Tumor-reactive T cells appear to heavily infiltrate the tumor microenvironment of patients who failed previous CPI treatment. These patients can still respond to an infusion of unselected autologous TILs. Our results warrant further testing of novel immune re-activation strategies in melanoma patients who failed multiple CPI therapy.

Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune‐related adverse events (irAEs). Immunosuppression with high dose corticosteroids, or tumor necrosis factor (TNF) antagonists in refractory cases, is the mainstay of treatment for irAEs. It is currently unknown what impact corticosteroids and anti‐TNF have on the activity of antitumor T cells. In our study, the influence of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10–125 mg prednisolone) and infliximab (anti‐TNF) on the activation and killing ability of tumor‐infiltrating lymphocytes (TILs) was tested in vitro. Overall, dexamethasone at low or intermediate/high doses impaired the activation (−46 and −62%, respectively) and tumor‐killing ability (−48 and −53%, respectively) of tumor‐specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T cell activation (−20%) and tumor killing (−10%). A 72‐hr resting period after withdrawal of dexamethasone was sufficient to rescue the in vitro activity of TILs, while a short withdrawal did not result in a full rescue. In conclusion, clinically relevant doses of infliximab only had a minor influence on the activity of tumor‐specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. However, the activity of TILs could be restored after withdrawal of steroids. These data indirectly support steroid‐sparing strategies and early initiation of anti‐TNF therapy for the treatment of irAEs in immuno‐oncology.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.

Abbreviations: CTCAE Common Terminology Criteria for Adverse Events ICI Immune checkpoint inhibitors irAE Immune-related adverse event PBL Peripheral blood lymphocyte PBMC Peripheral blood mononuclear cell REP Rapid expansion TIL Tumor-infiltrating lymphocyte TNF Tumor necrosis factor 2 Abstract:Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune-related adverse events (irAEs). Immunosuppression with high doses of corticosteroids or, in refractory cases, with tumor necrosis factor (TNF) antagonists, are the mainstay of treatment for irAEs. It is currently unknown what is the impact of corticosteroids and anti-TNF on the activity of antitumor T cells. In this study, the influences of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10 to 125 mg prednisolone) and infliximab (anti-TNF) on the activation and killing ability of tumor-infiltrating lymphocytes (TILs) was tested in vitro. Overall, dexamethasone at low or intermediate/high dose impaired the activation (respectively -46% and -62%) and tumor-killing ability (respectively -48% and -53%) of tumor-specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T cell activation (-20%) and tumor killing (-10%). A brief resting following exposure to dexamethasone was sufficient to rescue the in vitro activity of TILs. In conclusion, clinically-relevant doses of infliximab only influenced to a lesser extent the activity of tumor-specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. These data support steroid-sparing strategies and early initiation of anti-TNF for the treatment of irAEs in immuno-oncology.

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Background: Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune-related adverse events (irAEs). Immunosuppression with high doses of corticosteroids or, in refractory cases, with tumor necrosis factor (TNF) antagonists, are the mainstay of treatment for irAEs. It is currently unknown what is the impact of corticosteroids and anti-TNF on the activity of antitumor T-cells. Methods: The influence of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10 to 125 mg prednisolone) and infliximab (anti-TNF) on the activation and killing capacity of tumor-infiltrating lymphocytes (TILs) was tested in vitro. Results: Overall, dexamethasone at low or intermediate/high dose impaired the activation (respectively -46% and -62% in n=8) and tumor-killing ability (respectively -48% and -53% in n=6) of tumor-specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T-cell activation and tumor killing (respectively -20% in n=8 and -10% in n=6). A brief resting following exposure to dexamethasone was sufficient to rescue the in vitro activity of TILs. Conclusions: Clinically relevant doses of infliximab only influenced to a lesser extent the activity of tumor-specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. These data support steroid-sparing strategies and early initiation of anti-TNF for the treatment of irAEs in immuno-oncology. Citation Format: Marco Donia, Troels H. Borch, Haja D. Radic, Christopher Chamberlain, Aishwarya Gokuldass, Inge Marie Stentoft Svane, Arianna Draghi. Differential effects of corticosteroids and anti-TNF on tumor-specific immune responses— Implications for the management of irAEs [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B154.

Background: Pembrolizumab monotherapy was approved for first-line (1L) treatment of metastatic non-small cell lung cancer (NSCLC) with PD-L1 tumor proportion score (TPS) 50% in October 2016 in the US, based on the findings from KEYNOTE-024 trial. A recent update of the trial with 25.2 months median follow-up demonstrated median pembrolizumab treatment duration of 7.9 months. The objective of our study is to estimate real-world time on treatment (rwToT) for 1L pembrolizumab monotherapy in a clinically matched real-world NSCLC database. Methods: This retrospective study utilized the Flatiron Health EHR-derived advanced NSCLC database from which patients with stage IV diagnosis, TPS 50%, at least one dose of 1L pembrolizumab monotherapy, and ECOG performance status (PS) 0-2 were included. Patients with missing or ECOG PS > 2 were excluded. rwToT was defined as the length of time between first and last administration date of pembrolizumab. Patients with a record of next line of therapy, or death, or whose last activity date was 120 days from the last administration date were considered discontinued; others were censored. The Kaplan-Meier (KM) estimates were generated for median rwToT, restricted mean (rMean) rwToT at maximum time point where at least 10% of patients have not discontinued, and landmark on-treatment rates. rMean rwToT at 24 months was also computed using the parametric function that provided best fit based on statistical testing and visual inspection criterion. All results were stratified into ECOG PS 0-1 (similar to KEYNOTE-024) and ECOG PS 2. Flatiron dataset from 31-Jul-2018 with median follow-up of 10.8 months was utilized.