A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Ra strengthens T cell-mediated anti-tumor therapies

IL-2 is a cytokine approved for the treatment of melanoma and renal cell carcinoma and induced complete, durable tumor regression in some patients. ?However, its broader use in cancer immunotherapy has been limited by severe toxicity. A new generation of IL-2 therapies with decreased binding to IL-2 receptor alpha chain (IL-2Ra), intended to mitigate toxicity and Treg expansion, are being developed. However to date these have had limited clinical success. Here, we demonstrate that the ability to engage IL-2Ra is critical for the anti-tumor activity of a systemic IL-2 therapy. Despite inducing systemic expansion of CD8+ T cells and NK cells over Tregs, an IL-2 mutein with abolished IL-2Ra binding demonstrated limited anti-tumor efficacy compared to wild-type IL-2. Based on these findings, we developed a PD-1-targeted, receptor-masked IL-2 immunocytokine, PD1-IL2Ra-IL2, with attenuated systemic IL-2 activity but maintained the capacity to engage IL-2Ra on PD-1+ T cells. PD1-IL2Ra-IL2 (REGN10597) shows PD-1-targeting-dependent IL-2 activity in vitro and drives selective expansion of tumor-infiltrating PD-1+ CD8+ T cells with vigorous effector profiles in vivo. PD1-IL2Ra-IL2 treated mice displayed no signs of systemic toxicities observed with unmasked IL-2 treatment, yet achieved robust tumor growth control. Finally, we demonstrate that PD1-IL2Ra-IL2 can be effectively combined with several other T cell-mediated immunotherapies (anti-PD-1, CD3 bispecific antibodies, and CAR-T cells) to potentiate antitumor responses. Collectively, these results provide unique insights into the functional mechanism of IL-2 based therapeutics and highlight the therapeutic potential of PD1-IL2Ra-IL2 as a novel targeted, receptor masked, and “a-maintained” IL-2 therapy for cancer treatment. Overall design: To prepare TIL samples for single cell RNA-seq and TCRseq, human PD-1 knock-in mice were subcutaneously implanted with MC38 tumor cells on both left and right flanks. When tumors were established, mice were randomized and treated on days 10 and 13 post tumor engraftment with the indicated molecules through intraperitoneal injection. On day 16 post tumor engraftment, mice were euthanized, and tumors were dissected and dissociated into single cell suspensions using a tumor isolation kit (Miltenyi 130-096-730). Right and left flank tumors from the same mouse were pooled. Single-cell suspensions were enriched for CD45+ tumor-infiltrating lymphocytes using positive selection using magnetic beads (Miltenyi 130-110-618) to achieve >95% CD45+ purity in live cells as assessed by flow cytometry (Miltenyi 130-110-797).