Background/Purpose: Regulatory T cells (Treg) are a population of lymphocytes with immunosuppressive function. Activation and expansion of Treg is an attractive therapeutic approach for autoimmune diseases currently being evaluated clinically. In addition to inducing Treg activation and expansion, an effective Treg-directed therapy needs to generate cells with a stable immunosuppressive phenotype that is resistant to conversion to T effector function under inflammatory conditions. Nevertheless, current clinical approaches to stimulate Treg to treat autoimmunity expand Treg by increasing homeostatic proliferation (e.g., via IL-2 muteins) without improving Treg stability.

TNFR2 agonism induces Treg proliferation and activation. We hypothesized that TNFR2 stimulation will also increase Treg stability based on the phenotype of TNFR2 knockout mice in which Treg are unstable and convert into effector cells. To test this hypothesis, we developed multivalent anti-TNFR2 VHH proteins with agonist activity on TNFR2 and assessed their impact on Treg proliferation, activation, immune suppressive function and stability compared to IL-2 muteins.

Methods: VHHs specific for human TNFR2 were obtained by immunization of alpacas, followed by panning of phage-display VHH libraries against recombinant protein. VHH binding to TNFR2 was characterized by surface plasma resonance and flow cytometry. VHH identified as binders were formatted as multivalent fusion proteins. Human naïve Treg treated with anti-TNFR2 VHH or IL-2 mutein (PT-101) were evaluated to detect effects on proliferation, activation, suppressive function and stability in inflammatory conditions. The effects of anti-TNFR2 VHH agonists on spleen lymphocyte population in human TNFR2 knock in mice were assessed by flow cytometry 5 days after a single injection.

Results: Agonistic TNFR2 VHH induced the expansion of naïve human Treg, increased the expression of activation markers and stimulated suppressive effects against CD4+ T effector cells. Consistent with these in vitro findings, administration to mice increased Treg by 3-fold, without increasing conventional effector T cells. TNFR2 VHH constructs, but not IL-2 muteins, increased human Treg stability in the presence of proinflammatory cytokines based on sustained presence of FOXP3 bright cells and prevention of conversion of Treg into IL-17A- or IFNg-secreting cells.

Conclusion: Anti-TNFR2 VHH agonistic proteins are effective at expanding Tregs that have a more stable immunosuppressive phenotype compared to IL-2 muteins and may provide a durable and disease modifying therapy for multiple autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-tnfr2-vhh-agonistic-antibodies-promote-and-stabilize-treg-immunosuppressive-activity/