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.

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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