Olivier, S.

Abstract Chimeric antigen receptor T (CART) cell therapy has shown unprecedented success in the treatment of hematological malignancies. However, the immunosuppressive tumor microenvironment of both solid and liquid tumors is a major barrier for CART efficacy. Immunosuppressive myeloid cells inhibit CART efficacy. Colony-stimulating factor 1 receptor (CSF1R) promotes macrophage proliferation, survival, and anti-inflammatory response and is upregulated on myeloid cells in many cancers, making CSF1R an appealing therapeutic target. We aimed to test the interactions between CSF1R, myeloid cells, and CART response, and to study CSF1R targeting in the context of CART cell therapy. We utilized CD19-targeted CART cell therapy (CART19) in lymphoma preclinical models and thyroid-stimulating hormone receptor (TSHR)-targeting CART cell therapy (TSHR-CART) in thyroid cancer models. We first measured CSF1R expression on myeloid cells in vitro. Suppressive myeloid cells, polarized from healthy donor monocytes using an anaplastic thyroid cancer (ATC) cell line, 8505c, or mantle cell lymphoma cell line, JeKo-1, significantly upregulate CSF1R (p=0.0002) compared to media control. After polarization, myeloid cells significantly inhibit TSHR-CART and CART19 antigen-specific proliferation compared to freshly isolated monocytes (p=0.012, p<0.01, respectively) indicating that suppressive myeloid cells contribute to CART failure in multiple tumor types. We then performed single cell RNA sequencing on peripheral blood taken at baseline from patients with mantle cell lymphoma treated with CART19. We found a significant increase of CSF1R on monocyte subsets in CART19 non-responders vs responders (padj=0.0037). Next, we determined the impact of CSF1R blockade on CART cell therapy in vivo. Using lymphoma models, B6 mice were engrafted with the luciferase+ CD19+ murine lymphoma cell line TBL-12 (1x106 cells i.v. 1 day after 250 mg/kg cyclophosphamide). Tumor burden was significantly reduced after 7 days of treatment with low dose CART19 (0.5x106 i.v.) + anti-mouse CSF1R antibody (16mg/kg i.p. twice weekly) vs CART19 + IgG control (p=0.0146). We then tested the impact of CSF1R blockade in mice experiencing tumor relapse. After TBL-12 engraftment, mice were treated with anti-mouse CSF1R or IgG control antibody followed two days later with high dose CART19 (1x106 i.v.) to induce remission. Mice were re-challenged with TBL-12 14 days later. Mice treated with anti-mouse CSF1R antibody showed increased tumor control and significantly higher survival (p=0.0416). Using thyroid cancer models, first we identified high macrophage infiltration in biopsy specimens of patients with ATC compared to normal thyroid tissue and benign thyroid tumors through immunohistochemistry (IHC) of patient samples (p<0.01, p<0.0001, respectively). To test if macrophages are associated with CART failure in ATC, NSG mice were subcutaneously engrafted with 1x106 TSHR+ THJ529 tumors, an ATC cell line derived from patient biopsy samples from the Mayo Clinic thyroid cancer biobank, followed by i.v. treatment with 1x107 TSHR-CART or untransduced T cell control (UTD). IHC staining of tumor tissue collected at experimental endpoints revealed a dose-dependent, significant infiltration of human CD3+ T cells into the tumors which inversely correlated with murine F4/80+ myeloid cell and murine YM-1+ M2 macrophage infiltration. This led us to hypothesize that myeloid cell targeting with CSF1R blockade can improve TSHR-CART efficacy. We utilized the same ATC model described above to test CSF1R targeting. When TSHR+ THJ529 tumors reached approximately 100 mm3, mice were randomized by tumor volume and treated with CSF1R inhibitor PLX5622 (600mg/kg) or vehicle control, followed by either UTD or TSHR-CART cells 5x106 i.v. Mice treated with TSHR-CART + PLX5622 showed significantly improved tumor control vs TSHR-CART + vehicle control (p<0.0001) or UTD + PLX5622 (p<0.0001). Together, our results highlight immunosuppressive myeloid cells which express CSF1R as a major cell type limiting CART efficacy in both solid and liquid tumors. We also identified a strategy to overcome myeloid-mediated suppression of CART cells by targeting CSF1R with antibody or small molecule inhibitors.

Abstract Description This study dissects mechanisms of spontaneous tumor rejection leading to better understanding of successful immune responses to cancer. Cancers persist despite expression of tumor-specific neoantigens promoting ongoing anti-tumor immune responses. While some occult tumor cells likely are cleared by effective anti-tumor immune responses, the targeted antigens are not easily identifiable as those cells spontaneously disappear. Congenic mouse breast carcinoma models expressing a defined protein mimicking neoantigens were used to dissect the protective spontaneous immune response. BALB/c (MHC H-2d) mice challenged with BALB/c breast tumors expressing the rat-erbB2 oncoprotein succumb to their tumors despite demonstrated tumor immunogenicity, mimicking known patient responses to cancer. Meanwhile, H-2b positive BALB.B and H-2d/H-2b F1 hybrid mice spontaneously eliminate genetically matched tumors in an MHC-II dependent manner. CD4+ T-cells and B-cells or the rat-erbB2-specific antibodies they produce are crucial mediators of this protective response. Differences in the tumor microenvironments in rat-erbB2+ tumor-bearing BALB.B and BALB/c mice at the time of fate decision in the models reflect the differences between effective and ineffective anti-tumor immunity. Identifying the nature of the optimal MHC-II antigens, how they activate protective CD4+ T-cells, and the interactions that promote protective antibodies will better inform future cancer therapies. Funding Sources Bernie Fineman Research Fund Mayo Clinic Center for Biomedical Discovery Mayo Clinic Department of Immunology Mayo Clinic Graduate School of Biomedical Sciences NIH Traning Grant 5 R25 GM 55252 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Background Cancers persist despite expression of immunogenic neoantigens and ongoing antitumor immune responses. While some occult tumors likely are cleared by effective antitumor immune responses, the targeted antigens are not easily identifiable as those tumors spontaneously disappear. Methods We used mouse models with a defined antigenic protein mimicking tumor-specific neoantigens to address the nature of these spontaneous anti-tumor immune responses. Results BALB/c (H-2d ) mice challenged with BALB/c breast tumors expressing the rat-erbB2 oncoprotein succumb to their tumors despite ongoing immune responses targeting tumor-specific model antigens. Meanwhile, congenic BALB.B (H-2b ) and H-2d/H-2b F1 hybrid mice spontaneously eliminate genetically matched tumors in a major histocompatibility complex (MHC)-II dependent manner. Adoptive transfer and immune cell depletion strategies revealed CD4+ T cells and CD20+ B cells are crucial mediators of the protective response in H-2b mice. Furthermore, passive transfer of immune serum from mice rejecting their tumors confers resistance in tumor antigen-tolerant animals with an inversely proportional relationship between tumor outgrowth and the amount of rat-erbB2 specific antibody present in tumor-bearing mice. Introduction of the rat-erb2 ectodomain into other H-2b tumor models also promotes their spontaneous tumor rejection. Notably, the tumor microenvironments differ in rat-erbB2+ tumor-bearing BALB.B and BALB/c mice at the time of fate decision in the models reflecting the differences between effective and ineffective tumor immune responses. Conclusions We find that the effective antitumor immunity targeting neoantigens in these breast cancer models is determined by MHC-II-restricted presentation of optimal cancer-associated antigens. These responses are dependent on CD4+ T cells, B cells, and antigen-specific antibodies.