Roundabout (ROBO) 1 and 2 are transmembrane receptors that bind secreted SLIT ligands through their extracellular domains (ECDs) and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. SLIT-ROBO signaling regulates pathological ocular neovascularization, which is a major cause of vision loss worldwide, but pharmacological tools to prevent SLIT-ROBO signaling are lacking. Here, we developed human monoclonal antibodies (mAbs) against the ROBO1 and ROBO2 ECDs. One antibody that inhibited in vitro SLIT2 signaling through ROBO1 and ROBO2 (anti-ROBO1/2) also reduced ocular neovascularization in oxygen-induced retinopathy (OIR) and laser-induced corneal neovascularization (CNV) mouse models in vivo. Single-cell RNA sequencing of OIR retinas revealed that antibody treatment affected several cell types relevant to physiological and pathological angiogenesis, including endothelial cells, pericytes, and a heterogeneous population of myeloid cells. mAb treatment improved blood-retina barrier integrity and prevented pathological pericyte activation in OIR. SLIT-ROBO signaling inhibition prevented pathological activation of myeloid cells and increased neuroprotective myeloid populations normally seen in the developing retina. Microglia/infiltrating macrophage–specific ablation of Robo1 and Robo2 or knockout of the downstream effector phosphatidylinositol 3-kinase ( Pik3cg ) encoding PI3Kγ in both OIR and CNV models phenocopied anti-ROBO1/2 treatment, further demonstrating the key role of myeloid cells as drivers of ocular neovascular diseases. ROBO1/2 blocking antibodies may thus provide a promising strategy to combat inflammation in blinding eye diseases.

中文翻译：

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阻断 Roundabout 1 和 2 信号转导的单克隆抗体通过骨髓细胞靶向病理性眼部新生血管形成


Roundabout （ROBO） 1 和 2 是跨膜受体，通过其胞外结构域 （ECD） 结合分泌的 SLIT 配体，并通过其细胞质结构域发出信号，以调节细胞骨架并调节细胞迁移、粘附和增殖。SLIT-ROBO 信号传导调节病理性眼部新生血管形成，这是全球视力丧失的主要原因，但缺乏预防 SLIT-ROBO 信号传导的药理学工具。在这里，我们开发了针对 ROBO1 和 ROBO2 ECD 的人单克隆抗体 （mAb）。一种通过 ROBO1 和 ROBO2 抑制体外 SLIT2 信号传导的抗体（抗 ROBO1/2）也减少了氧诱导视网膜病变 （OIR） 和激光诱导角膜新生血管 （CNV） 小鼠模型中的眼部新生血管形成。OIR 视网膜的单细胞 RNA 测序显示，抗体治疗影响了与生理和病理血管生成相关的几种细胞类型，包括内皮细胞、周细胞和异质性髓系细胞群。mAb 治疗改善了血视网膜屏障完整性并防止了 OIR 中的病理性周细胞活化。SLIT-ROBO 信号抑制阻止了骨髓细胞的病理性激活，并增加了通常在发育中的视网膜中可见的神经保护性髓细胞群。在 OIR 和 CNV 模型中，编码 PI3Kγ 的小胶质细胞/浸润巨噬细胞特异性消融 Robo1 和 Robo2 或敲除编码 PI3Kγ 的下游效应物磷脂酰肌醇 3-激酶 （Pik3cg） 表型复制抗 ROBO1/2 治疗，进一步证明了髓系细胞作为眼部新生血管疾病驱动因素的关键作用。因此，ROBO1/2 阻断抗体可能为对抗致盲性眼病的炎症提供了一种有前途的策略。