CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

Background: A strategy to broaden the applicability of checkpoint inhibitors is the combined use with antibodies targeting the immune stimulatory receptors CD40 and 41BB. However, the use of anti-CD40 and anti-41BB antibodies as agonists is problematic in two ways. First, anti-CD40 and anti-41BB antibodies need plasma membrane-associated presentation by FcγR binding to exert robust agonism but this obviously limits their immune stimulatory efficacy by triggering ADCC, CDC or anti-inflammatory FcγRIIb activities. Second, off tumor activation of CD40 and 41BB may cause dose limiting systemic inflammation. Methods: To overcome the FcγR-dependency of anti-41BB and anti-CD40 antibodies, we genetically fused such antibodies with a PDL1-specific blocking scFv as anchoring domain to enable FcγR-independent plasma membrane-associated presentation of anti-CD40- and anti-41BB antibodies. By help of GpL-tagged variants of the resulting bispecific antibodies, binding to their molecular targets was evaluated by help of cellular binding studies. Membrane PDL1-restricted engagement of CD40 and 41BB but also inhibition of PDL1-induced PD1 activation were evaluated in coculture assays with PDL1-expressing tumor cell lines and 41BB, CD40 and PD1 responsible cell lines or T-cells. Results: The binding properties of the bispecific antibody fusion proteins remained largely unchanged compared to their parental molecules. Upon anchoring to membrane PDL1, the bispecific antibody fusion proteins activated CD40/41BB signaling as efficient as the parental anti-CD40/anti-41BB antibodies when bound to FcγRs or cells expressing membrane-bound CD40L/41BBL. PD1 inhibition remained intact and the anti-41BB fusion protein thus showed PDL1-restricted costimulation of T-cells activated in vitro with anti-CD3 or a BiTe. Conclusions: Targeting of anti-CD40 and anti-41BB fusion proteins to membrane PDL1 with a blocking PDL1 scFv links PD1-PDL1 checkpoint blockade intrinsically with engagement of CD40 or 41BB.