Abstract 1546: Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy
Authors: Kumar Rajesh , Sullivan Peter , Li Wei , Zhang Haifeng , Hoglund Virginia , Wang Lingyang , Zhang Yue , Sorensen Poul H. , Dimitrov Dimiter S. , Orentas Rimas J. 
Published: July 2021

Abstract
IL1RAP (Interleukin 1 receptor accessory protein) is differentially expressed in both Ewing sarcoma (EWS) and select hematologic malignancies (CML, AML). Using single domain antibody-like binders generated against IL1RAP to create chimeric antigen receptors (CARs) we demonstrated highly specific cytokine production and cytolytic activity against multiple EWS cell lines in vitro. Preliminary in vivo functionality of IL1RAP CAR-T indicates that additional approaches will be required to increase the efficacy of IL1RAP CAR-T for orthotopic EWS tumor models. To optimize CAR-T cell therapy for EWS, we used model cell lines to define tumor- and stromal-derived factors that must be overcome to obtain effective treatment. The human cell line TC71 (fusion gene positive) was administered both i.v. and i.m. to NSG mice, and subsequently treated with IL1RAP-specifc CAR-T cells. Tumor engraftment was not seen in the i.v. model, however the orthotopic i.m. model in NSG mice was successful, generating tumor and metastatic lesions surrounded by a collagen rich stromal element when CAR-T or control activated T cells were administered. This was not seen in control untreated tumors. Histochemical analysis of i.m. tumors demonstrated that T cells (CD3 positive) were confined to the tumor stromal boundary in proximity to cells of myeloid lineage (CD11b positive). We therefore analyzed cell surface and soluble factors produced by TC71 by flow cytometry, ELISA, and NanoString mRNA panels. TC71 was cultured alone or with interferon-gamma (IFNG), to model the presence of activated T cells and was found to constitutively express transcripts for both canonical and non-canonical chemokines: GPI, MIF, IL-32, TGFβ1; whereas CSF1, CXCL9, CXCL10 and CXCL11 were only induced by IFNG. MIF and TGF-β1 production were confirmed by ELISA. Flow cytometry revealed that numerous CAR-T targets (IL1RAP, CD276, EGFR and FGFR4) were expressed, as were the immune inhibitory molecules: PDL1, PDL2, Galectin-9, and CTLA-4. MHC class I and II and PDL1 were strongly induced by IFNG. We also observed transcripts for collagen related genes in TC71: COL16A1, 3A1, 5A3, LOXL1, P4HB and SERPINH1 both in vitro and in vivo. Our results indicate that tumor surface expression of immune inhibitory signals, PDL-1, PDL-2, CTLA4, as well as the soluble factors MIF, and TGF-b1, must be considered in armoring CAR-T for effective immune destruction of EWS, along with strategies designed to increase the transmigration of T cells through matrix proteins. Moreover, the chemokines produced by tumor alone, as well as those induced by IFNG, indicate a complex network for recruitment of local and bone marrow-derived stromal cells that create a physical as well as cellular barrier to adoptive immunotherapy which needs to be addressed for effective CAR-T therapy of this solid tumor.
Citation Format: Rajesh Kumar, Peter Sullivan, Wei Li, Haifeng Zhang, Virginia Hoglund, Lingyang Wang, Yue Zhang, Poul H. Sorensen, Dimiter S. Dimitrov, Rimas J. Orentas. Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy [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 1546.