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The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.

The impact of radiation on TREG cells remains controversial. Radiation has been shown by our lab, and others, to alter the phenotype of human tumor cell by enhancing their expression of the co-stimulatory molecules OX-40L and 4-1BBL. Both OX-40 and 4-1BB signaling have been reported to influence TREG frequency and function, and we hypothesized that radiation-induced changes in expression of these molecules could influence the frequency of TREG cells within tumors. We evaluated three widely used murine tumor models for these experiments. 4T1 (mammary), MC38 (colon), or CT26 (colon) cells were implanted s.c. into mice and treated locally with 8-10Gy of radiation. Several days after treatment the frequency of tumoral T cells, including CD4+Foxp3+ TREG cells, was evaluated by fluorescent IHC and flow cytometry. We found that radiation differentially impacted the frequency of TREGS among the three tumor models evaluated and appeared to correlate with expression levels of 4-1BBL and OX-40L on the tumor cells post-IR. The level of tumor control post-RT also mirrored expression levels of 4-1BBL and OX-40L on tumor cells. Interestingly, 4T1 tumors contained more total CD4+ cells within tumors, but contained less TREG cells within the CD4 population, after radiation as compared to MC38 tumors. 4T1 tumor-bearing mice also contained more CD4+41BB+ cells as compared to MC38 tumor-bearing mice. These differences may contribute to differences in the therapy responses of various murine models utilizing radiation and may serve as useful biomarker to predict antitumor responses post-IR. Citation Format: Samantha Simon, Charlie Garnett-Benson. Direct and indirect effects of low-dose radiation on Tregs and antitumor efficacy among diverse tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A06.

Meeting abstracts The use of sub-lethal radiation has been shown to alter cell phenotype and gene expression. T regulatory (Treg) cells are phenotypically defined as being CD4+CD25+ Foxp3+ T cells and are known to suppress the function of CD8+ cytotoxic T lymphocytes (CTLs). Inhibiting the