Background: Ig-Like Transcript 2 (ILT2, LILRB1) is an inhibitory receptor prominently expressed on resting macrophages, monocytes, and dendritic cells (DCs), with variable expression on CD4+ T cells, CD8+ T cells, B cells, NK cells, and NKT cells in the tumor microenvironment. Interaction of ILT2 with its ligands, HLA-G and classical MHC class I molecules, is reported to impair cytotoxic activity of NK and effector T cells, attenuate B cell function, inhibit antigen-presentation by dendritic cells, and promote immunosuppressive activity of myeloid cells. Further, high ILT2 expression has been associated with poor prognosis in several human malignancies. As a result, ILT2 has been proposed as a therapeutic target to promote antitumor immunity and to overcome resistance to checkpoint blockade. Here, we report that AGEN1571, a fully human anti-ILT2 monoclonal IgG4κ antibody, reversed ILT2-mediated immunosuppression and promoted activation of NK, NKT, T cells, and myeloid cells in a series of preclinical studies.

Methods: AGEN1571 binding to ILT2 was analyzed by SPR and flow cytometry. HDX-MS was used to identify the AGEN1571 binding epitope on ILT2. Mechanisms of AGEN1571-mediated modulation of immune cell function were evaluated using reporter cell lines and primary cell-based in vitro and ex vivo assays.

Results: AGEN1571 bound to recombinant ILT2 with an approximate monovalent binding affinity of 0.38 nM and to cell-expressed ILT2 with an EC50 of 0.07 - 0.55 nM, depending on cell type and donor. AGEN1571 did not cross-react with related LILRA and LILRB family members except with weak reactivity to ILT4 (~370 nM). Consistent with its binding epitope, AGEN1571 potently blocked ILT2 binding to HLA-A, -B, -C, and -G and restored FcγR signaling inhibited by ILT2-HLA-G engagement in dose-dependent manner. AGEN1571 also augmented NFκB and ISRE activation induced by TLR, FcγR, and STING agonists in myeloid cells, and promoted polarization of primary human macrophages towards a pro-inflammatory phenotype. ILT2 blockade with AGEN1571 alone and in combination with a PD-1 antagonist enhanced secretion of pro-inflammatory cytokines and activation of NK, NKT and CD8+ T cells in a PBMC tumor cell co-culture assay. AGEN1571 was designed to have minimal binding to FcγR and complement which has been confirmed by lack of FcγR signaling and complement cascade induction in vitro. An in silico T cell epitope screen predicted AGEN1571 to be non-immunogenic, while AGEN1571 showed no evidence of inducing systemic cytokine release in a cytokine release assay using whole blood and PBMC cultures.

Conclusion: AGEN1571 is a novel high-affinity ILT2 antagonist antibody that effectively antagonizes ILT2 to enhance T cell, NK cell, NKT cell, and myeloid activity. These data support clinical development of AGEN1571 as a therapeutic agent for patients with solid tumors.

Citation Format: Olga Udartseva, Malgorzata Pupecka-Swider, Pilar Garcia-Broncano, Naxin Guo, Spencer Campbell, Elizabeth Klakus, Rebecca Ward, Priyadarshini Iyer, Christine Brittsan, Beth Wensley, Benjamin Morin, Andrew Slee, Dhan Chand, Claire Galand, Olga Ignatovich, Emmanuel Briend, David Savitsky. AGEN1571 is a novel high-affinity ILT2 antagonist antibody that promotes adaptive and innate immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2906.