Tumor Associated Macrophages (TAM) Antagonize Chimeric Antigen Receptor (CAR) T Cell Therapy in Peritoneal Solid Tumors and Are Reprogrammed to Fight the Tumor By Therapeutic Apoptotic Cells
Authors: Karbian Netanel , Amore Raja , Mevorach Dror 
Published: October 2019

Abstract
Background. CAR genetically engineered T cells carry MHC-independent specific antigen receptors and co-stimulatory molecules that can activate an immune response to a cancer-specific antigen. Excellent results from treatment in hematological malignancies were not replicated in solid tumors, possibly due to lack of antigens, poor trafficking, and a hostile tumor microenvironment. Here we describe the crucial effect of TAM and microbiome, on CAR T cell therapy on a human peritoneal solid tumor model in SCID-Bg mice.
Methods. To follow tumor growth in vivo, HeLa-CD19 were stably transduced with pLenti-PGK-V5-Luc-Neo. For CAR preparation, fresh mononuclear cells (MNC) were isolated from peripheral blood of healthy donors, introduced with CD28+CD3+ beads and transfected with 3rd generation CD19-CAR plasmids. For apoptotic cell preparation, enriched MNC fractions were collected by leukapheresis from healthy eligible human donors, and prepared by Enlivex (Mevorach et al. BBMT 2014), and gamma irradiated (Allocetra-OTS). For Intra-peritoneal solid tumor model: SCID-Bg mice were injected intra-peritoneally (IP) with human HeLa-CD19 or HeLa-CD19-luciferase cells, with 10×106 human apoptotic cells or vehicle, and 10×106 CD19-CAR T cells or mock T cells. Mice were monitored daily for clinical signs and peritoneal fluid accumulations and weekly for tumor growth (in vivo imaging). Cell and macrophage subpopulations were characterized and survival analysis (Kaplan-Meier log rank test) was done in remaining mice. Survival endpoint was scored by severe peritoneal fluid accumulation and reduced mobility, which correlated to a large peritoneal Hela cell accumulation. Peritoneal cells were evaluated by flow-cytometry. Macrophage subpopulations were characterized into large peritoneal macrophages (LPM) and small peritoneal macrophages (SPM). Tumors were examined for bacterial presence by Immunohistochemistry staining with anti-lipoteichoic acid (LTA) and anti-lipopolysaccharide (LPS). Single cell analysis was performed for macrophage subpopulations.
RESULTS. Mice survived 30±5 days (range 27-37), dying or sacrificed from a solid tumor in the peritoneal cavity with accumulation of bloody peritoneal fluid and clinical deterioration (Figure). The results were verified using IVIS of intraperitoneal HeLaCD19-Luc cells. Mock treatment ameliorated non-significantly their survival to 34±4 days (range 30-38, NS), probably due to some T cell activity. CAR T cell therapy significantly ameliorated their survival to 55±11 days (range 34-76, p<0.05 vs MOCK). However, when mice received co-administration of apoptotic cells and CAR T cells, survival increased to 70±20 days (range 48-90, P<0.05 vs CAR T cells alone). Furthermore, 20% of mice treated with apoptotic cells were disease free for 150 days (end of experiment). On days 17 and 26 of the experiment, peritoneum was washed, and cells were centrifuged and stained to characterize cell subpopulations. During intraperitoneal tumor progression, marked disappearance of LPM and appearance of recruited and reprogrammed macrophages was seen. With CAR T cell treatment, with/without apoptotic cells, there was LPM restoration. Tumors were found sterile by staining with anti LTA and anti LPS and no role for intra-tumor bacteria was found in this model.
Conclusion: During intraperitoneal tumor progression and with no relation to presence of bacteria, TAM are reprogrammed to support tumor growth. CAR T cell therapy partially antagonize the TAM effect. Following interaction with apoptotic cells, TAM are further reprogrammed for anti-tumor and pro-CAR T cell cytotoxicity.
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Disclosures
Mevorach: Enlivex Ltd: Consultancy, Patents & Royalties.