BACKGROUND: Interleukin-2 (IL-2) immunotherapy can induce durable tumor remissions, but its clinical performance has been limited by significant drawbacks such as short serum half-life and high toxicity. Administration of IL-2 in complex with certain anti-IL-2 antibodies (IL-2cx) enhances circulation half-life while also selectivity directing the cytokine to particular immune cell subsets. In particular, IL-2cx has been developed that targets either cells expressing the CD25-containing high-affinity IL-2 receptor (ie, CD25-biased IL-2cx) or cells expressing the CD25-lacking intermediate-affinity IL-2 receptor (ie, CD25-blocking IL-2cx). Since regulatory T (Treg) cells primarily express the high-affinity IL-2 receptor whereas naïve effector T and natural killer cells mainly express the low-affinity IL-2 receptor, CD25-blocking IL-2cx have traditionally been considered as potential cancer therapeutics, particularly in combination with immune checkpoint inhibitors (ICIs). METHODS: Stimulation of antigen-primed T cells by IL-2cx in the absence or presence of ICIs was evaluated through adoptive transfer of primed ovalbumin-specific T cells and analysis of expansion. Effects of IL-2cx on Treg cell-mediated inhibition of CD8+ T cells were assessed by flow cytometry and thymidine incorporation. Tumor-bearing mice received combination treatments comprizing IL-2cx and ICIs, where complexes were delivered either before or after ICIs. Tumor growth and mouse survival were monitored, and immune cell phenotyping was performed. Toxicity was determined by tracking body weight, temperature, and lung edema. Substitution of IL-2cx with single-agent cytokine/antibody fusion proteins (immunocytokines, ICs) was also explored. RESULTS: We showed that CD25-biased IL-2cx and ICs synergize with ICIs to completely eradicate large, established tumors despite robust Treg cell expansion. Importantly, we found that timing is crucial, as administration of IL-2cx after (but not before) ICIs led to profound antitumor effects. Mechanistically, CD25-biased IL-2cx selectively stimulated expansion and effector functions of tumor-specific CD8+ T cells in a CD25-dependent manner, overcoming Treg cell-mediated suppression. Moreover, CD25-biased IL-2cx showed much lower toxicity than CD25-blocking IL-2cx, enabling a larger therapeutic window. Furthermore, we demonstrated that administration of a human IL-2-based IC significantly enhanced the antitumor activity of ICIs, establishing the translational relevance of our work. CONCLUSIONS: Our findings support the temporally optimized use of CD25-biased IL-2-based therapeutics in combination with ICIs for cancer immunotherapy.

• Keywords
• Cytokine, Immune Checkpoint Inhibitor, Immunotherapy, T cell, T regulatory cell - Treg,
• MeSH
• Immunotherapy methods   MeSH
• Immune Checkpoint Inhibitors * pharmacology   therapeutic use   MeSH
• Interleukin-2 * agonists   pharmacology   MeSH
• Humans MeSH
• Mice MeSH
• Interleukin-2 Receptor alpha Subunit * metabolism   agonists   MeSH
• T-Lymphocytes, Regulatory * immunology   drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Immune Checkpoint Inhibitors * MeSH
• Interleukin-2 * MeSH
• Interleukin-2 Receptor alpha Subunit * MeSH

Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins' limitations as drugs. The IL-2 cytokine is a promising immune stimulant for cancer treatment but is limited by its concurrent activation of both pro-inflammatory immune effector cells and antiinflammatory regulatory T cells, toxicity at high doses, and short serum half-life. One approach to improve the selectivity, safety, and longevity of IL-2 is complexing with anti-IL-2 antibodies that bias the cytokine toward immune effector cell activation. Although this strategy shows potential in preclinical models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multiprotein drug and concerns regarding complex stability. Here, we introduced a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine toward immune effector cells. We optimized IC construction and engineered the cytokine/antibody affinity to improve immune bias. We demonstrated that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared with natural IL-2, both alone and combined with immune checkpoint inhibitors. Moreover, therapeutic efficacy was observed without inducing toxicity. This work presents a roadmap for the design and translation of cytokine/antibody fusion proteins.

• Keywords
• Cancer immunotherapy, Cytokines, Drug therapy, Immunology, Therapeutics,
• MeSH
• Cytokines metabolism   MeSH
• Interleukin-2 * immunology   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplasms immunology   therapy   drug therapy   MeSH
• Protein Engineering methods   MeSH
• T-Lymphocytes, Regulatory immunology   drug effects   MeSH
• Recombinant Fusion Proteins * pharmacology   immunology   administration & dosage   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokines MeSH
• Interleukin-2 * MeSH
• Recombinant Fusion Proteins * MeSH

The therapeutic potential of interleukin (IL)-2 in cancer treatment has been known for decades, yet its widespread adoption in clinical practice remains limited. Recently, chimeric proteins of an anti-PD-1 antibody and suboptimal IL-2 variants were shown to stimulate potent antitumor and antiviral immunity by inducing unique effector CD8+ T cells in mice. A similar subset of cytotoxic T cells is induced by depletion of regulatory T cells (Tregs), suggesting IL-2 sequestration as a major mechanism through which regulatory T cells suppress activated CD8+ T cells. Here, we present our view of how IL-2-based biologicals can boost the antitumor response at a cellular level, and propose that the role of Tregs following such treatments may have been previously overestimated.

• Keywords
• IL-2, PD-1, cancer, cytotoxic T cells, immunotherapy, regulatory T cell,
• MeSH
• CD8-Positive T-Lymphocytes * MeSH
• T-Lymphocytes, Cytotoxic MeSH
• Phenotype MeSH
• Immunotherapy MeSH
• Interleukin-2 * therapeutic use   metabolism   MeSH
• Mice MeSH
• T-Lymphocytes, Regulatory MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Interleukin-2 * MeSH