Preclinical Characterization Of Novel Anti-Ctla-4 Prodrug Antibodies With An Enhanced Therapeutic Index

Background: Blockade of the CTLA-4 pathway with ipilimumab (IPI) as monotherapy or in combination with nivolumab (anti–PD-1) is an effective treatment for a variety of cancers. To enhance the therapeutic index of CTLA-4–directed therapy, a proprietary Probody® therapeutics (PB-Tx) technology platform was used to generate anti–CTLA-4 monoclonal antibodies (mAbs) with a masking peptide to attenuate CTLA-4 binding in the periphery. Increased protease activity in the tumor microenvironment is hypothesized to preferentially cleave the masking peptide. Here, we describe the preclinical characterization of 2 novel anti–CTLA-4 PB mAbs: anti–CTLA-4 PB (BMS-986249) is a peptide-masked version of IPI, and anti–CTLA-4 nonfucosylated (NF) PB (BMS-986288) is a peptide-masked version of anti–CTLA-4 NF, which has enhanced antibody-dependent cellular cytotoxicity (ADCC) and regulatory T-cell (Treg) depletion compared with IPI. Methods: Antibody binding to CD16 was studied by surface plasmon resonance. IL-2 release from staphylococcal enterotoxin B (SEB)–stimulated normal human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells was measured; ADCC function was evaluated by IL-2–activated, NK-cell–induced lysis of CD4+ T cells or activated CD4+Foxp3+ Tregs. Tumor growth was measured in transgenic (human CTLA-4 knock-in) mice implanted with MC38 tumors. Peripheral pharmacodynamic (PD) markers (ICOS, Ki-67) of anti–CTLA-4 activity were assessed using Tregs and effector T cells from the spleens of treated mice. PD was evaluated in cynomolgus (CYNO) macaques after administration of customized adenovirus-5 vector vaccines. Results: With the masking peptide intact, both anti–CTLA-4 PB and anti–CTLA-4 NF PB showed decreased activity vs IPI and anti–CTLA-4 NF, respectively, in non–protease containing in vitro assays. Anti–CTLA-4 PB exhibited a 40-fold reduction in CTLA-4 binding affinity vs IPI and decreased activity in SEB-activated PBMCs. Anti–CTLA-4 NF PB showed decreased ADCC activity vs anti–CTLA-4 NF in vitro. When tested in a MC38 tumor model, anti–CTLA-4 PB and anti–CTLA-4 NF PB showed equivalent antitumor activity to IPI and anti–CTLA-4 NF, respectively. Both anti–CTLA-4 PB and anti–CTLA-4 NF PB showed equivalent intratumoral PD activity and reduced peripheral PD activity relative to their parental mAbs. Similarly, anti–CTLA-4 PB and anti–CTLA-4 NF PB resulted in reduced inflammation and peripheral PD responses relative to their parental mAbs in CYNO macaques. Conclusion: These data demonstrate the potential of the PB-Tx technology platform to improve the therapeutic indices of anti–CTLA-4 PB and anti–CTLA-4 NF PB relative to their parental mAbs. The safety and antitumor activity of anti–CTLA-4 PB (NCT03369223) and anti–CTLA-4 NF PB (NCT03994601) are being investigated in patients with advanced solid cancers in ongoing phase 1 studies. Citation Format: John Engelhardt, Rahima Akter, John Loffredo, Natalie Bezman, Paula So, Kimberly Tipton, Bryan Irving, James West, Wendy Freebern, Todd Bunch, Karen Price. Preclinical characterization of novel anti-CTLA-4 prodrug antibodies with an enhanced therapeutic index [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 4551.