs for the preferential recognition of CEACAM5/6-high-expressing cancer cells, and one CD3 binding domain that permits CEACAM5/6-mediated CD3 crosslinking and T cell activation. To reduce off-target T cell stimulation there is no CH2 in this format to eliminate Fc receptor binding, while an anti-HSA VHH was included into this construct for half-life extension. Binding affinity and specificity were studied by flow cytometry, IHC and on the Retrogenix Cell Microarray Technology platform, a human membrane/secreted protein binding array. The immunomodulatory functions were evaluated using luciferase reporter cell assays, cell killing assays in vitro and via PBMC-based Hu-NSG mouse models in vivo. Results: Our CEACAM5/6 binding antibody only interacted with CEACAM5 and CEACAM6 on the Retrogenix Cell Microarray against over 5,500 proteins. Importantly, it showed a similar sensitivity to recognize CRC tumor tissues in an IHC array as the benchmark (BMK), but significantly better selectivity at recognition of tumor tissues vs. normal tissues. Next, in vitro testing illustrated CEACAM5/6-mediated T cell activation by the TCE, and such stimulation also correlated with the binding affinity of the anti-CD3 domain. The lead candidate with the optimized CD3 binding affinity, showed better anti-tumor efficacy than the BMK in mice. Furthermore, this molecule displayed a typical PK as a conventional antibody with a mean T1/2 of around