Fig 1: Induction of glomerular disease in male mice with high levels of suPAR via intranasal inhalation of 2019-nCov spike S1 protein.a Experimental scheme. I/N, intranasal (n = 8 mice per group, all male). The illustrations of SARS-Cov-2 and mouse were created with BioRender.com. b Proteinuria. 2019-nCov spike S1 protein inoculation induced proteinuria in mouse soluble urokinase receptor transgenic (suPAR-Tg) mice, but not in wild-type B6, or mouse urokinase receptor (uPAR) deficiency (Plaur-/-) mice (n = 8 per group, male). Alb albumin. Creat creatinine. c Transmission electron microscopy (TEM) analyses. TEM images showing glomerular filtration barrier and foot process effacement (red arrow). Representative of ten images per group. Blue scale bar, 1 µm. d Foot process count per µm GBM. FP foot process. GBM glomerular basement membrane (n = 10 replicates per group). e Blocking suPAR with uPAR monoclonal antibody (Mab) as shown in treatment scheme reduced proteinuria in male suPAR-Tg mice upon 2019-nCov spike S1 protein inoculation (n = 6 per group). IgG isotype IgG control. D day. f Vaccination with single dose BNT162b2 as shown in the treatment scheme prevented 2019-nCov spike S1 protein induced proteinuria in male suPAR-Tg mice (n = 6 per group). Saline, phosphate-buffered saline (PBS). q.d. once a day, I.M, intramuscular. I/N, intranasal. In (b), (e) and (f), two-way analysis of variance with Tukey’s multiple comparisons test was conducted. d One-way analysis of variance with Tukey’s multiple comparisons test was performed. Data are presented as mean ± SD. ***P < 0.001, ****P < 0.0001.
Fig 2: Specificity of VH/VH-Fc with human uPAR on cell surface (A) Anti-uPAR VH and VH-Fc binding to recombinant human uPAR measured by ELISA (left), and competition BLItz of VH 3 with VH 115 at 500nM for binding to uPAR (right); (B) Cell surface detection of uPAR by commercial mouse anti-uPAR antibody, VH 3 and 115 (1μM), and VH-Fc 3 and 115 (50nM) on 293T, 293T-uPAR, and A375 cells measured by Flow cytometry; (C) Dose-dependent cell surface binding of VH-Fc 3 and 115 on 293T-uPAR cells. (D) Aggregation evaluation of VH 3 and VH-Fc 3 (left), VH 115 and VH-Fc 115 (right) measured by SEC.
Fig 3: In vitro cytotoxicity of T cells to uPAR-expressing cells by anti-uPAR DbTE. (A, B) DbTE 3 and 115 binding to uPAR (A) and CD3 (B) measured by ELISA. (C) Cell binding of 100nM DbTE 3 and 115 on A375 cells and T cells tested by flow cytometry. (D-F) Percent relative lysis of 293T cells (D), 293T-uPAR cells (E), and A375 cells (F) by T cells mediated by DbTE 3 and 115, respectively. T cells and target cells were added at E: T ratio of 10:1 and simultaneously treated with serially diluted DbTE antibodies for 24h. Experiment was repeated two times. Values were reported as the mean of percent relative lysis ± SD. Significance was tested by using two-way ANOVA, followed by Tukey’s multiple comparisons test. ****p<0.0001.
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