Background
TGF-β (Transforming Growth Factor-beta) plays a significant role in cancer progression and has immunosuppressive effects within the tumor microenvironment (TME), inhibiting the function of T cells and thereby suppressing their anti-tumor response. Blocking or inhibiting TGF-β signaling may alleviate this immunosuppression and create a more favorable environment for T cell infiltration. However, pan-inhibition of TGF-β1/2/3 isoforms were reported to induce adverse effects that include cardiotoxicity. Selective inhibition of TGF-β1/3 may overcome the adverse effects caused by TGF-β2 blockade. To address this issue, we engineered AR148, a TGF-β SelecTrapTM that incorporates extracellular domains of TGFBR2 that is able to provide enhanced selective inhibitory effects that are specific to the targeted TGF-β isoforms.
Methods
To fine-tune the specific binding, crystal structures of TGFBR2 and TGFβ isoforms were compared to identify residues with specific binding affinity. Mutant TGF-β-traps were generated by substituting selected residues with different amino acids, and their binding affinity to TGF-β isoforms was measured using ELISA. . Binding kinetics between mutant TGFβ-traps and TGF-β isoforms were measured using BLI. Blocking potency on the Smad-dependent TGF-β signaling pathway was assessed using Western blot and HEK-Blue TGF-β cells (InvivoGen). IFN- produced by T cells in response to TGF-β and AR148 were measured using ELISA. In vivo toxicity based on concentration levels and combination/monotherapy efficacy in vivo was confirmed using a syngeneic mouse model.
Results
AR148 shows effective binding kinetics to TGF-β1/3 that are similar to wild-type tetra-receptor TGF-β trap but a 10-fold decrease in binding to TGF-β2. TGF-β1/3-induced Smad phosphorylation was effectively inhibited by AR148. Furthermore, the inhibition efficacy of AR148 against TGF-β1/3 was confirmed using reporter cells. Assessment of IFN- levels using ELISA show T cell activity was not suppressed by TGF-β-inhibition. Efficacy was demonstrated in vivo using mice when combined with anti-PD-1 with good safety profile.
Conclusions
AR148 demonstrated specific binding and inhibition potency towards TGF-β1/3 but low affinity and inhibitory effect towards TGF-β2. AR148 selectively inhibits the TGF-β1/3 isoforms and exhibited minimal toxicity in animal models. Efficacy assessment of AR148 in combination with immune checkpoint inhibitors (anti-PD-1) in vivo suggests promising potential as a therapeutic strategy.