Fig. 2: QMP engineering in HSA yields FIX-HSA fusion proteins with favorable FcRn binding properties and FcRn-mediated cellular handling. | Nature Communications

Fig. 2: QMP engineering in HSA yields FIX-HSA fusion proteins with favorable FcRn binding properties and FcRn-mediated cellular handling.

From: Tailored collagen binding of albumin-fused hyperactive coagulation factor IX dictates in vivo distribution and functional properties

Fig. 2

a Illustration of the ELISA-based binding assay used to study binding between hFcRn or mFcRn and FIX-HSA variants at pH 5.5. Soluble truncated FcRn (light gray) was captured on IgG1 MST/HN (blue) coated in the well, before FIX-HSA (red/black) was added to the wells, and the HSA region of the fusion protein was detected by an ALP-conjugated anti-HSA antibody (dark gray). b, c Results from the ELISA-based hFcRn and mFcRn binding assays performed at pH 5.5. Data represent the mean ± SD of technical duplicates from one representative experiment. d Illustration of the SPR experiment used to study the interaction between FcRn (gray) and FIX-HSA (orange/black) performed by immobilizing FIX-HSA fusion proteins on a CM5 sensor chip before injecting a truncated form of soluble FcRn in a concentration gradient. Representative sensorgrams from SPR performed by injecting soluble truncated hFcRn (e–h) or mFcRn (i–l) over immobilized FIX-HSA variants at pH 5.5, showing one out of three independent experiments performed. The dotted lines show curves fitted by the 1:1 Langmuir binding model. m An illustration of the HERA setup designed to study hFcRn-mediated cellular (n), uptake and (o), recycling of FIX-HSA fusion proteins. Data are presented as relative to FIX-HSAWT (=1, dotted line) and represent the group mean ± SD of three independent experiments performed in technical triplicates (uptake: n = 9, except n = 8 for FIXKA-HSAWT and FIXKR-HSAWT, n = 6 for PaduaKR-HSAQMP; recycling: n = 9, except n = 7 for FIXKA-HSAWT). Statistical significance was tested by unpaired two-tailed Student’s t tests with 95% confidence level (precise p-values are given in Supplementary Table 1). FIX-HSAWT, black; Padua-HSAQMP, gray; FIXKA-HSAWT, blue; FIXKR-HSAWT, orange; PaduaKA-HSAQMP, pink; PaduaKR-HSAQMP, purple. Source data are provided as a Source Data file. a, d, m Created in BioRender. Hovden Aaen, K. (2025) https://BioRender.com/auh41ln.

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