Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics Angela Schocha, Hubert Kettenbergerb, Olaf Mundiglc, Gerhard Winterd, Julia Engertd, Julia Heinricha, and Thomas Emricha,1 aLarge Molecule Bioanalytical Research and Development, Pharmaceutical Sciences, bLarge Molecule Research Biochemical and Analytical Research, Large Molecule Research, and cLarge Molecule Research Discovery, Large Molecule Research, Roche Pharmaceutical Research and Early Development, Roche Innovation Center Penzberg, 82377 Penzberg, Germany; and dInstitute of Pharmaceutical Technology and Biopharmaceutics, Ludwig Maximilians University, 81377 Munich, Germany Edited by Jeffrey V. Ravetch, The Rockefeller University, New York, NY, and approved April 3, 2015 (received for review May 20, 2014) Here, we investigated the influence of the variable fragment (Fv) characteristics at different pHs may strongly influence circulation of IgG antibodies on the binding to the neonatal Fc receptor (FcRn) half-lives of IgGs (21). as well as on FcRn-dependent pharmacokinetics (PK). FcRn plays a In addition to the specific interaction of the Fc region with key role in IgG homeostasis, and specific manipulation in the FcRn, the Fab regions have also been suggested to contribute crystallizable fragment (Fc) is known to affect FcRn-dependent PK. to FcRn binding (22–24). For example, Fab-mediated residual Although the influence of the antigen-binding fragment (Fab) on binding at near physiological pH was correlated with the phar- FcRn interactions has been reported, the underlying mechanism is macokinetic properties of a set of therapeutic antibodies, indi- hitherto only poorly understood. Therefore, we analyzed the two cating that IgGs with excessive binding to FcRn at pH 7.3 suffer IgG1 antibodies, briakinumab and ustekinumab, that have similar from reduced terminal half-lives (23). Recently, Schlothauer et al. Fc parts but different terminal half-lives in human and systemat- (24) have described a novel pH gradient FcRn affinity chroma- ically engineered variants of them with cross-over exchanges and tography method that closely mimics physiological conditions for the dissociation between FcRn and IgGs. Furthermore, they varied charge distribution. Using FcRn affinity chromatography, showed that IgGs with identical Fc regions differ in their dis- molecular dynamics simulation, and in vivo PK studies in human sociation from FcRn, thereby indicating the influence of the Fab FcRn transgenic mice, we provide evidence that the charge distri- region on FcRn binding. However, the underlying mechanism bution on the Fv domain is involved in excessive FcRn binding. This – how the distal Fab region influences FcRn binding is hitherto excessive binding prevents efficient FcRn IgG dissociation at physi- only poorly understood. ological pH, thereby reducing FcRn-dependent terminal half-lives. To systematically investigate the influencing factors of the Fab Furthermore, we observed a linear correlation between FcRn column region to FcRn-mediated IgG homeostasis, we used the antibody retention times of the antibody variants and the terminal half-lives pair briakinumab (Ozespa) and ustekinumab (Stelara) as a in vivo. Taken together, our study contributes to a better under- model system. Both briakinumab and ustekinumab are fully standing of the FcRn–IgG interaction, and it could also provide pro- human monoclonal IgG1 antibodies that specifically bind to the found potential in FcRn-dependent antibody engineering of the human p40-subunit of interleukin 12 and interleukin 23 (25) and variable Fab region. not to the corresponding mouse interleukin 12 and interleukin 23. Both antibodies have nearly identical constant IgG1 domains, FcRn | pharmacokinetics | antibody | charge | engineering with minor differences in several allotype-specific amino acids in this region (Fig. S1). However, these different amino acids are uman IgGs contain two antigen-binding (Fab) regions that outside of the cognate FcRn-binding regions and therefore Hconvey specificity for the target antigen and a constant (Fc) are considered to play no significant role in FcRn-dependent region that is responsible for interactions with Fc receptors (1, 2). Human IgGs of subclasses 1, 2, and 4 have an average serum half- Significance life of 21 days (d), which is longer than that of any other known serum protein (3). This long half-life is predominantly mediated by Therapeutic antibodies of the immunoglobulin G (IgG) isotype the interaction with the neonatal Fc receptor (FcRn) (4, 5) and is show a pharmacokinetic (PK) profile that is strongly mediated one of the reasons why IgGs or Fc-containing fusion proteins are by the interaction with the neonatal Fc receptor (FcRn). There- widely