The activity of ipilimumab as a single agent and in combination with nivolumab (anti-PD-1) in melanoma, as well as the use of ipilimumab/nivolumab combinations in other malignancies, has confirmed the importance of CTLA-4 blockade in immunotherapy. The antitumor effect of this treatment also results in significant immune-related adverse events that limit dosing and result in patient discontinuation. We have taken two approaches to alter the activity of ipilimumab so as to improve its potency and its safety profile. One approach is to enhance the antibody-dependent cellular cytotoxicity (ADCC) activity of ipilimumab in order to increase the potential for Treg depletion at the tumor site; this would be expected to increase the activity of the antibody. The second approach is to produce a prodrug form of ipilimumab (an anti-CTLA-4 Probody therapeutic) that will have reduced activity systemically, but will become proteolytically cleaved at the tumor site to produce the fully functional antibody; the goal of this approach is to reduce the adverse event profile while retaining the antitumor activity of ipilimumab. It has previously been shown that antitumor activity of anti-CTLA-4 antibodies in mouse models of cancer is dependent on the ability of the antibody to bind activating FcγRs and mediate ADCC against Tregs at the tumor site (1, 2). Although human IgG1 Abs have been shown to be effective mediators of ADCC in patients with hematologic malignancies, it is still unclear whether ipilimumab mediates Treg depletion in solid tumors. Using in vitro ADCC assays, we have found that a nonfucosylated (NF) version of ipilimumab (ipilimumab-NF) has increased activity compared to ipilimumab. Ipilimumab-NF also demonstrates increased IL-2 secretion in peripheral mononuclear cells treated with the superantigen SEB as compared to ipilimumab. Transgenic mice that express human FcγRs in the place of mouse FcγRs were used to investigate the activity of anti-mouse surrogate CTLA-4 antibody engineered with either a human IgG1 or human IgG1-NF Fc region in a mouse tumor model. In these mice, the IgG1-NF version of anti-mouse CTLA-4 was found to significantly increase antitumor activity and Treg depletion at the tumor site compared to the IgG1. These data suggest that the clinical activity of ipilimumab could be enhanced by use of the nonfucosylated version of the Ab. In addition, ipilimumab-NF was tested for its ability to enhance a vaccine response in Mauritian cynomolgus macaques. Ipilimumab-NF was shown to result in increased vaccine-induced T-cell responses compared to ipilimumab using two replication-incompetent adenovirus serotype 5 viral vectors encoding SIV antigens as assessed by MHC-I tetramers and IFN-gamma ELISPOT in Mauritian cynomolgus macaques expressing the common allele, Mafa-A1*063. In a second approach, using Probody platform technology developed by CytomX, we have developed an anti-CTLA-4 Probody therapeutic (Probody Tx) based on ipilimumab. Probody Txs utilize a masking peptide that binds to the antigen-binding site of the Ab to reduce target binding. The mask extends from the light chain of the Ab via a linker sequence that contains cleavage sites for proteases preferentially active at the tumor site relative to healthy tissue. The ipilimumab Probody Tx binds to CTLA-4 with significantly lower affinity than the parental antibody and has reduced activity in in vitro assays. When tested in a mouse tumor model using human CTLA-4 KI mice, the ipilimumab-Probody Tx has comparable antitumor activity and Treg depletion at the tumor compared to ipilimumab. In contrast, ipilimumab-Probody Tx-treated mice show reduced levels of activated peripheral Tregs compared to ipilimumab-treated mice, even at doses 8-fold higher than are required for antitumor efficacy, consistent with reduced activity of the Probody Tx outside the tumor microenvironment. The development of next-generation anti-CTLA-4 antibodies holds promise for improving the utility of ipilimumab for single-agent or combination therapy. The two improvements to ipilimumab outlined above could each lead to a superior therapeutic outcome and merit further investigation. References 1. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, et al. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res 2013;1:32-42. 2. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 2013;210:1695-710. 1. Citation Format: Alan J. Korman, John Engelhardt, John Loffredo, Jose Valle, Rahima Akter, Raja Vuyyuru, Natalie Bezman, Paula So, Robert Graziano, Kimberly Tipton, James West, Bryan Irving, Mark Selby. Next-generation anti-CTLA-4 antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY09-01. doi:10.1158/1538-7445.AM2017-SY09-01

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. Antibody-dependent cellular cytotoxicity (ADCC) is a cellular process through which effector immune cells that express an Fc receptor (FcR) recognize and eliminate antibody-bound targets. Anti–CTLA-4 has been shown to mediate the ADCC depletion of tumor-infiltrating regulatory T cells (Tregs), which play an important role in suppressing antitumor immune response in the tumor microenvironment. A version of IPI that is nonfucosylated (NF) in the Fc region (BMS-986218; anti–CTLA-4 NF) was created to increase binding affinity to activating Fcγ receptors (FcγR, CD16) and improve ADCC, thus increasing intratumoral Treg depletion. 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 CD16) mice implanted with MC38 tumors. Peripheral pharmacodynamics (PD) was evaluated in cynomolgus (CYNO) macaques after administration of customized adenovirus-5 vector vaccines. Results: Binding affinity to human CD16 was enhanced by BMS-986218 compared with IPI. BMS-986218 induced greater NK cell–mediated lysis of CD4+ T cells (37%) and Tregs (100%) compared with that induced by IPI (7% and 58%, respectively). Anti–CTLA-4 NF was also more effective than IPI in enhancing IL-2 release from SEB-stimulated PBMCs. Addition of an anti-CD16 antibody blocked the IL-2 response, supporting the concept that CD16 was necessary for anti–CTLA-4 NF activity. In mice, anti–CTLA-4 NF produced greater dose-dependent tumor growth inhibition (99%) than anti–CTLA-4 (61%). Tumors from anti–CTLA-4 NF–treated mice compared with anti–CTLA-4–treated mice had a greater decrease in Treg levels (70% vs 30%, respectively) and a larger increase in CD8+ T-cell levels (82% vs 62%, respectively) and no changes in Treg or CD8+ cell levels in the spleen. A more pronounced vaccine-induced T-cell response in anti–CTLA-4 NF–treated vs anti–CTLA-4–treated CYNO macaques was observed over a 3-week period. Conclusions: Nonfucosylation of an anti–CTLA-4 antibody increased binding affinity to CD16, induced depletion of Tregs while increasing T-effector cells in mouse tumors, and enhanced tumor growth inhibition in a dose-dependent manner, thus demonstrating improved ADCC compared with IPI. An ongoing phase 1/2 study is evaluating the safety and antitumor activity of anti–CTLA-4 NF alone and in combination with nivolumab (NCT03110107) in patients with advanced solid cancers. Citation Format: John Engelhardt, Rahima Akter, John Loffredo, Paula So, Natalie Bezman, Karen Price. Preclinical characterization of BMS-986218, a novel nonfucosylated anti–CTLA-4 antibody designed to enhance antitumor activity [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 4552.

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.

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