Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF- and PD-L1 enhances antitumor responses

T cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-beta play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-beta inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-beta, termed AxF (scFv)2, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)2 antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3xEGFR TriTE with the AxF (scFv)2 delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-beta by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression.