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. 2021 Jan-Dec;13(1):1893888.
doi: 10.1080/19420862.2021.1893888.

Extended plasma half-life of albumin-binding domain fused human IgA upon pH-dependent albumin engagement of human FcRn in vitro and in vivo

Simone Mester  1   2 , Mitchell Evers  3 , Saskia Meyer  4 , Jeannette Nilsen  1   2 , Victor Greiff  1 , Inger Sandlie  1   5 , Jeanette Leusen  3 , Jan Terje Andersen  1   2
Affiliations

Extended plasma half-life of albumin-binding domain fused human IgA upon pH-dependent albumin engagement of human FcRn in vitro and in vivo

Simone Mester et al. MAbs. 2021 Jan-Dec.

Abstract

Albumin has a serum half-life of 3 weeks in humans. This feature can be used to improve the pharmacokinetics of shorter-lived biologics. For instance, an albumin-binding domain (ABD) can be used to recruit albumin. A prerequisite for such design is that the ABD-albumin interaction does not interfere with pH-dependent binding of albumin to the human neonatal Fc receptor (FcRn), as FcRn acts as the principal regulator of the half-life of albumin. Thus, there is a need to know how ABDs act in the context of fusion partners and human FcRn. Here, we studied the binding and transport properties of human immunoglobulin A1 (IgA1), fused to a Streptococcus protein G-derived engineered ABD, in in vitro and in vivo systems harboring human FcRn. IgA has great potential as a therapeutic protein, but its short half-life is a major drawback. We demonstrate that ABD-fused IgA1 binds human FcRn pH-dependently and is rescued from cellular degradation in a receptor-specific manner in the presence of albumin. This occurs when ABD is fused to either the light or the heavy chain. In human FcRn transgenic mice, IgA1-ABD in complex with human albumin, gave 4-6-fold extended half-life compared to unmodified IgA1, where the light chain fusion showed the longest half-life. When the heavy chain-fused protein was pre-incubated with an engineered human albumin with improved FcRn binding, cellular rescue and half-life was further enhanced. Our study reveals how an ABD, which does not interfere with albumin binding to human FcRn, may be used to extend the half-life of IgA.Abbreviations: ABD - Albumin binding domain, ADA - anti-drug-antibodies, ADCC - Antibody-dependent cellular cytotoxicity, ELISA - Enzyme-linked Immunosorbent assay, FcαRI - Fcα receptor, FcγR - Fcγ receptor, FcRn - The neonatal Fc receptor, GST - Glutathione S-transferase, HC - Heavy chain, HERA - Human endothelial cell-based recycling assay, Her2 - Human epidermal growth factor 2, HMEC - Human microvascular endothelial cells, IgG - Immunoglobulin G, IgA - Immunoglobulin A, LC - Light chain, QMP - E505Q/T527M/K573P, WT - Wild type.

Keywords: Immunoglobulin A (IgA); albumin-binding-domain (ABD); half-life; human FcRn transgenic mice; human serum albumin (HSA); the neonatal Fc receptor (FcRn).

