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. 2021 Jun 14;10:e62927. doi: 10.7554/eLife.62927

Figure 2. Development of NC410, a LAIR-2 Fc protein that blocks LAIR-1-collagen interaction.

(A) LAIR-2 overexpression is associated with improved overall survival in some tumors: head and neck squamous cell carcinoma (HNSC), thyroid carcinoma (THCA), thymoma (THYM) and skin cutaneous melanoma (SKCM). Patients were grouped in low 25% quantile (black) and high 25% quantile (red) of LAIR-2 mRNA expression for overall survival analysis. Hazards ratio indicating if high expression is associated with poor survival (HR(High)), and p-value (p(High)) indicating the significance of association was determined using Wald test as indicated. (B) NC410 is a biologic fusing LAIR-2 with a functional IgG1 to generate a dimeric fusion protein. (C) Avidity characterization of NC410 to human, mouse and rat collagen I and III as measured by Octet analysis. (D) Indicated amounts of collagen I were plate coated, and the binding of soluble LAIR-1 was inhibited by NC410. Asterisks indicate statistical significance (****p<0.0001, two-way ANOVA). (E, F) The human LAIR-1 (hLAIR-1) extracellular domain was fused with CD3z and stably expressed in a cell line containing an NFAT-GFP pathway reporter. LAIR-1 ligation and CD3 ligation induce NFAT-GFP signaling. A parental cell line containing the CD3 NFAT-GFP reporter without LAIR-1 was used as control (WT). NC410 protein was added at increasing concentrations and inhibited human collagen I (5 µg/mL)-mediated NFAT-GFP signaling through LAIR-1 binding by (E) FACS analysis and (F) Incucyte microscopy. Total green integrated intensity of WT and hLAIR-1 reporter cells is shown over time. Points represent the median of n = 3 (with experimental triplicates in each independently performed experiment), and the whiskers indicate the 95% confidence interval (CI). Isotype control was used at the highest concentration (100 µg/mL) and showed no inhibition of NFAT-GFP signaling. Anti-human LAIR (8A8 clone) and anti-mouse CD3 were used as positive controls. Closed circles in (F) indicate NC410 treatment, and open circles indicate control treatment. Significant differences between different treatment groups of hLAIR-1 reporter cells are indicated (and tested using a two-way ANOVA with Dunnett’s correction). In all plots: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 2.

Figure 2—figure supplement 1. NC410 blocks LAIR-1 functional interactions with collagen.

Figure 2—figure supplement 1.

NC410 dose-response measured by Incucyte imaging during 24 hr. Representative microscopic images (10×) of WT and hLAIR-1 reporter cells over time. NC410 protein was added at increasing concentrations and inhibited collagen I (5 µg/mL)-mediated NFAT-GFP signaling through LAIR-1 binding. Isotype control was used at the highest concentration (100 µg/mL) and showed no inhibition of NFAT-GFP signaling. Pictures were taken every 1 hr for 24 hr. Representative images from one out of three independently performed experiments (each with experimental triplicates).