553 Depletion of effector regulatory T cells drives major response to induction dual immune checkpoint blockade (ICB) in patients with oropharyngeal carcinoma (OPC)

<h3>Background</h3> Pathological response to neoadjuvant ICB is established as a biomarker of long-term disease control in several solid tumors.¹ To determine tumor microenvironment (TME) features associated with pathological response, we performed single-cell profiling on matched baseline and on-treatment tumor biopsies from patients with newly diagnosed HPV-positive OPC enrolled in a phase 2 trial of a 6-week cycle of induction ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) prior to radiotherapy.^{2 3} <h3>Methods</h3> Five 2-millimeter cores from tumor biopsies from 35 patients were formalin-fixed and paraffin-embedded (FFPE) or immediately processed for scRNA-seq and scTCR-seq on the 10X Genomics and Illumina platforms. A pathologist masked to clinical response evaluated FFPE for percent tumor viability, and a major histological response (MHR) was defined as &lt;10% residual tumor viability on treatment. After stringent clustering and annotation of single-cell data, 76,319 CD8+ and 78,622 CD4+ T cells with paired TCR-seq data were identified for comprehensive transcriptomic, TCR repertoire and clonotype analysis and associations with MHR. <h3>Results</h3> Co-primary endpoints achieved included 6-month complete metabolic response rate (94%) and 2-year progression-free survival (84%). Induction yielded a 46% MHR rate. Single-cell profiling revealed MHRs had higher baseline intratumoral NK cells expressing Fc Gamma Receptor IIIa (Fcg+) (figure 1). Decreases in effector regulatory T (eTreg) cells, which highly expressed CTLA4, occurred only in MHRs, suggesting ipilimumab-dependent depletion⁴ by Fcg+ NK cells (figure 1). eTreg depletion correlated with increased Interferon Gamma (IFNG)+ effector⁵ CD8+ T cells (figure 2). CD8+ T-cell clonotypes transitioned from tissue resident memory (TRM), a phenotype more enriched in MHRs at baseline, to effector memory (TEM) and IFNG+ effector cells in MHRs, whereas clonotypes transitioned to exhausted TRM and proliferating cells in nonMHRs (figure 3). Basal TME features including Fcg+ NK proportion, CD8+ cytotoxicity score and immune checkpoint score are associated with percent reduction in tumor viability (R² =0.89) in a predictive lasso regression model (figure 4). <h3>Conclusions</h3> We identified TME features associated with tumor cell death in HPV-positive OPC. Of them, eTreg depletion is critical for major response to induction dual PD-1 and CTLA-4 ICB and potentially induced by Fcg+ NK mediated ADCC. Additionally, a pre-existing antitumor immune response mediated by CD8+ T cells was associated with MHR to the dual ICB. Treatment led to Treg depletion and expansion of CD8+ T cell clonotypes with TRM and TEM phenotypes, revealing a remarkable degree of plasticity of distinct T cell clonotypes. <h3>Acknowledgements</h3> This study was funded by CPRIT. MLG is a CPRIT Scholar in Cancer Research. <h3>Trial Registration</h3> NCT03799445. <h3>References</h3> Stein JE, <i>et al</i>. Major pathologic response on biopsy (MPRbx) in patients with advanced melanoma treated with anti-PD-1: evidence for an early, on-therapy biomarker of response. <i>Ann Oncol</i> 2019;<b>30</b>:589–596. Jiang X, <i>et al</i>. Depletion of effector regulatory T cells drives major response to induction dual immune checkpoint blockade.<i> medRxiv</i> 2024.2001.2004.23300616 (2024). Vos JL, <i>et al</i>. Neoadjuvant immunotherapy with nivolumab and ipilimumab induces major pathological responses in patients with head and neck squamous cell carcinoma.<i> Nat Commun</i> 2021;<b>12</b>:7348. Romano E, <i>et al</i>. Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients.<i> Proc Natl Acad Sci U S A</i> 2015;<b>112</b>:6140–6145. Ivashkiv LB. IFNgamma: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. <i>Nat Rev Immunol</i> 2018;<b>18</b>:545–558. <h3>Ethics Approval</h3> The study was approved by MD Anderson institution’s Ethics Board, approval numbers 2018-0381 and PA19-0470.