An Fc-inert mouse-human chimeric variant of DuoBody®-CD40x4-1BB exhibits in vivo antitumor activity and peripheral immune modulation

DuoBody®-CD40x4-1BB (GEN1042/BNT312) is an investigational bispecific antibody designed to conditionally activate costimulatory receptors CD40 and 4-1BB on immune cells to induce antitumor immunity. Comprehensive in vitro studies demonstrated that dual target engagement by DuoBody-CD40x4-1BB is required to enhance antigen presentation and T-cell priming. As the expected primary sites of action of DuoBody-CD40x4-1BB are the tumor-draining lymph nodes and the tumor microenvironment, we developed a chimeric bispecific surrogate antibody (chi-CD40x4-1BB) containing the VH/VL sequences of DuoBody-CD40x4-1BB in an Fc-inert mouse IgG2a backbone to enable mechanistic studies in vivo. Chi-CD40x4-1BB retained the functional and pharmacokinetic properties of DuoBody-CD40x4-1BB and showed antitumor activity in MC38 and B16F10-OVA syngeneic tumor models in double knock-in mice that express extracellular domains of human CD40 and 4-1BB. In the MC38 tumor model, chi-CD40x4-1BB elicited peripheral immunomodulation, including activation of CD8⁺ and CD4⁺ T cells as well as B cells. Transcriptomic profiling of tumors and lymph nodes revealed induction of gene expression consistent with CD40 and 4-1BB engagement mainly in the secondary lymphoid organs. Combination of chi-CD40x4-1BB with PD-1 blockade resulted in enhanced antitumor activity beyond the single agents. These findings establish chi-CD40x4-1BB as a relevant surrogate to evaluate the activity and mechanism of action of DuoBody-CD40x4-1BB in vivo. Chi-CD40x4-1BB induces CD40 and 4-1BB costimulation, promoting coordinated innate and adaptive immunity, resulting in antitumor activity that can be potentiated in combination with PD-1 blockade. These results may provide context for clinical investigations of DuoBody-CD40x4-1BB, as monotherapy and in combination with PD-1 inhibitors, in solid tumors.