Scavenger receptors are a superfamily of membrane-bound receptors that recognize both self and non-self targets. Scavenger receptor class A (SR-A) has five known members (SCARA1 to 5 or SR-A1 to A5), which are type II transmembrane proteins that form homotrimers on cell surface. SR-A members recognize various ligands and are involved in multiple biological pathways. Among them, SCARA5 can function as a ferritin receptor; however, the interaction between SCARA5 and ferritin has not been fully characterized. Here, we determine the crystal structures of the C-terminal scavenger receptor cysteine-rich (SRCR) domain of both human and mouse SCARA5 at 1.7 and 2.5Å resolution, respectively, revealing three Ca2+ binding sites on the surface. Using biochemical assays, we show that the SRCR domain of SCARA5 recognizes ferritin in a Ca2+-dependent manner, and both L- and H-ferritin can be recognized by SCARA5 through the SRCR domain. Furthermore, the potential binding region of SCARA5 on the surface of ferritin is explored by mutagenesis studies. We also examine the interactions of ferritin with other SR-A members and find that SCARA1 (SR-A1, CD204) and MARCO (SR-A2, SCARA2), which are highly expressed on macrophages, also interact with ferritin. By contrast, SCARA3 and SCARA4, the two SR-A members without the SRCR domain, have no detectable binding with ferritin. Overall, these results provide a mechanistic view regarding the interactions between the SR-A members and ferritin that may help to understand the regulation of ferritin homeostasis by scavenger receptors.

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Background: Dendritic cells (DCs) are vital for initiating antigen-specific T cell-dependent antitumor immune responses. However, emerging evidence reveals that local DCs within the tumor microenvironment (TME) are tolerized to facilitate immune evasion. Reprogramming of DCs is recognized as a promising strategy for tumor immunotherapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor belonging to the LILR family which is mainly expressed on normal myeloid cells and myeloid-derived malignant cells. It is upregulated on tolerogenic DCs (tolDCs), which exhibit low expression levels of costimulatory molecules and resistance to DC maturation. Targeting LILRB4 to reprogram tolDCs has been reported to be a promising strategy for cancer treatment. Here we report the preclinical development of an LILRB4 antagonist antibody, ATG-034. Method: The protein-based and cell-based binding affinity of ATG-034 were measured using SPR, ELISA and FACS analysis. Fc receptor (FcR) stimulation assay was used to evaluate the ability of ATG-034 to block the interaction of LILRB4 with its ligand fibronectin. ATG-034-mediated enhancement of antigen presentation and co-stimulation ability of DCs was determined by FACS analysis for the change of surface expression of HLA-DR, HLA-ABC, CD86, and CD206. The mixed lymphocyte reaction (MLR) assay was used to assess the immunomodulatory potential of ATG-034. The in vivo antitumor efficacy of ATG-034 was evaluated in a radiation therapy-resistant murine Lewis lung carcinoma (LLC) syngeneic model. Results: ATG-034 binds to LILRB4 protein with a single-digit nM affinity. It potently reversed the fibronectin-mediated inhibition of FcR-driven TNF-α cytokine production. TolDCs were reprogrammed by ATG-034 to immunogenic DCs with significant upregulation of the expression of HLA-DR, HLA-ABC, CD86 and downregulation of the expression of CD206. Furthermore, ATG-034 remarkably reinvigorated tolDCs for priming T cell activation with noticeable upregulation of the expression of CD25 and the secretion of IFN-γ, enhancing antitumor immunity. In addition, 10 mg/kg ATG-034 significantly inhibited the LLC tumor growth in vivo with a TGI of 40.28%, while a benchmark antibody only demonstrated a 24.43% TGI. Conclusion: Our data show that ATG-034 reprograms tolDC, enhancing anti-tumor immunity, and demonstrates potent in vivo anti-tumor efficacy. Therefore, ATG-034 may be a promising strategy for the treatment of cancer. Citation Format: Ao Sun, Enlin Zheng, Huili Cao, Mengshi Cao, Suya Bai, Peng Chen, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6384.