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Figures

Figure 1.
Figure 1.
Recycling model of IgAABD through binding to albumin and indirect rescue by FcRn. A) Illustration of the two ABD-fused human IgA1 formats studied; IgA1-LCABD and IgA1-HCABD. B) (1) IgA1-LCABD and IgA1-HCABD will be taken up in complex with albumin via fluid-phase pinocytosis and enter early endosomes. (2) FcRn predominantly resides within acidified endosomes, where the lower pH will trigger binding of albumin bound IgA1ABD to the receptor. As such, the IgA1 fusions will be indirectly bound to FcRn. (3–4) The ligands bound to the receptor will then be recycled back to the cell membrane, where the physiological pH of blood (pH 7.4) results in release of the ligands back into the circulation. (5) Proteins that do not bind FcRn in the sorting endosomes, such as naked IgA, will end in lysosomes for degradation. C) Illustrations of wild-type (WT) and engineered (QMP) human albumin variants. The figure was made with BioRender
Figure 2.
Figure 2.
Human albumin binds IgA1-fused ABD in a pH-independent manner but human FcRn in a pH-dependent manner. A-B) ELISA showing binding of titrated amounts of ABD-fused IgA1 variants to human albumin at both pH 5.5 and 7.4. The numbers represent the mean±s.d. of duplicates. C-D) ELISA showing pH-dependent binding of ABD-fused IgA1 in complex with human albumin to human FcRn. The numbers represent the mean±s.d. of duplicates. Figure A and C were made with bioRender
Figure 3.
Figure 3.
Reversible human FcRn binding to albumin in complex with ABD-fused IgA1. A) SPR setup with immobilized IgA1-HCABD, followed by injection of human albumin and soluble monomeric human FcRn at pH 5.5. The figure was made with bioRender. B) Sensorgram showing binding of human albumin after multiple injections over immobilized IgA1-HCABD, followed by injections of human FcRn. C) Close-up of the sensorgram showing binding of monomeric human FcRn to human albumin captured on ABD-fused IgA1-HCABD.
Figure 4.
Figure 4.
FcRn-mediated recycling in a cellular system. A) A schematic illustration of the HERA protocol. Briefly, HMEC-1 cells overexpressing human FcRn are seeded one day prior to the experiment. Cells are then washed and starved before samples are added and incubated for 4 hours. Before incubation ON, the medium is replaced. Recycling was defined as amount of protein taken up after 4 hours of incubation and further released back into the medium after ON incubation . The amounts of proteins present in the medium are then quantified in ELISA. The figure was made with BioRender. B) IgA1, IgA1-LCABD and IgA1-HCABD pre-incubated with human albumin were added to the cells. The amounts of the human IgA1 variants in collected medium samples were quantified by ELISA. The numbers given represent duplicates in ELISA of triplicates in HERA (SD). ns > 0.05, * = p 0.0136, **** = p < .0001, by two-tailed analysis using unpaired T-test (df = 10, t = 2.99, 11.05)
Figure 5.
Figure 5.
Extended plasma half-life in human FcRn transgenic mice in the presence of human albumin. A) Schematic illustration of the in vivo experimental setup, where IgA1 variants were pre-incubated with excess amounts of human albumin and given by intravenous (i.v.) injection to human FcRn transgenic mice that lack mouse albumin. Blood samples were collected up to day 23 post injection. The figure was made with BioRender. B-C) Log-linear changes in plasma levels (%) of B) IgA1 and IgA1-LCABD given with and without human albumin, and C) IgA1-HCABD given with and without human albumin. The variants were given to 5 mice per group. ns > 0.05,**** = p < .0001, by two-tailed analysis using unpaired T-test (df = 8, t = 7.45–17.17)
Figure 6.
Figure 6.
Engineered human albumin enhances human FcRn binding of ABD-fused IgA1. A-B) ELISA showing binding of titrated amounts of IgA1ABD variants to WT and QMP albumin at pH 5.5 and 7.4. The numbers represent the mean±s.d. of duplicates. C-D) ELISA showing binding of human FcRn to IgA1ABD variants pre-incubated with WT or QMP albumin at pH 5.5 and 7.4. The numbers represent the mean±s.d. of duplicates
Figure 7.
Figure 7.
Enhanced cellular recycling and half-life of ABD-IgA1 fusion in the presence of engineered albumin. A) IgA1ABD variants were pre-incubated with QMP or WT albumin, and then given to HMEC-1 cells overexpressing human FcRn. The data is presented as relative recycling where IgA1 is set to 1.0. The numbers given represent duplicates in ELISA of triplicates in HERA (SD). ns > 0.05, **** = p < .0001, by two-tailed analysis using unpaired T-test (df = 10, t = 2.99, 11.05). B) Log-linear changes in plasma level (%) of IgA1-LCABD when given together with WT or QMP, and, IgA1-HCABD when given together with WT or QMP, in human FcRn transgenic mice lacking expression of mouse albumin. The variants were given by i.v. injections to 5 mice per group. ns > 0.05, * = p 0.0342, by two-tailed analysis using unpaired T-test (df = 8, t = 2.23, 2.55)
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This work was supported by the Horizon 2020 Framework Programme [825821]; KWF Kankerbestrijding [7650]; Norges Forskningsråd [230526]; Norges Forskningsråd [179573]; Norges Forskningsråd [300740]; South-Eastern Norway Regional Health Authority [40018]; Research Council of Norway [179573, 230526, 287927]; South-Eastern Norway Regional Health Authority [40018].
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