Extended Data Fig. 5: Pharmacokinetics and immunogenicity of Crunch.
From: Phagocytic clearance of targeted cells with a synthetic ligand
a, Effect of vitamin K on Crunch. Engulfment assay of MerTK+ NIH3T3 cells targeting thymocytes from ROSA26GFPm-OVA mice, in the presence of 5% FBS and 10 µg/ml GFPNb-Crunch produced with or without vitamin K (Extended Data Fig. 1j). b-d, Plasma half-life of Crunch. 150 µg of GFPNb-Crunch was administered intravenously (i.v.) or intraperitoneally (i.p.) to mice (b). Plasma was collected at various time points (10 min to 120 hr), and GFPNb-Crunch levels were quantified by ELISA (c). The plasma remaining after 12 hr was used to calculate the half-life (d) (n = 4 mice). e, Antibody epitope prediction of Crunch. Mouse Protein S (mProS) and GFPNb-Crunch antibody epitopes were predicted by BepiPred-2.0. The epitope score was shown as threshold 0.5. f-g, Anti-drug antibody (ADA) assay. Mice were intravenously injected with 150 µg of GFPNb-Crunch or GFP, and plasma was collected 24 days later. Antibodies against GFP, GFPNb-Crunch, GFPNb, and Gla domain-deleted mProS (f) were measured by ELISA (Crunch n = 4, GFP n = 3). h, Macrophage stimulation. Macrophages were stimulated with 10 µg/ml GFPNb-Crunch, mouse IgG2a, 10% FBS, or 10 ng/ml LPS. RNA expression of GAPDH, IFNγ, IL-6, CCL5, IL-10, and Arg1 was measured by RT-PCR (n = 3, independent biological samples). All data are shown as mean ± S.D. Statistical analysis in g used Student’s unpaired t test; h used one-way ANOVA with Tukey-Kramer t-test. *P < 0.05 indicates significance.
