Targeting IL-21 to tumor-reactive T cells enhances memory T cell responses and anti-PD-1 antibody therapy

Immune checkpoint blockade, specifically PD-1/PD-L1 blockade, has shown remarkable success in treating advanced cancers by reactivating tumor-reactive CD8+ T cells. However, this success is limited to a subset of patients. The failure of PD-1 blockade in many cases is linked to inefficient T cell activation. Cytokines, particularly IL-2, IL-15, and IL-21, play a crucial role in regulating CD8+ T cell responses. While IL-2 is widely used, IL-21 is particularly important for memory CD8+ T cell development and maintenance, exhibiting synergistic effects with IL-7. Tumor-reactive T cells expanded with IL-21 show superior antitumor activity compared to those expanded with other cytokines. However, systemic cytokine administration leads to broad effects and side effects due to activation of counter-regulatory pathways and off-target effects. This study explores the concept of targeting IL-21 specifically to tumor-reactive T cells, leveraging the fact that PD-1 is highly expressed on these cells, acting as a marker for identifying and targeting them. The hypothesis was that fusing IL-21 to an anti-PD-1 antibody would effectively deliver IL-21 to the desired T cells, thereby enhancing efficacy while minimizing side effects.

The literature review extensively covers the role of PD-1 blockade in cancer immunotherapy, highlighting its limitations and the need for combination therapies. It discusses the importance of IL-21 in T cell development and function, emphasizing its role in generating memory T cells and enhancing antitumor responses. The review also explores the challenges of systemic cytokine administration and the advantages of targeted cytokine delivery. Existing studies demonstrating the efficacy of IL-21 in combination with other immunotherapies are reviewed, setting the stage for the current study's investigation into a targeted IL-21 delivery system.

The study involved the construction of a fusion protein, PD-1Ab21, by fusing IL-21 to a diabody form of an anti-PD-1 single-chain antibody fragment. This fusion protein was expressed in 293E cells and purified. In vitro studies assessed the binding of PD-1Ab21 to PD-1-expressing cells and its ability to activate T cells. The effects of PD-1Ab21 on the differentiation of CD8+ T cells were characterized using OVA-specific TCR transgenic CD8+ T cells (OT-I cells) and polyclonal CD8+ T cells. The in vivo antitumor effects of PD-1Ab21 were evaluated in CT26 and MC38 tumor models, comparing it to anti-PD-1 antibody alone, a combination of anti-PD-1 and IL-21, and control groups. The study also explored the combination of PD-1Ab21 with anti-Her2/neu antibody therapy and cancer vaccination. Flow cytometry, immunofluorescence staining, RNA sequencing, and ELISA were employed to analyze T cell differentiation, proliferation, cytokine production, and tumor-specific T cell responses. Specific details on cell lines, reagents, antibody preparation, in vitro assays, in vivo tumor models, size-exclusion chromatography, bioassays, flow cytometry protocols, PD-1 receptor occupancy assays, RNA-seq procedures, ELISA methods, immunofluorescence staining techniques, and statistical analyses were provided in the methods section.

The key findings demonstrate that the PD-1Ab21 fusion protein effectively targets IL-21 to PD-1+ T cells in vitro and in vivo. In vitro experiments showed PD-1Ab21 induced differentiation of naïve-like T cells from activated CD8+ T cells, more effectively than IL-21 alone. In vivo studies showed that PD-1Ab21 treatment significantly enhanced the antitumor effect compared to anti-PD-1 antibody alone or combined anti-PD-1 and IL-21 treatment in several tumor models. PD-1Ab21 treatment also led to a significant expansion of tumor-specific memory CD8+ T cells (especially TSCM), as demonstrated by increased frequencies of these cells in the spleen, tumor-draining lymph nodes, and tumors. The enhanced antitumor effect was observed in different tumor models and when PD-1Ab21 was combined with anti-Her2/neu antibody or cancer vaccination. RNA sequencing analysis revealed distinct transcriptomic profiles of T cells generated by PD-1Ab21 compared to other treatments, indicating a unique mode of action. In summary, the results demonstrate that PD-1Ab21 enhances the therapeutic efficacy of immune checkpoint blockade by simultaneously targeting cytokines to tumor-reactive T cells, promoting the differentiation of memory T cells and leading to superior antitumor effects.

The study successfully demonstrated a novel strategy to enhance the efficacy of immune checkpoint blockade by targeting IL-21 to tumor-reactive T cells using a fusion protein, PD-1Ab21. This approach addressed the limitations of systemic cytokine administration by precisely delivering IL-21 to activated T cells, improving the therapeutic index and reducing side effects. The superior antitumor activity of PD-1Ab21 compared to combined PD-1 blockade and IL-21 infusion highlights the importance of targeted delivery. The findings contribute significantly to the field by providing a potential approach for improving the response rates of patients to PD-1 blockade, a major challenge in cancer immunotherapy. Further research should focus on translating this approach to human clinical trials and investigating potential mechanisms of resistance to this targeted therapy.

This study successfully demonstrated a novel approach to enhance cancer immunotherapy by creating a fusion protein (PD-1Ab21) that targets IL-21 to tumor-reactive T cells. PD-1Ab21 demonstrated superior antitumor effects compared to standard treatments in several preclinical models. The study highlights the potential of targeted cytokine delivery to improve the efficacy of immune checkpoint blockade therapies. Future research should focus on clinical translation and further mechanistic studies to optimize the approach.

The study was conducted in preclinical mouse models, and the results may not directly translate to human cancers. Further research is needed to evaluate the efficacy and safety of PD-1Ab21 in human clinical trials. The study focused on specific tumor models and may not be generalizable to all cancer types. The mechanisms underlying the superior antitumor effects of PD-1Ab21 require more in-depth investigation.