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Importance of isoelectric point (pI) of antibodies

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One important characteristic of monoclonal antibodies (mAbs) is their isoelectric point (pI), which essentially is the pH at which the antibody has no net electrical charge, and its value depends on the charged amino acids the antibody contains. If the pH of the surrounding environment is below the antibody’s pI, then the molecule carries a net positive charge, whereas the antibody will carry a net negative charge when the pH is above the pI.

When assessing pharmacokinetic (PK) properties of therapeutic antibodies not only the target-mediated drug disposition (TMDD) but also non-target-related mechanisms influence overall PK behavior and pI can be an important factor of the latter. Since the surface of most cells is negatively charged, antibodies need to be positively charged for efficient fluid-phase endocytosis (pinocytosis), therefore the environmental pH needs to be below the pI of the antibody. Therapeutic antibodies with pI values in the range of 8-9 are taken up adequately after administration since the physiological pH is 7.4, however, in some cases antibodies have a pI outside of this range or have been manipulated to achieve increased or decreased pI values (cationization or anionization, respectively). It is generally observed that increases in net positive charge of antibodies result in increased blood clearance and increased tissue retention with shorter half-life, whereas antibodies with lower pI generally have decreased tissue uptake and longer half-life (1-3), although observations can be conflicting regarding correlation between mAb clearance and pI (4). Even subtle manipulation such as molecular surface remodeling to disrupt positive patch regions can influence PK properties (5). In summary, pI of mAbs is known to have a substantial effect on PK behavior independent of recycling mediated by the neonatal Fc receptor, FcRn. Small changes of pI during the routine manufacturing of mAb charge variants, however, are not expected to result in dramatic changes and may not require extensive analyses (6). In any case, it is encouraged that pI values are reported in studies as an important factor influencing antibody behavior. Furthermore, since selecting the best candidates during early preclinical phases of product development can substantially decrease time and cost of development, it is of great importance to consider de-risk strategies and tools during drug discovery and development, including antibody variable region charge and antibody pI analyses (7-9). A new analytical platform has been recently described and validated on seven mAbs to rapidly assess and rank mAb charge variants during early stage development, which can be a useful screening technique during early stage development (10).

References:

1 Boswell et al, Bioconjug Chem. 2010 Dec 15;21(12):2153-63.
2 Igawa et al, Protein Eng Des Sel. 2010 May;23(5):385-92.
3, Li et al, MAbs. 2014;6(5):1255-64.
4, Hötzel et al, MAbs. 2012 Nov-Dec;4(6):753-60.
5, Datta-Mannan et al, MAbs. 2015;7(3):483-93.
6, Khawli et al, mAbs, 2010;(2)6:613-624.
7, Bumbaca Yadav et al, J Biol Chem. 2015 Dec 11;290(50):29732-41.
8, Dostalek et al, MAbs. 2017 May 2:1-11.
9, Jarasch et al, J Pharm Sci. 2015 Jun;104(6):1885-98.
10, Wagner-Rousset, J Chromatogr A. 2017 May 19;1498:147-154.

FcRn: are we done yet?

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The story of the neonatal Fc receptor (FcRn) started as a hypothesis made by F. W. Rogers Brambell more than half a century ago, when he predicted the existence of a saturable receptor responsible for protecting IgG molecules from degradation and the same or a similar system involved in IgG transport from mother to newborns. After its initial identification in neonatal rat intestine, FcRn indeed turned out to be the receptor responsible for these functions, but its further roles in albumin homeostasis and antigen presentation were also subsequently discovered (1-6).

While these properties have been widely exploited in the development of therapeutic and diagnostic reagents, the possible functions of FcRn are even broader. For example, further investigation of the immune functions of FcRn has revealed a role in anti-tumor responses. Intestinal dendritic cells expressing FcRn were shown to cross-present antigen derived from immune complexes, thereby activating tumor-specific T cells and eliciting immunity against colorectal cancer (7). More recently, it was also demonstrated that the presence of FcRn-expressing immune cells (macrophages and dendritic cells) in tumor samples was associated with a better prognosis in non-small cell lung cancer patients (8). A role for FcRn in cellular metabolism through its ability to recycle albumin has also been recently described. Specifically, low FcRn levels in tumor cell lines were associated with intracellular albumin accumulation and growth increase of tumor xenografts, whereas enforced expression of FcRn had the reverse effect (9). Despite these new results, FcRn likely has even more interesting features waiting to be revealed by enterprising scientists.


1, Rath et al, J Clin Immunol. 2013 Jan;33 Suppl 1:S9-17.
2, Challa et al, Curr Top Microbiol Immunol. 2014;382:249-72.
3, Sand et al, Front Immunol. 2015 Jan 26;5:682.
4, Pyzik et al, J Immunol. 2015 May 15;194(10):4595-603.
5, Stapleton et al, Immunol Rev. 2015 Nov;268(1):253-68.
6, Cervenak et al, Immunol Rev. 2015 Nov;268(1):269-87.
7, Baker et al, Immunity. 2013 Dec 12;39(6):1095-107.
8, Dalloneau et al, Oncotarget. 2016 Aug 23;7(34):54415-54429.
9, Swiercz et al, Oncotarget. 2017 Jan 10;8(2):3528-3541.

Free virtual issue featuring articles on neonatal Fc receptor is online now

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In a special issue of mAbs, Guest Editor Zita Schneider, D.V.M., Ph.D., Texas A&M Health Science Center, has compiled 12 articles describing recent results obtained about the neonatal Fc receptor, FcRn. All articles can be freely downloaded for a limited time. As a regulator of immunoglobulin G (IgG) and albumin homeostasis and a modulator of immune functions, this receptor has been attracting the interest of the scientific community and has become an unavoidable factor for consideration in the development of IgG- and albumin-based diagnostic and therapeutic reagents.

Burvenich et al. identified important amino acids playing a role in FcRn-IgG interaction, whereas Hironiwa et al. showed efficient antigen drop-off and IgG recycling utilizing calcium-dependent antigen-antibody binding. Ying and colleagues provided a tool to increase the transcytosis and half-life of engineered antibody domains through an FcRn-binding motif, whereas Meyer et al. utilized the albumin-binding characteristics of FcRn to generate IgA molecules with elongated half-life. Adams et al. used an anti-albumin Fv domain to extend the half-life of an Fab fragment, and Davé et al. used this domain to generate an Fab-dsFv bispecific antibody format.

FcRn can also serve as a screening tool for antibody selection as demonstrated by Souders et al. who developed a novel FcRn-binding biolayer interferometry assay, while Fan and colleagues used online peptide immune-affinity chromatography coupled with high resolution mass spectrometry to determine human FcRn expression levels in transgenic (Tg) mice and Avery et al. characterized these human FcRn Tg mice with respect to mAb pharmacokinetics (PK) prediction. Unverdorben et al. provided details of the PK of various Fc fusions compared to IgG molecules, Kelly et al. proved that FcRn-independent antigen-independent nonspecific interactions can also play a role in antibody PK, and Datta-Mannan et al. investigated several properties of mAbs in order to optimize PK parameters.

These articles provide valuable details to explore the possibilities offered by utilizing the functions of FcRn.

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