Abstract LB-319: Establishment of automated multiplex immunofluorescence T-cell panel for baseline screening of syngeneic mouse models and evaluation of T-cell infiltration in A20 syngeneic tumors treated with TAK-981, a first-in-class SUMO inhibitor
Authors: Ghasemi Omid , Zawadzka Agatha , Luongo Evan , Tse Aimy , Chouitar Johara , Casson Cierra , Ganno-Sherwood Michelle , He Xingyue , LeRoy Patrick , Xega Kristina , Grossman Stephan , Song Keli , Berger Allison , Galvin Katherine , Huszar Dennis , Shinde Vaishali 
Published: August 2019

Abstract
Mouse syngeneic tumor models play a crucial role in the translationally oriented process of assessing the preclinical activity and mechanism of action of emerging novel immunotherapies and immunotherapy combinations. Recent advancements in fluorescent multiplex technology provide a potential opportunity to improve the evaluation and identification of specific subsets of mouse immune cells using a panel approach. This technology may provide the ability to complement more traditional means of immunophenotyping such as flow cytometry.
We established Opal™ multiplex immunofluorescence (IF) assays for the evaluation of T-cells (CD4, CD8, CD25, FOXP3 and tumor marker) in mouse using a Leica Bond RX autostainer. Images were acquired by Vectra® Polaris™ multispectral scanner. HALO™ image analysis was used to assess identification, quantification, and spatial localization of the immune cell subsets within tumors. The multiplex IF assays were used to carry out baseline characterization of the T-cell panels in multiple syngeneic models (A20, CT26, 4T1, EMT6, B16F10, LLC1, Renca and MC38). Notably, integrated data from the Opal™ multiplex IF assay enabled characterization and validation of “hot vs. cold” tumors across multiple syngeneic models. In addition, using PAX5 as a tumor marker, the Opal™ multiplex IF assay T-cell panel was used to specifically evaluate A20 lymphoma tumors from BALB/c mice treated with TAK-981, an investigational, first-in-class small ubiquitin-like modifier (SUMO) inhibitor.
Currently, TAK-981 is being evaluated in a first-in-human, Phase I trial (NCT03648372) in patients with relapsed/refractory lymphomas. In pre-clinical models, A20 tumors demonstrated a dose-dependent increase in cytotoxic T cell numbers in response to TAK-981, consistent with previously generated flow cytometry data in animal models and indicative of induction of an adaptive anti-tumor immune response by TAK-981.
In conclusion, across a range of syngeneic mouse models, an Opal™ multiplex IF assay can successfully enable characterization of “hot vs. cold” tumors. In tumors derived from a specific syngeneic mouse model treated with a novel, first-in-class SUMO inhibitor, the Opal™ multiplex IF assay was also capable of recapitulating the characterization of the T-cell repertoire within the tumor microenvironment previously determined by flow cytometry. Together, this suggests multiplex IF can contribute to ongoing efforts driving early translational research in immuno-oncology.
Citation Format: Omid Ghasemi, Agatha Zawadzka, Evan Luongo, Aimy Tse, Johara Chouitar, Cierra Casson, Michelle Ganno-Sherwood, Xingyue He, Patrick LeRoy, Kristina Xega, Stephan Grossman, Keli Song, Allison Berger, Katherine Galvin, Dennis Huszar, Vaishali Shinde. Establishment of automated multiplex immunofluorescence T-cell panel for baseline screening of syngeneic mouse models and evaluation of T-cell infiltration in A20 syngeneic tumors treated with TAK-981, a first-in-class SUMO inhibitor [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 LB-319.