Abstract
Targeting various combinations of tumor antigens and immune cell receptors is of increasing importance in antibody-based cancer immunotherapy. Here, we present a novel modular P329G-engager platform that enables rapid combination of primary tumor-targeting and secondary immune effector antibodies. The platform utilizes two antibodies, each selected from: 1) a set of tumor-targeting adaptor antibodies, bearing P329G mutations in the Fc region, and 2) a set of P329G-targeting (bispecific) cell engagers, including innate and T cell engagers, costimulators and immunocytokines. Specifically, upon defining a tumor-associated cell surface target, a primary adaptor - tumor antigen-binding IgG1 antibody with Fc-silencing P329G L234A L235A mutations - is administered. Subsequently, a secondary antibody recognizing the P329G mutation is chosen from a panel of effector cell engagers with different modes of action - ADCC-competent P329G-innate cell engagers (P329G-ICE), P329G-T cell bispecifics (P329G-TCB), P329G-costimulators (P329G-CD28/4-1BBL), or P329G-immunocytokine (P329G-IL2v). In vitro assays showed that all P329G-targeting modalities induce anti-tumoral and/or immunomodulatory activity when both components were combined. In vivo, tumor shrinkage and T cell infiltration were confirmed in tumor-bearing humanized mice treated with P329G-mutated CEACAM5 adaptor IgG and P329G-TCB. Individually, neither the adaptor nor the P329G-TCB induced efficacy, validating the requirement for primary and secondary antibody assembly for T cell-engaging activity. These results provided evidence for the in vivo assembly and subsequent pharmacological activity, and provide preclinical proof-of-concept for the P329G-engager platform as an efficacious tool in drug discovery. Ultimately, this modular approach may enable mix-and-match drug assembly as a novel therapeutic principle in immunotherapy.