Abstract

Background IL-2 plays a critical role in regulating the immune system by stimulating the proliferation and activation of T cells, and thus has the potential to enhance anti-tumor immunity. Nevertheless, the clinical use of IL-2 is severely limited by significant systemic toxicity and a narrow therapeutic window. To overcome these limitations, we developed an antibody-cytokine fusion protein, termed anti-PD1-IL2Att, that is composed of an anti-PD1 antibody fused to a highly attenuated variant of IL-2. This construct was designed to deliver attenuated IL-2 specifically to PD-1-expressing cells. Anti-PD1-IL2Att selectively stimulates PD-1+ effector T cells, while not inhibiting PD-1 receptor function. Since PD-1 expression is significantly higher in tumor-infiltrating T cells compared to circulating and tissue-resident T cells, targeting IL-2 to PD-1+ T cells may boost anti-tumor immunity, while avoiding systemic IL-2-related adverse effects. Here we describe the impact of anti-PD1-IL2Att activity on tumor progression and tumor-infiltrating lymphocytes in pre-clinical tumor models. Methods The anti-tumor effect of anti-PD1-IL2Att was evaluated using a mouse surrogate antibody-cytokine fusion protein (mAPD1-IL2Att) in a mouse model of MC38 colorectal adenocarcinoma in a dose escalation study, a repeated-dose administration study and a long-term immunity rechallenge study. Additionally, the ability of a human anti-PD1-IL2Att, TEV-56278, to induce anti-tumor immune responses was evaluated in humanized immune-system mouse models engrafted with human melanoma tumor cells. All models employed flow cytometry to characterize T cell population variations within the tumor microenvironment after treatment. Results Administration of mAPD1-IL2Att in the MC38 mouse model resulted in dose-dependent reduction in tumor growth following single dose administration and complete tumor regression following repeated administration. Furthermore, mice responsive to mAPD1-IL2Att treatment were fully protected from secondary tumor re-challenge, indicating induction of long-term anti-tumor immunity. Additionally, TEV-56278 administration to humanized immune system mice engrafted with human melanoma tumors led to tumor growth inhibition similar to that observed in the MC38 model. Flow cytometry analysis of tumor-infiltrating lymphocytes revealed a shift in the immune response in both models following treatment. Specifically, we observed a significant increase in the proportion of effector T cells, accompanied by a decrease in regulatory T cells. Moreover, in the MC38 model terminally exhausted CD8+ T cells (CD8+CD44+CD62L-CD69+ki67+PD-1+Tim3+) were decreased following treatment. These findings collectively suggest a favorable immune-activated anti-tumor response. Conclusions Anti-PD1-IL2Att treatment led to tumor regression, increased infiltration of T cells into the tumor, and establishment of durable immune memory. Our preclinical models highlight the potential of anti-PD1-IL2Att as an effective anti-tumor immunotherapy. Ethics Approval The in vivo animal studies described in the abstract were approved by the following ethics review boards: IACUC #: NPC-TE-IL-2307-447 (Israel Ministry of Health); IACUC ASP #: 980701 (Charles River) and IACUC ASP #: 980702 (Charles River).