Abstract 2288: Investigational CD25 x CTLA-4 bispecific DART® molecule for depletion of tumor infiltrating Tregs via an enhanced Fc-dependent effector mechanism
Authors: Berezhnoy Alexey , Shah Kalpana , Liu Daorong , Jill Rillema , Fieger Claudia , Smith Douglas , Chen Francine , Tamura James , Bonvini Ezio , Diedrich Gundo , Moore Paul A. 
Published: September 2020

Background: Tregs are suppressive cells whose presence in the tumor microenvironment (TME) has been correlated with tumor progression and may be a major impediment in the effectiveness of tumor immunotherapy. Removal of Tregs has been shown to induce cancer regression in animal models; however, selective depletion of tumor-infiltrating Tregs (TI-Tregs) is complicated as they share phenotypic markers with Tregs in healthy tissues as well as with tumor-infiltrating effector cells. To overcome cross-reactivity and achieve enhanced targeting specificity, we employed a bispecific strategy to simultaneously target two cell surface antigens (CD25 and CTLA-4) with overlapping, high level expression on TI-Tregs. Furthermore, we incorporated Fc engineering to enable target-cell depletion via enhanced Fc-mediated effector function.

Methods: CD25 x CTLA-4 DART proteins were constructed based on CD25 and CTLA-4 mAbs and incorporating an Fc domain with enhanced binding to the activating Fc-gamma RIIIa receptor (CD16A) and reduced binding to the inhibitory Fc-gamma RIIb receptor (CD32B). Depletion of FoxP3-positive cells ex vivo was monitored by flow cytometry. Immunomodulatory effects of the DART proteins were evaluated in cultured PBMC or dissociated tumor cells in various in vitro immune assays, including mixed lymphocytes reaction (MLR).

Results: Consistent with their design, we observed increased binding of CD25 x CTLA-4 DART molecules to cells when both target antigens were engaged compared to single antigen engagement. The bispecific DART molecule depleted in vitro induced FoxP3+ CD4+ TI-like Tregs cells by an Fc-dependent mechanism, with minimal effect on the viability of FoxP3-negative T cells. A molecule bearing a null Fc was ineffective. The extent of FoxP3+ T-cell depletion by the Fc-enhanced CD25 x CTLA-4 DART molecule was greater than that exhibited by the combination of its monovalent components, with the depleting activity requiring both arms of the bispecific molecule. The DART molecule had only minor effect on the capacity of effector T cells to secrete IL-2 and interferon-gamma upon restimulation or to exert cytotoxic function. In contrast the combination of CD25 and CTLA-4 mAbs reduced the ability of effector T cells to produce cytokines in a restimulation assay. An increased response in the presence of the Fc-enhanced CD25 x CTLA-4 DART molecule was also observed in various immune assays, consistent with Treg suppression and preservation of T-cell effector function.

Conclusions: A CD25 x CTLA-4 DART molecule was effective in depleting activated FoxP3+ CD4+ Tregs in vitro, with reduced effects on resting Tregs or activated FoxP3- effector cells. Targeting CD25 and CTLA-4 in a bispecific molecular format with enhanced Fc-dependent effector function may be suitable for specifically depleting TI-Tregs.

Citation Format: Alexey Berezhnoy, Kalpana Shah, Daorong Liu, Rillema Jill, Claudia Fieger, Douglas Smith, Francine Chen, James Tamura, Ezio Bonvini, Gundo Diedrich, Paul A. Moore. Investigational CD25 x CTLA-4 bispecific DART® molecule for depletion of tumor infiltrating Tregs via an enhanced Fc-dependent effector mechanism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2288.