Abstract 1671: Evaluation of total PD-1 expression using multi-color flow cytometry in metastatic non-small Cell lung cancer patients treated with multi-neoantigen vector (ADXS-503) alone and in combination of pembrolizumab to assess T-cell & T-cell memory subsets
Authors: Mohanram Venkat , Belkina Natalya , Bisconte Angelina R. , Goldman Jonathan W. , Gerstner Gregory J. , Haigentz Missak , Stinchcombe Thomas , Halmos Balazs , Vangala Surya , Kabala Victor , Simkhada Dinesh , Metran Cristiane , Davis Darren , Parsi Megan , Gutierrez Andres A. , Phippard Deborah , Ramalingam Suresh S. 
Published: July 2021

Abstract
Precision for Medicine (Precision) developed and qualified two 17 color flow immunophenotyping assays to be used as pharmacodynamic biomarkers for Advaxis clinical studies in patients with Metastatic Non-Small Cell Lung Cancer treated with ADXS-503 alone and in combination with Pembrolizumab (Pembro). ADXS-503 (A503) is an off-the-shelf, attenuated Listeria monocytogenes (Lm)-based immunotherapy bioengineered to elicit potent T-cell responses against 22 tumor antigens commonly found in NSCLC. Pembrolizumab (Pembro) is a programmed death receptor-1 (PD-1)-blocking antibody approved for the treatment of advanced lung cancer. A503 and Pembro have complementary mechanisms of immune activation and reversal of immune tolerance. Here, we qualified two multi-color flow cytometry assays to quantify total PD-1 expression in cryopreserved peripheral blood mononuclear cells (PBMCs) from individuals that were treated either with A503 only or with A503 in combination with Pembro. The detection of free PD-1 and Pembro-bound PD-1 was achieved by co-staining a partially competing αPD-1 antibody (clone PD1.3.1.3) with a biotinylated αHu-IgG4 antibody. The robustness of the assay was demonstrated using a nine-point half-log serial dilution of Pembro, where the highest concentration was 10µg/mL and the lowest concentration was 0.001µg/mL, including a no drug control. The assay conditions were optimized for sensitivity, optimal signal:noise ratio, detection of free and drug bound receptor by titrating and testing various commercial αPD-1 antibody clones and tertiary reagents to detect biotinylated αHu-IgG4. The Pembro bound receptor was detected using a biotinylated αHu-IgG4 antibody, while the free receptors were quantified using a commercial αPD-1 antibody. The assay was able to quantify free and drug bound PD-1 in the intended immune cell types without compromising the staining of other cell surface and intra-nuclear markers. Majority of the evaluable patients, 6 out of 8, had increased counts of NK, CD4+ and CD8+ T-cells, including TCM, TEM and memory stem cells after the administration of ADXS-503 ± Pembro. PD1 expression on circulating CD4+, CD8+ and NK T-cells was also increased while PD-L1 expression was elevated in on-therapy tumor biopsies in some of these patients. Measuring total PD-1 in T-cells can be more challenging in patients on Pembrolizumab therapy as no known commercial non-competing αPD-1 antibody clones are available. This novel assay will facilitate the evaluation of total PD-1 expression as a pharmacodynamic biomarker in T-cells when PD-1 blockade is being used. These results also support that combination of ADXS-503 with PD-1 blockade could lead to enhancement of efficacy of anti-tumor immunotherapy.
Citation Format: Venkat Mohanram, Natalya Belkina, Angelina R. Bisconte, Jonathan W. Goldman, Gregory J. Gerstner, Missak Haigentz, Thomas Stinchcombe, Balazs Halmos, Surya Vangala, Victor Kabala, Dinesh Simkhada, Cristiane Metran, Darren Davis, Megan Parsi, Andres A. Gutierrez, Deborah Phippard, Suresh S. Ramalingam. Evaluation of total PD-1 expression using multi-color flow cytometry in metastatic non-small Cell lung cancer patients treated with multi-neoantigen vector (ADXS-503) alone and in combination of pembrolizumab to assess T-cell & T-cell memory subsets [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 1671.