Targeted erythrophagocyte reprogramming of Kupffer cells halts cancer immunotherapy associated liver toxicity [Multiplexed_CD40]

Agonistic CD40 monoclonal antibodies have emerged as promising immunotherapeutic compounds with impressive anti-tumoral effects. However, one of the main limitations of their use in patients is the severe liver necro-hepatitis they can induce. Up to this point, no effective treatment for anti-CD40 liver toxicity that does not hinder anti-tumor efficacy has been found. In the present study , we show that anti-CD40 liver toxicity is dependent on liver macrophages and recruitment of monocytes and neutrophils. We specifically reprogrammed the phenotype of liver macrophages to anti-inflammatory erythrophagocytes by using repeated injection of anti-erythrocyte murine Ter119 (mTer119) antibody. mTer119 administration induced phagocytosis of erythrocytes by liver macrophages and their transformation to Hmoxhigh/Marcohigh/MHC-IIlow erythrophagocytes. mTer119 treatment prevented anti-CD40 induced liver toxic side-effect, with significantly reduced elevation of plasma transaminases level and area of liver necrosis on histology, while preserving the anti-tumoral efficiency of anti-CD40. Our study offers a novel targeted therapeutic approach to treat immune-related liver adverse side effects of immunotherapies. C57BL/6J mice were injected with 20 mg/kg anti-CD40 antibody (InVivoPlus BP0016-2) intraveinously or saline (Control mouse). At 7, 14 or 22 hours post injection, mice were euthanized and their liver digested into a non-parenchymal single-cell suspension. Cells were enriched for leucocytes using CD45+ coated magnetic beads (MagniSort™ Mouse CD45 Positive Selection Kit, 8802-6865-74), stained with Totalseq B hashtag antibodies (Biolegend) and pooled together at equal cell count before processing for single-cell RNA sequencing (Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein).

激动型CD40单克隆抗体（agonistic CD40 monoclonal antibodies）已成为极具潜力的免疫治疗化合物，展现出优异的抗肿瘤效应。但其临床应用的主要限制之一在于可诱发严重的肝脏坏死性肝炎。截至目前，尚未发现既能缓解抗CD40相关肝毒性，又不影响抗肿瘤疗效的有效治疗手段。本研究证实，抗CD40相关肝毒性依赖于肝脏巨噬细胞以及单核细胞和中性粒细胞的招募。我们通过重复注射抗红细胞小鼠Ter119（mTer119）抗体，将肝脏巨噬细胞的表型特异性重编程为抗炎性红细胞吞噬细胞。mTer119给药可诱导肝脏巨噬细胞吞噬红细胞，并使其转化为Hmox高表达/Marco高表达/MHC-II低表达的红细胞吞噬细胞。mTer119治疗可预防抗CD40诱导的肝脏毒性不良反应，显著降低血浆转氨酶水平升高幅度及组织病理学下的肝脏坏死面积，同时保留抗CD40的抗肿瘤效能。本研究为治疗免疫治疗相关的肝脏免疫不良反应提供了一种全新的靶向治疗策略。C57BL/6J小鼠经静脉注射20 mg/kg抗CD40抗体（InVivoPlus BP0016-2）或生理盐水（对照组小鼠）。于注射后7、14或22小时处死小鼠，将其肝脏消化为非实质细胞单细胞悬液。采用CD45包被的磁珠（MagniSort™ 小鼠CD45阳性分选试剂盒，8802-6865-74）富集白细胞，使用Totalseq B标签抗体（Biolegend）进行染色，按等量细胞数混合后，采用搭载细胞表面蛋白特征条形码技术的Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index)进行单细胞RNA测序。