Abstract 4999: Evaluation of anti-Gr1 antibody for depletion of MDSC in preclinical NSG mouse models of pediatric sarcoma
Authors: Kailayangiri Sareetha , Altvater Bianca , Urban Katja , Meltzer Jutta , Greune Lea , Farwick Nicole , Jamitzky Silke , Rossig Claudia 
Published: September 2020

Abstract
Preclinical in vivo studies of chimeric antigen receptor (CAR) T cells often rely on NOD-scid gamma (NSG) mouse models which lack T cells, B cells and NK cells and thereby allow for reliable engraftment of human tumor xenografts. But murine myeloid cells present in the NSG mouse strain can affect the tumor microenvironment as well as the function of adoptively transferred human immune effector cells. Long et al. (Cancer Immunol Res. 2016 Oct;4(10):869-880) reported that pediatric sarcoma xenografts in NSG mice induce in vivo expansion of murine CD11b+ myeloid-derived suppressor cells (MDSC), and that this cell population suppresses human CAR T cell proliferation in vitro. We investigated a strategy to avoid inhibitory effects of murine MDSCs on Ewing sarcoma xenografts in NSG mice by in vivo pretreatment with murine antibody against two cell surface antigens, Ly6C/Ly-6G (granulocyte-differentiation antigen-1, Gr-1), expressed on murine MDSC. Analysis of the CD11b+ myeloid cell populations in NSG mice 17-37 days after subcutaneous transplantation of the Ewing sarcoma cell line TC-71 showed a noticeable increase of CD11b+ cells in the peripheral blood compared to non-tumor bearing NSG mice (median 3.4 × 103, range 0.7-15.5 × 103 cells/ml, n=8 vs median 2.1 × 103, range 1.2-4.0 × 103 cells/ml, n=7). The increase was even more pronounced in the spleens, with a median absolute number of 2.6 × 106 CD11b+ cells (range 0.2-21 × 106 cells) per spleen in sarcoma-bearing mice versus 0.5 × 106 CD11b+ cells (range 0.2-0.8 × 106 cells) per spleen in mice without tumors. The granulocytic MDSC subset coexpressing Ly6Gpos was the most prominent subpopulation in spleen and blood. To eliminate murine MDSCs, we treated sarcoma-bearing mice twice weekly with 200 µg anti-Gr1 antibody RB6-8C5 over a period of 2 weeks, starting at tumor volumes of 100-200 mm3. While the combined percentage of the Ly6Gpos and Ly6Cpos cell populations in the spleens decreased compared to untreated mice (median 55%, range 36-75%, n=9 versus median 74%, range 72-86%, n=8), the total numbers of CD11b+ cells further increased (median 3.1 × 106, range 0.4 × 106-11 × 106 cells/spleen). Our data suggest that anti-Gr1 antibody pretreatment leads to blockade of the Ly6C/Ly6G receptors rather than eliminating MDSC subsets. To optimize preclinical pediatric sarcoma models, other methods for depletion or functional inactivation of mouse MDSCs will need to be evaluated.
Citation Format: Sareetha Kailayangiri, Bianca Altvater, Katja Urban, Jutta Meltzer, Lea Greune, Nicole Farwick, Silke Jamitzky, Claudia Rossig. Evaluation of anti-Gr1 antibody for depletion of MDSC in preclinical NSG mouse models of pediatric sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4999.