Abstract 4079: EGL-001 is a novel immunocytokine designed to specifically target and disarm T regulatory cells in the tumor microenvironment
Authors: Kotsias Fiorella , Janot-Sardet Caroline , Caudana Pamela Pamela , Russo Erica , Dubois Marine , Zucchetti Andres , Tkach Mercedes , Lemoine Sebastien , Mamedov Ilgar Z. , Lladser Alvaro , Denizeau Jordan , Meola Annalisa , Sedlik Christine , Borcoman Edith , Rey Felix , Guardado-Calvo Pablo , Vanhove Bernard , Gostissa Monica , PIaggio Eliane 
Published: March 2024

Abstract
Introduction Immune checkpoint blockade (ICB) therapies targeting PD-1/PD-L1 or CTLA-4 have greatly improved clinical outcome in many cancers. However, multiple mechanisms of resistance to ICB severely limit the number of patients experiencing long-term survival benefits. Overcoming resistance to ICB represents a significant challenge for drug development. Regulatory T cells (Tregs) in the tumor microenvironment (TME) prevent effector T cells (Teffs) from mounting a productive immune response and have been implicated in ICB resistance. Novel therapeutic strategies aimed at disarming Tregs in the TME could restore anti-tumor responses and sensitize patients to ICB therapy. Treg survival is dependent on interleukin-2 (IL-2), which signals in Tregs via the high affinity IL-2 receptor (IL-2R). Tumor-associated Tregs differentiate from tumor-associated Teffs and from peripheral Tregs by higher expression of CD25, the IL-2R alpha chain. Tregs in the TME also express higher surface levels of CTLA-4 as compared to peripheral Tregs. Thus, CD25 and CTLA-4 overexpression could allow preferential targeting of tumor-associated Tregs.
Methods and Results We used single-cell RNA sequencing data from cancer patients and flow cytometry analysis of human tumor samples to verify that CD25 and CTLA-4 are selectively overexpressed in tumor-associated Tregs as compared with peripheral Tregs or other T cells. Based on these findings, we developed EGL-001, a novel immunocytokine (ICK) comprising a humanized IgG1 directed against CTLA-4 fused to a Treg-specific IL-2R antagonist IL-2 mutein, to selectively target tumor infiltrating Tregs. EGL-001 IgG1 moiety binds membrane CTLA-4, as demonstrated by flow cytometry, and competes with CTLA-4-CD80/86 interaction, as evaluated by ELISA. EGL-001 IL-2 mutein moiety binds to CD25, as demonstrated by bio-layer interferometry, and specifically blocks high-affinity IL-2R signaling in Tregs. Signaling blockade is further potentiated by a CTLA-4 dependent CD25 downmodulation process evidenced by confocal microscopy and flow cytometry. We demonstrate that EGL-001 unique tumor-associated Treg-targeted dual mechanism of action results in increased Treg apoptosis and decreased Treg frequencies in multiple in vitro, ex vivo and in vivo systems. Importantly, EGL-001 only minimally affects IL-2 signaling and survival of Teffs, which are then expected to be unleashed from Treg cell-mediated suppression. Accordingly, EGL-001 induces anti-tumor responses in mice in combination with anti-PD1 therapy.
Conclusions EGL-001 is an ICK with a novel and unique mechanism of action combining ICB and Treg selective IL-2 starvation, designed to specifically disarm and deplete tumor-associated Tregs. Based on our preclinical data, EGL-001 is being developed as a cancer immunotherapeutic to improve patients’ response to ICB and counteract ICB resistance.
Citation Format: Fiorella Kotsias, Caroline Janot-Sardet, Pamela Pamela Caudana, Erica Russo, Marine Dubois, Andres Zucchetti, Mercedes Tkach, Sebastien Lemoine, Ilgar Z. Mamedov, Alvaro Lladser, Jordan Denizeau, Annalisa Meola, Christine Sedlik, Edith Borcoman, Felix Rey, Pablo Guardado-Calvo, Bernard Vanhove, Monica Gostissa, Eliane PIaggio. EGL-001 is a novel immunocytokine designed to specifically target and disarm T regulatory cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4079.