Abstract 4043: Application of high complexity flow cytometry for Monitoring activated T-cells, T and B regulatory subsets in combination immunotherapy trials, melanoma case study

Abstract

Abstract Background Ever increasing knowledge of the interplay between immune composition and a novel drugs’ treatment efficacy or the lack thereof leads to continual advancements in immunotherapies. Given the exponential increase in combination therapies, tools to determine the most promising regimens are critical. Immunotherapeutic agents either reverse T-cell inhibitory pathways (e.g., PD-1) or activate the immune system to induce cytotoxic T-cell activation and proliferation through agonistic stimulation (e.g., Cytokines). These treatment strategies have proven to be efficacious by enhancing the balance between immune-effector and suppressive/regulatory cells that can directly affect the outcome of immunotherapy. To concurrently monitor multiple immune cell subsets responsible for either immuno-suppression or activation, we developed a high-complexity (13-color) flow cytometry panel to reliably enumerate activated T-cells, Tregs (A & B subsets) and Bregs for clinical trial application. Methods The panel includes CD3, CD4, CD8, CD19, FoxP3, CD25, CD127, CD45RA, HLA-DR, CD38, CD24, and CD27 to monitor total Tregs, Treg-subsets, Bregs and T-cell activation. After robust analytical method validation following industry guidelines for exploratory biomarker assessments, we confirmed the clinical utility of this panel in stage III or IV melanoma patients (N= 14) enrolled in a randomized phase II clinical trial receiving high-dose of IL-2 with or without Afliberecept (VEGF inhibitor). Results We observed up-to 20-fold increase in activated CD8+ T-cells following treatment. Interestingly, while no discernible change was observed in the naive Treg-A cells (CD45RAhiFoxp3lo), a remarkable 5-fold expansion of effector Treg-B cells (CD45RAloFoxp3hi) was observed following treatment. Additionally, frequencies of Bregs (CD24hiCD38hi) remained unchanged after treatment. Conclusions In summary, we demonstrated the utility of a powerful high complexity clinical flow cytometry tool for precisely monitoring immune cell subsets involved in tumor escape such as Tregs and Bregs or those involved in tumor killing like CD8+ cytotoxic T-cells. More notably, this tool may be utilized to deprioritize agents that cause immunosuppression and focus on combination agents that lead to additive immune activation. Citation Format: Jennifer Tsau, Michael Abadier, Brittney Atzmiller, Christine Vaupel, Naveen Dakappagari, Ghanashyam Sarikonda, Ahmad Tarhini. Application of high complexity flow cytometry for Monitoring activated T-cells, T and B regulatory subsets in combination immunotherapy trials, melanoma case study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4043.