Figure 1.

Generation of mouse-reactive Fc-inert mbsAb-PD-L1×4-1BB and characterization in vitro.

(a) Generation of mbsAb-PD-L1x4-1BB by controlled Fab arm exchange (cFAE) of Fc-silenced analogues of PD-L1 clone MPDL3280A (mAb-PD-L1) and 4-1BB clone 3H3 (mAb-4-1BB). The parental antibodies contain double matched point mutations in the CH3 domain (F405L/R411T in one and T370K/K409R in the other [EU numbering]) that drive heterodimerization of the Fab arms and formation of bispecific molecules during cFAE, as well as L234A and L235A Fc-silencing mutations that abrogate binding to FcγR and C1q. (b) Blockade of PD-1/PD-L1 interaction by mbsAb-PD-L1×4-1BB or the indicated control antibodies was assessed using a mouse PD-1/PD-L1 blockade bioassay. RLU, relative light units. (c) Induction of 4-1BB signaling by mbsAb-PD-L1×4-1BB or the indicated control antibodies was assessed using a mouse 4-1BB reporter assay. (d) CFSE-labeledCD8⁺ T cells from C57BL/6 mice were co-cultured with autologous BMDCs (T-cell:DC ratio 15:1) and stimulated with 5 ng/mL anti-CD3, or left unstimulated (w/o anti-CD3). mbsAb-PD-L1×4-1BB or the indicated control antibodies were added at 1 µg/mL each. Proliferation was measured by CFSE dilution after four days (pooled data are shown in upper panel) and expressed as expansion index (a measure of T-cell expansion at population level). IFN-γ and TNF-α concentrations in the supernatants were measured 48 hours after initiation of the culture (one representative experiment is shown in the middle and lower panels). (e) CFSE-labeledCD8⁺ T cells were co-cultured with autologous BMDCs and stimulated with anti-CD3 in the presence of increasing concentrations of mbsAb-PD-L1×4-1BB. Proliferation and IFN-γ concentration were measured as in (d). Data shown are mean (± SD) of triplicate wells of one representative experiment. *, P < .05; One-way ANOVA with Sidak’s multiple comparisons test.