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Objective-The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates antiinflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. Approach and Results-Apoe −/− (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1r e/e) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe −/− Mc1r e/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe −/− controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe −/− Mc1r e/e mice showed a defect in bile acid metabolism that aggravated high-fat dietinduced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6C high monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6C high monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. Conclusions-The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation. Visual Overview-An online visual overview is available for this article.

BackgroundWe have previously demonstrated adaptive antibody responses targeting public tumor antigens in cancer patients. ATRC-101, a clinical stage, engineered version of an antibody identified in such a patient, displays robust single-agent activity in syngeneic tumor models requiring Fc receptors (FcRs) expressed by innate immune cells and the presence of CD8+ T cells. The novel target of ATRC-101 was found to be a tumor-restricted ribonucleoprotein (RNP) complex, and because RNP complexes drive T cell responses in infectious and autoimmune disease via innate immune cells, we further characterized the mechanism-of-action of ATRC-101. Here we describe changes in immune cell populations in a tumor model proximal to treatment initiation with ATRC-101.MethodsMice bearing EMT6 tumors received ATRC-101 beginning on day 7 post-tumor inoculation. Tissues were harvested between days 7 and 14 and analyzed by flow cytometry and immunohistochemistry. Transcriptome analysis was performed using RNA sequencing on whole tumors taken on days 7, 9, and 12.ResultsThe earliest significant changes induced by ATRC-101, relative to vehicle, were noted just 24 hours after dosing: increased numbers of cDC1 cells in blood, and decreased numbers of cDC2 cells in blood and M-MDSCs in tumor. A significant increase of CD8+ T cells was observed in blood 48 hours after dosing and in tumor 96 hours after dosing. Increased numbers of NK cells were also observed in blood and tumor at this later time. Multiplex analysis of circulating cytokines demonstrated a very early increase in myeloid chemo-attractants, such as MCP1 and MIP1a.Whole exome sequencing of tumor samples showed that ATRC-101 dosing drives a significant increase, relative to vehicle, in the expression of interferon-stimulated genes. Co-culturing experiments demonstrated that induced, bone marrow-derived dendritic cells are activated by ATRC-101 and its target in a dose-dependent fashion.ConclusionsDosing with ATRC-101 in the EMT6 syngeneic tumor model, in which ATRC-101 displays notable single-agent activity, leads to changes in immune cell composition in the blood and tumor, with the earliest changes observed in myeloid or myeloid-derived cell populations, and to the early appearance of myeloid chemo-attractants. We believe these data indicate that ATRC-101 acts proximally on the myeloid cell populations in the tumor, leading to a remodeling of the tumor environment and an adaptive immune response that includes CD8+ T cells driving tumor regression. Our data demonstrate that ATRC-101, bound to its target which is an RNP complex, can activate myeloid cells and are consistent with this activation occurring via FcR and Toll-like receptor (TLR) pathways.

BackgroundWe have previously described ATRC-101, a fully human, engineered IgG1 antibody binding a tumor-restricted ribonucleoprotein (RNP) complex as its target. ATRC-101 is currently under evaluation in the clinic as a monotherapy for solid tumors. Following target engagement, ATRC-101 functions in an Fc-mediated fashion to deliver the target to the innate immune system, which modifies the tumor microenvironment and generates an adaptive immune response involving CD8+ T cells leading to anti-tumor activity in syngeneic mouse models. Binding of ATRC-101 appears restricted to malignant tissues in both mouse models and human, across a range of cancer histologic phenotypes, including carcinomas that are known candidates for anti-PD-1 treatment. In the EMT6 mouse model, representing a T cell-excluded phenotype in which anti-PD-1 agents display limited activity, ATRC-101 monotherapy was uniformly vigorous with persistent anti-tumor memory. When co-administered at a lower dose with anti-PD-1, the combination of therapy demonstrated a robust and heightened anti-tumor response relative to either agent dosed as monotherapy at similar concentrations.MethodsTo gain insight into the mechanisms that contribute to the anti-tumor effect with combination therapy, in vivo experiments in the EMT6 syngeneic mouse model were performed to determine temporal and spatial patterns of infiltrates and assessed tumors by using whole exome sequencing following administration of ATRC-101 vs. vehicle control. Within naive human tumor samples, coincident immunoreactivities of ATRC-101 and PD-L1 were also characterized.ResultsIn mice treated with ATRC-101, analysis by immunofluorescence revealed a significant increase in the percentage of PD-1 reactive T cells within the tumor microenvironment. Elevated transcripts for PD-L1 also were detected in tumors from mice administered ATRC-101 vs baseline levels or vehicle control. When human tumor tissues were characterized for coincident expression of these targets, a high prevalence of ATRC-101 immunoreactivity was noted in both PD-L1 reactive and non-reactive tumor cores. Across multiple indications, ATRC-101 immunoreactivity was apparent in > 50% of PD-L1+ cores.ConclusionsIn situ studies suggest the target of ATRC-101 may co-locate with PD-L1, and in vivo studies indicate that ATRC-101 administration increases PD-L1 transcripts and PD-1-positive infiltrates in mouse tumor. Altogether, our data support studies to combine ATRC-101 with agents targeting PD-1 in the clinical treatment of solid tissue malignancies.AcknowledgementsWe acknowledge the significant effort and contributions of our colleagues from the clinical, in vivo pharmacology, translational sciences, in vitro pharmacology, and cell biology groups. This includes Mark Armanini, Erin Brosey, Chantia Carroll, Sean M. Carroll, Nicole Haaser, Benjamin Haugen, Dongkyoon Kim, Beatriz Millare, Yann Chong Tan, Danhui Zhang, and Patricia Zuno.Trial RegistrationNCT04244552Ethics ApprovalThe study was approved by WIRB (Western Institutional Review Board) on Jun 11, 2013. The WIRB study number is 20130121.ReferenceDeFalco J, Harbell M, Manning-Bog A, et al. Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens. Clinical Immunology 2018; 187:37–45.