Background: Immune suppression in the tumor microenvironment is known to contribute to tumor immune evasion. ILT2 (aka LILRB1) and ILT4 (aka LILRB2) are distinct ITIM-containing immunosuppressive receptors that recognize the shared ligand MHC-I. Both ILT2 and ILT4 are highly expressed on tumor infiltrating myeloid cells, while ILT2 is also expressed on tumor-associated T and NK cells. Given their common expression on myeloid cells, we anticipated that ILT2 and ILT4 blockade would be redundant. However, assays showed distinct functional activities of ILT2 or ILT4, with limited overlap. Here we explore the relative contribution of ILT2 and ILT4 on in vitro immune activation and in vivo anti-tumor activity.

Methods: Anti-ILT2 and anti-ILT4 antibodies were assessed on T cells, NK cells and various myeloid cell subsets, including monocytes, macrophages, myeloid-derived suppressor cells (MDSC) and dendritic cells. ILT2 and ILT4 do not have clear murine orthologs; thus, to characterize in vivo activity, we utilized humanized murine tumor models, including PBMC-reconstituted and CD34-reconstituted mice. Mice were engrafted with tumors and treated with anti-ILT2, anti-ILT4, dual antagonist NGM707 and/or anti-PD-1 antagonist antibodies.

Results: The in vitro and in vivo biology of ILT2 and ILT4 was explored using mono-specific antagonist antibodies, as well as the ILT2/ILT4 dual antagonist NGM707. Surprisingly, ILT2- and ILT4-specific antibodies showed distinct activities on myeloid cell populations, despite expression of both receptors. Using assays designed to induce optimal MHC-I engagement, both ILT2 and ILT4 contributed to myeloid suppression. ILT2 blockade further enhanced macrophage phagocytosis, CD8+ T cell cytolytic activity and activation of NKG2C+ memory-like NK cells. Humanized mouse models were used to characterize the in vivo anti-tumor activity of ILT2 and ILT4 blockade. Consistent with our in vitro findings, we observed a distinct effect of ILT2 and ILT4 blockade on tumor growth inhibition. Combination with an anti-PD-1 antibody further enhanced tumor suppression.

Conclusions: Despite high expression of ILT2 and ILT4 on myeloid cell populations, evidence from standard in vitro assays suggested that ILT2 and ILT4 have distinct functional activities. Here, we provide evidence that both ILT2 and ILT4 play key roles in myeloid immune suppression, and that blockade of these two receptors can be additive or synergistic. ILT2 blockade additionally enhanced T cell and NK cell function. Furthermore, in vivo studies delineated a role for both ILT2 and ILT4 blockade in promoting anti-tumor immune responses and demonstrated that blockade of ILT2 and ILT4 is complementary to PD-1 inhibition. These data support the clinical evaluation of the dual ILT2/ILT4 antagonist antibody NGM707 alone and in combination with PD-1 blockade.

Citation Format: Jane Tian, Samir Qurashi, Christina Song, Lauren Rocha, Kalyani Mondal, Suzanne Crawley, Sara Medfisch, Peirong Krisney Chen, Julie Roda, Jon Sitrin, Carmence Ho, Jonathan Aguayo, Lee Rivera, Jiawei Huang, Vicky Lin, David Shen, Yan Wang, Hui Tian, Alan Kutach, James Sissons, Daniel D. Kaplan, Geoffrey W. Stone. Immune inhibitory receptors ILT2 and ILT4 exhibit both distinct and overlapping biology in vitro and in vivo [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 664.