Figure 1.

Humanized 219 (h219) is an antagonist anti-LAIR1 monoclonal antibody. (A) Affinity of h219 (VH9/VK2) to human LAIR1 was determined by Octet RED96. ForteBio’s data analysis software was used to fit the data to a 1:1 binding model to extract an association rate and dissociation rate. The Kd was calculated using the ratio k_dis/k_on. (B) h219 blocks LAIR1 and collagen I interaction in ELISA. Collagen I was coated on 96-well plate at 1 μg/ml. Pre-incubated LAIR1-hFc (2 μg/ml) and h219, h94, or hIgG (5 μg/ml) were added to the plate after blocking with 5% non-fat milk. HRP conjugated goat anti-human IgG Fc was used to detect the binding of LAIR1-hFc to Collagen I. (C) h219 blocks LAIR1 and Collagen I interaction performed by Octet RED96. NTA biosensors were used to capture his tagged human LAIR1 recombinant protein (20 μg/ml). After loading of h219 or control antibody h94 (40 μg/ml), the collagen I (80 μg/ml) binding signal was detected. (D) h219 blocks LAIR1 activation induced by the indicated molecules as determined by the LAIR1 chimeric receptor reporter cell assay. (E) Comparison of the activities of control, h94, and h219 antibodies with wildtype Fc or with LALA-PG mutation in Fc that does not have Fc-mediated effector function (see Supplementary Figure 7 ) to block collagen-induced LAIR1 activation. Immobilized collagen (left) or collagen stably expressed on K562 cells (right) was used to induce LAIR1 activation in LAIR1 chimeric receptor reporter cells. One-way ANOVA test for IgG-ctrl, h94, and h219, p<0.0001. One-way ANOVA test for IgG-LLG ctrl, h94-LLG, and h219-LLG, p<0.0001, ** p < 0.01.