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.