Anti-tumor activity of a novelLAIR1antagonist in combination with anti-PD-1to treat collagen-rich solid tumors (GeoMx). Anti-tumor activity of a novelLAIR1antagonist in combination with anti-PD-1to treat collagen-rich solid tumors (GeoMx)

We recently reported that resistance to PD-1-blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and thus we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. Here, we report LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist monoclonal antibody, elicits myeloid inflammation and allogeneic T cell responses by binding to LAIR1 and blocking collagen engagement. Further, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic. Overall design: Freshly cut sections were first confirmed to contain adequate RNA quality using RNAscope LS 2.5 Probe Hs-PPIB (Advanced Cell Diagnostic, 313908). Adjacent tissue sections were then subjected to a whole transcriptome assay using guidelines from the manufacturer (GeoMx Human Whole Transcriptome Atlas; manual reference MAN-10115-05). Briefly, slides were deparaffinized and rehydrated in DEPC-treated water before being subjected to antigen retrieval using Tris-EDTA pH 9. Antigen retrieval was performed for 15 minutes at 99oC in a steamer using prewarmed buffer, and slides were then washed in PBS at room temperature for 5 minutes. Slides were then transferred to a 1 ug/mL proteinase K in PBS solution prewarmed to 37oC and incubated for 15 minutes, then washed in PBS for 5 minutes, and finally postfixed in 10% neutral buffered formalin (NBF) for 5 minutes. Slides were then washed twice in NBF stop buffer, and once in PBS. Slides were wiped and 200 µuL hybridization solution was then applied. Hybridization was allowed to proceed overnight at 37oC for 16 hours. After hybridization, slides were subjected to 2 stringent washes at 25 minutes each at 37oC, then washed twice in 2X SSC buffer. Cells were then blocked with Buffer W at room temperature for 30 minutes in the dark, then subjected to staining with the morphology panel for 1 hour in the dark at room temperature. The morphology panel consisted of Syto-13 (ThermoFisher, S7575), anti-smooth muscle actin A488 clone 1A4, anti-pan cytokeratin A594 clone AE-1/AE-3, and anti-CD68 A647 clone KP1. Slides were then washed twice in 2X SSC and immediately transferred to the GeoMx instrument for stromal and tumor nest ROI selection. CD68+ cells were masked within these ROIs, and 8 um-wide contour-based TME masks spaced 2 µum away from the edge of the CD68 masks were then applied using ImageJ (Fig S2A). Probes from all masks were collected and subjected to library preparation and subsequent sequencing using an Illumina HiSeq 4000. FASTQ files were then processed into DCC files using GeoMx NGS pipeline implemented at NGM.

我们此前报道，难治性肺癌来源模型中针对PD-1阻断治疗的耐药性与胶原蛋白沉积增加，以及胶原蛋白结合型抑制性受体白细胞相关免疫球蛋白样受体1（leukocyte-associated immunoglobulin-like receptor 1, LAIR1）的表达上调相关，因此我们推测LAIR1与胶原蛋白协同抑制肿瘤的治疗应答。
本研究报道LAIR1与肿瘤间质密切相关，并在人类肿瘤及小鼠癌症模型的瘤内髓系细胞中呈高表达状态。间质相关髓系细胞呈现免疫抑制表型，且与人类癌症组织中的LAIR1表达水平呈正相关。NGM438是一种新型人源化LAIR1拮抗剂单克隆抗体，可通过结合LAIR1并阻断其与胶原蛋白的结合，诱导髓系炎症反应及同种异体T细胞应答。此外，一款可与小鼠靶标结合的NGM438替代抗体，可使难治性KP小鼠肺肿瘤模型对抗PD-1治疗产生致敏性，并提升瘤内CD8+ T细胞的浸润量及炎症相关基因的表达水平。上述研究结果表明LAIR1处于肿瘤间质与抑制性髓系细胞的交互节点，并支持LAIR1/胶原蛋白轴阻断有望在临床中解决检查点抑制剂治疗耐药性的观点。
整体实验设计：首先通过RNAscope LS 2.5探针Hs-PPIB（Advanced Cell Diagnostic, 313908）确认新鲜切割的组织切片具备合格的RNA质量。随后参照制造商操作指南（GeoMx人类全转录组图谱；手册参考编号MAN-10115-05），对相邻组织切片开展全转录组分析。具体步骤简述如下：将玻片脱蜡并经焦碳酸二乙酯（DEPC）处理的水复水，随后使用pH 9的Tris-EDTA缓冲液进行抗原修复：在蒸汽锅中以99℃预热缓冲液，孵育玻片15分钟；之后在室温下用PBS洗涤玻片5分钟。将玻片转移至预热至37℃的1 μg/mL蛋白酶K PBS溶液中孵育15分钟，随后用PBS洗涤5分钟，最后用10%中性缓冲福尔马林（NBF）进行后固定5分钟。之后用NBF终止缓冲液洗涤玻片两次，再用PBS洗涤一次。擦拭玻片后滴加200 μL杂交液，于37℃下杂交过夜16小时。杂交完成后，将玻片置于37℃下进行两次严格洗涤，每次25分钟，随后用2×SSC缓冲液洗涤两次。随后在黑暗环境中用Buffer W室温封闭30分钟，再用形态学标记组合在室温黑暗中染色1小时。该形态学标记组合包括Syto-13（ThermoFisher, S7575）、抗平滑肌肌动蛋白A488克隆1A4、抗泛细胞角蛋白A594克隆AE-1/AE-3以及抗CD68 A647克隆KP1。随后将玻片用2×SSC洗涤两次，立即转移至GeoMx仪器中进行间质区域与肿瘤巢的感兴趣区域（region of interest, ROI）选取。在这些ROI中对CD68+细胞进行掩膜标记，随后使用ImageJ创建距离CD68掩膜边缘2 μm的8 μm宽基于轮廓的肿瘤微环境（tumor microenvironment, TME）掩膜（补充图S2A）。收集所有掩膜区域的探针，进行文库制备，并使用Illumina HiSeq 4000开展后续测序。随后使用NGM部署的GeoMx NGS分析流程将FASTQ文件处理为DCC文件。