used as therapeutic proteins. fore, modulating the FcRn–IgG interaction allows altering PK FcRn is a membrane-associated receptor involved in both IgG characteristics of therapeutic antibodies. So far, engineering and albumin homeostasis, in maternal IgG transport across the the crystallizable fragment (Fc) is known to affect PK, and, al- placenta, and in antigen–IgG immune complex phagocytosis (6– though the influence of the antigen binding fragment (Fab) on 8). Human FcRn is a heterodimer consisting of the glycosylated FcRn interactions has been reported, the underlying mecha- class I major histocompatibility complex-like protein (α-FcRn) nism remains unknown. Here, we demonstrate that the charge and a β2 microglobulin (β2m) subunit (9). FcRn binds to a site in distribution in the distal variable fragment (Fv) of IgGs is in- the CH2-CH3 region of the Fc region (10–13), and two FcRn volved in excessive binding to the FcRn, thereby reducing FcRn- molecules can bind to the Fc region simultaneously (14, 15). The dependent terminal half-lives in vivo. These findings contribute – affinity between FcRn and Fc is strongly pH dependent, showing to a better understanding of the FcRn IgG interaction. SCIENCES nanomolar affinity at endosomal pH of 5–6 and negligible binding at a physiological pH of 7.4 (12, 16, 17). The underlying Author contributions: A.S., H.K., O.M., G.W., J.H., and T.E. designed research; A.S., H.K., APPLIED BIOLOGICAL mechanism conveying long half-life to IgGs can be explained by and O.M. performed research; A.S. and T.E. analyzed data; and A.S., H.K., O.M., J.E., and three fundamental steps. First, IgGs are subject to unspecific T.E. wrote the paper. pinocytosis by various cell types (18, 19). Second, IgGs encounter The authors declare no conflict of interest. and bind FcRn in the acidic endosome at a pH of 5–6, thereby This article is a PNAS Direct Submission. protecting IgGs from lysosomal degradation (10, 17, 20). Finally, Freely available online through the PNAS open access option. IgGs are released in the extracellular space at physiological pH 1To whom correspondence should be addressed. Email: [email protected]. of 7.4 (4). This strict, pH-dependent bind-and-release mecha- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nism is critical for IgG recycling, and any deviation of the binding 1073/pnas.1408766112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1408766112 PNAS | May 12, 2015 | vol. 112 | no. 19 | 5997–6002 Downloaded by guest on October 5, 2021 pharmacokinetics (PK) (10). Briakinumab is an IgG1λ antibody A C Fab with variable heavy and light chain domains of the VH3 and Vλ1 Fab‘ germ-line families. Ustekinumab is an IgG1κ antibody with var- FcRn‘ iable heavy and light chain domains of the VH5 and Vκ1D germ- line families. Interestingly, ustekinumab has a median terminal FcRn half-life of 22 d (26) whereas briakinumab has a terminal half-life Fc of only 8–9 d (25, 27, 28). Therefore, we used briakinumab and ustekinumab as our model system to elucidate the role of the Fab β2m region on FcRn-dependent PK. BD70 Briakinumab Here, we report that the charge distribution in the variable 50 fragment (Fv) domain is involved in excessive FcRn binding, Ustekinumab thereby reducing FcRn-dependent terminal half-lives. To eluci- 30 date which structural element of the Fab region and how the Fab 10 -10 45678910 region influences FcRn binding, we engineered nine variants of Net charge (electrons) briakinumab and ustekinumab. Furthermore, we analyzed all 11 -30 antibodies using a combination of in vitro FcRn affinity chro- pH matography, molecular dynamics (MD) simulation, subcellular Fig. 1. Charge distribution and pH-dependent net charge. Isopotential sur- trafficking analysis by confocal microscopy, and in vivo PK studies faces of the proteins protonated at pH 7.4 and contoured at 2 kBT/e. Blue, in human FcRn transgenic mice. positive; red, negative. (A) Briakinumab. The light chain is shown in yellow, and the heavy chain is shown in orange. Views of the Middle and Right images are Results related to the view in the Left panel by a rotation about a vertical and a hor- Charge Distribution and pH-Dependent Net Charge. Using the pub- izontal axis, respectively. (B) Ustekinumab. Light and heavy chains are colored in lished crystal structure of ustekinumab (29) and a homology light and dark green, respectively. The views are identical to A.(C) Homology β β model of briakinumab, we observed that briakinumab exhibits an model of briakinumab in complex with two human FcRn/ 2 microglobulin ( 2m) heterodimers, shown as molecular surface. The surface is colored according to unusual, nonuniform charge distribution at physiological pH of ± 7.4. Briakinumab