Abstract Immunocytokines fusing anti-PD-1 antibody with interleukin-2 (IL-2) have demonstrated therapeutic potential in clinical settings in overcoming resistance to PD-(L)1 inhibitors. However, clinical response rates were varied, highlighting the need for improved efficacy, particularly in tumors with primary resistance to PD-1 blockade expressing high levels of LAG-3. Emerging evidence indicates LAG-3, a key immune checkpoint, acts synergistically with PD-1 to promote T cell exhaustion, while dual blockade of LAG-3 and PD-1 can reprogram CD8+ T/Tregs to enhance antitumor immunity. Here, we present AR166, a novel tri-specific immunocytokine engineered with novel Fc-silencing, delivering dual PD-1 and LAG-3 blockade while inducing PD-1-targeted CD8+ T cell activation via an optimized IL-2 variant (IL-2v). Patient-derived samples were profiled by flow cytometry and IHC. PD-1×LAG-3 bispecific activity and IL-2v cis-acting were assessed using PD-1/PD-L1 and LAG-3/MHC II blockade assays, and PD-1 and/or LAG-3-dependent CD8+ T cell pSTAT5 signaling. Fc-silencing was assessed using ADCC, ADCP, and CDC assays. Repeatedly stimulated PBMCs were co-cultured with tumor cells to determine T cell proliferation and activation in the presence of AR166. Patient-derived samples were treated with AR166 followed by immune cell profiling. Antitumor efficacy was evaluated in tumor-bearing (≥300 mm3) humanized syngeneic mouse models and tumor infiltrating lymphocytes were profiled. Complete responders were rechallenged with tumor cells to assess memory response. Pharmacokinetic and safety profiles were analyzed in hIL-2Rαβγ knock-in mice and cynomolgus monkeys. Expression of PD-1 and LAG-3 was confirmed in patient-derived tumor samples. AR166 effectively inhibited PD-1/PD-L1 and LAG-3/MHC II pathways while IL-2v showed cis-activity only when anchored to PD-1 and/or LAG-3. Assessment of Fc-silencing showed minimal Fc-dependent effector functions. AR166 elicited robust T cell proliferation and activation compared to a PD-1xIL2v competitor in an in vitro T cell exhaustion model, with similar results observed in patient-derived CD8+ T cells. AR166 showed remarkable tumor growth inhibition compared to anti-PD-1 and PD-1xIL-2v competitors in humanized mouse models that recapitulate anti-PD1 resistance with high LAG-3 expression, while immune profiling showed rapid expansion of PD-1+TCF1+CD8+ stem-like T cells. AR166 elicited durable tumor regression in a tumor rechallenge model, suggesting tumor-specific immune memory. AR166 achieved favorable safety and pharmacokinetic profiles in both mice and cynomolgus monkeys, supporting its broad therapeutic window. Our findings show that AR166, a first-in-class PD-1xLAG-3xIL-2v tri-specific, can exceed the efficacy of both PD-1 inhibitors and PD-1xIL-2v with good safety. Citation Format: Jaeho Song, Seon-mi Yu, Jae-Seok Lee, Jinkeol Mok, WooJeong Lee, Seo-Young Koo, Ye Rim Gu, Min-Young Park, Seoyun Yang, Juhan Yoon, Young Woo Park, Wooick Jang, Su-Hyung Park. AR166, a first-in-class PD-1xLAG-3xIL-2v tri-specific immunocytokine delivering Cis-acting IL-2v to overcome immune checkpoint inhibitor resistance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4332.
Song et al. (Fri,) studied this question.
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