Hirdler, Jacob B.

Abstract Seven different anti-PD-1 and PD-L1 monoclonal antibodies are now widely used in the US to treat a variety of cancer types, but there have been no clinical trials comparing them directly. Furthermore, because many of these antibodies do not cross react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by FDA-approved antibodies. To do this, we performed an in vivo T cell priming assay in which anti-PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor antigen (OVA), followed by subsequent B16-OVA tumor challenge. We found that anti-PD-1/L1-treated mice exhibited significantly better tumor rejection than controls, although both the control and antibody-treated mice generated comparable numbers of OVA-specific T cells at the time of priming. To determine what could mediate this strong antitumor immune response, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the anti-PD-1/L1-treated mice, the same subset of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of FDA-approved anti-PD-1/L1 monoclonal antibodies and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for future studies to optimize and better utilize anti-PD-1/L1 therapeutics.

AbstractImmune checkpoint inhibitors (ICIs), as a novel class of anticancer therapy, can be more efficacious and less toxic than chemotherapy, but their clinical success is confined to certain tumor types. Elucidating their targets, mechanisms and scope of action, and potential synergism with chemotherapy and/or targeted therapies are critical to widen their clinical indications. Treatment response to an ICI targeting programmed death‐1 (anti‐PD‐1) is sought to be understood here by conducting a preplanned correlative analysis of a phase II clinical trial in patients with small bowel adenocarcinoma (SBA). The cytolytic capacity of circulating immune cells in cancer patients using a novel ex vivo cytotoxicity assay is evaluated, and the utility of circulating biomarkers is investigated to predict and monitor the treatment effect of anti‐PD‐1. Baseline expression of Bim and NKG7 and upregulation of CX3CR1 in circulating T cells are associated with the clinical benefit of anti‐PD‐1 in patients with SBA. Overall, these findings suggest that the frequency and cytolytic capacity of circulating, effector immune cells may differentiate clinical response to ICIs, providing a strong rationale to support immune monitoring using patient peripheral blood.

Abstract Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

Abstract Cytotoxic T lymphocytes (CTL's) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. However, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, we sought to identify key factors associated with failure of PD-1 blockade in order to develop strategies that could significantly improve response rates. We performed single-cell RNA-seq analysis of peripheral CD8+ T cells from responding and non-responding patients and found that the non-responders had less NKG7 expression. Functional assays revealed that knock-down of NKG7 compromised cytolytic granule localization and trafficking within CTL's. Since decreasing NKG7 reduced the cytolytic efficiency of CD8+ T cells, we next hypothesized that by increasing expression of NKG7 we could promote better T cell killing of tumor and enhance the effects of immune checkpoint inhibitors. To test this, we utilized peripheral blood samples from melanoma patients who failed to respond to anti-PD-1 treatment and transfected the PBMC's with NKG7 mRNA. The addition of NKG7 mRNA significantly increased T cell killing of melanoma tumor cells in an in vitro cytotoxicity assay, and this effect was even more robust in combination with anti-PD-1 or anti-PD-L1 pre-treatment. To confirm that over-expression of NKG7 could improve CD8+ T cell killing in an antigen-specific manner, we transfected OT-1 transgenic T cells with murine sequence of NKG7 mRNA or control mRNA. OT-1 CD8+ T cells with increased NKG7 expression exhibited enhanced cytolytic ability when challenged with either EL4 cells pulsed with OVA peptide or B16-OVA tumor cells in vitro. In addition, NKG7 mRNA-transfected OT-1 CD8+ T cells suppressed the aggressive growth of B16-OVA cells in vivo significantly better than control OT-1 CD8+ T cells. These results indicate that deficits in a T cell-intrinsic cytolytic factor can lead to failure of PD-1/L1 blockade and establish NKG7 as a new therapeutic target for enhancing response to cancer immunotherapy. Citation Format: Whitney J. Barham, Ti Wen, Ying Li, Henan Zhang, Cindy Liu, Jacob Hirdler, Joanina Gicobi, Fabrice Lucien-Matteoni, Yiyi Yan, Haidong Dong. NKG7 mRNA therapy increases the anti-tumor cytotoxicity of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 504.