Improved Fc-mediated effector functions by an anti-CTLA-4 multivalent Fc agent

Numerous therapeutic monoclonal antibodies (mAbs) rely on antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) Fc effector functions to deplete target cells and achieve clinical efficacy. It has been previously shown that adding multiple Fc domains to Abs or afucosylating the Fc domain increases Fc effector functions. We have leveraged a proprietary Fc multimerization technology (SIF; selective immunomodulator of Fc receptors) to identify potential novel products designed to improve the immune system’s elimination of tumor and other pathogenic cells. These agents utilize the valency effect of Fc multimerization to increase the binding to Fc receptors and complement, enhancing immune-mediated cytotoxicity mechanisms. CTLA-4 is a clinically validated immune checkpoint inhibitor exemplified by the human IgG1 therapeutic mAb ipilimumab. CTLA-4 is induced upon activation on T cells and constitutively expressed on T regulatory cells (Tregs). Based on data from mouse models of cancer and clinical studies, the proposed mechanisms of action of anti-CTLA-4 mAbs, including ipilimumab, are to block the interaction of CTLA-4 with its ligands CD80 and CD86 resulting in T cell activation and to induce Fc-mediated ADCC of CTLA-4+ cells, mainly intratumoral Tregs. Therefore, we have produced an anti-CTLA-4 multivalent IgG1 Fc agent termed anti-CTLA-4 SIFbody with the purpose of enhancing binding to Fc gamma receptors (FcγRs) and complement and increasing Fc-mediated elimination of intratumoral Tregs. Data from avidity binding to low affinity FcγRs showed more than 100-fold increase with anti-CTLA-4 SIFbody as compared to ipilimumab. In in vitro functional assays using CTLA-4 transfected target cells and primary human effector cells, anti-CTLA-4 SIFbody showed more than 10-fold increase in potency in ADCC and more than 5-fold increase in ADCP as compared to ipilimumab. Anti-CTLA-4 SIFbody induced 80% cell lysis by CDC, whereas ipilimumab failed to show any activity. More importantly, we generated in vitro expanded Tregs with suppressive function and showed that anti-CTLA-4 SIFbody induced significant enhanced ADCC on these cells as compared to ipilimumab and to an afucosylated anti-CTLA-4 mAb. ADCP was also significantly increased on Tregs. Unexpectedly, Tregs were resistant to anti-CTLA-4 SIFbody induced CDC. Notably, the blocking activity of the SIFbody F(ab)s was not altered by the multivalent Fc structure as shown by similar CTLA-4/CD80 and CD86 blockade and induction of IL-2 production upon antigen stimulation of PBMC. In conclusion, these data demonstrate that our Fc multimerization technology applied to an anti-CTLA-4 mAb significantly improves Fc-dependent immune-mediated cytotoxicity and suggest that anti-CTLA-4 SIFbody may represent an optimized novel product to deplete intratumoral Tregs and enhance anti-tumor activity. Citation Format: Laura Rutitzky, Daniel F. Ortiz, Jonathan C. Lansing, Julia Brown, Joseph Paquette, Kevin Garofalo, Josephine D9Alessandro, Naveen Bhatnagar, Maurice Hains, Abhinav Gupta, Stan Lee, Radouane Zouaoui, Jason Wang, John Schaeck, Salvatore Marchese, Robin Meccariello, Nathaniel Washburn, Kimberly Holte, Carlos J. Bosques, Anthony M. Manning. Improved Fc-mediated effector functions by an anti-CTLA-4 multivalent Fc agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3244.