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. 2022 Jan 10;25(2):103746. doi: 10.1016/j.isci.2022.103746

Figure 4.

Figure 4

Cellular transport properties of ustekinumab and briakinumab

(A) Relative uptake of WT and Fc-engineered human IgG1 variants when 400 nM of each variant was added to the cells followed by 4 h incubation, washing, and lysis of the cells.

(B) Relative recycling of the Fc-engineered human IgG1 variants when 400 nM of each variant was added to the cells and incubated for 4 h followed by extensive washing and additional 4 h incubation before sample collection.

(C) Relative residual amount of WT and Fc-engineered human IgG1 variants. The same procedure as in (B) followed by lysis of the cells.

(D–F) Relative (D) uptake, (E) recycling, and (F) residual amount of ustekinumab and briakinumab in the absence or presence of IL-12. The amounts of IgG variants in all samples were quantified by ELISA, and obtained data are shown as mean ± SD of (A–C) five or (D–F) three independent experiments performed in (A–C) triplicates or (D–F) duplicates. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, by one-way ANOVA (Dunnett’s multiple comparison test for A–C and for D–F Sidak’s multiple comparisons test). (A–F) Values for ustekinumab were set to 1, and the other antibody variants relative to this.

(G–J) Uptake of (G) ustekinumab, (H) briakinumab, (I) ustekinumab-YTE, and (J) briakinumab-YTE at pH 7.4 when 400 nM of each was added to HMEC1-hFcRn cells treated with a mixture of control siRNA or siRNA targeting the hFcRn HC followed by incubation for 15 min, 30 min, 1 h, and 2 h. After extensive washing, the cells were lysed and added to ELISA for quantification of the amounts of antibody variants. Obtained data are presented as mean ± SD from one representative experiment performed in duplicates out of five independent experiments. ns, not significant and ∗∗∗∗p < 0.0001, by two-way ANOVA (Sidak’s multiple comparisons test).

(K) HERA score for the ustekinumab and briakinumab and the respective YTE variants were calculated from the data shown in A, C, H, and J. ns, not significant and ∗∗∗p < 0.001, by one-way ANOVA (Dunnett’s multiple comparison test).