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. 2024 Sep 16:15:1371156.
doi: 10.3389/fimmu.2024.1371156. eCollection 2024.

Impact of mAb-FcRn affinity on IgG transcytosis across human well-differentiated airway epithelium

Kohei Togami  1 , Whitney Wolf  1 , Lucas C Olson  1 , Madison Card  2 , Limei Shen  1 , Alison Schaefer  3 , Kenichi Okuda  2 , Larry Zeitlin  4 , Michael Pauly  4 , Kevin Whaley  4 , Raymond J Pickles  2   5 , Samuel K Lai  1   5
Affiliations

Impact of mAb-FcRn affinity on IgG transcytosis across human well-differentiated airway epithelium

Kohei Togami et al. Front Immunol. .

Abstract

Effective treatment and immunoprophylaxis of viral respiratory infections with neutralizing monoclonal antibodies (mAbs) require maintaining inhibitory concentrations of mAbs at the airway surface. While engineered mAbs with increased affinity to the neonatal Fc receptor (FcRn) are increasingly employed, little is known how increased affinity of Fc to FcRn influences basal-to-apical transepithelial transport (transcytosis) of mAbs across the airway epithelium. To investigate this, we utilized a model of well-differentiated human airway epithelium (WD-HAE) that exhibited robust FcRn expression, and measured the transepithelial transport of a mAb against SARS-CoV-2 Spike protein (CR3022) with either wildtype IgG1-Fc or Fc modified with YTE or LS mutations known to increase affinity for FcRn. Despite the marked differences in the affinity of these CR3022 variants for FcRn, we did not find substantial differences in basal-to-apical transport reflective of systemic dosing, or apical-to-basal transport reflective of inhaled dosing, compared to the transport of wildtype IgG1-Fc. These results suggest increasing FcRn affinity may only have limited influence over transcytosis rates of systemically dosed mAbs across the human airway epithelium over short time scales. Over longer time scales, the elevated circulating levels of mAbs with greater FcRn affinity, due to more effective FcRn-mediated recycling, may better resupply mAb into the respiratory tract, leading to more effective extended immunoprophylaxis.

Keywords: FcRn; airway; lung; monoclonal Abs; respiratory virus; transcytosis.

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Conflict of interest statement

Authors LZ, MP, and KW were employed by the company ZabBio. Author SL is founder of Mucommune, LLC and currently serves as its interim CEO. Author SL is also founder of Inhalon Biopharma, Inc, and currently serves as its CSO as well as on its Board of Director and Scientific Advisory Board. Author SL has equity interests in both Mucommune and Inhalon Biopharma; Author SL’s relationships with Mucommune and Inhalon are subject to certain restrictions under university policy. The terms of these arrangements are managed by UNC-CH in accordance with its conflict-of-interest policies. Author SL was inventor of intellectual property that has been licensed to Inhalon Biopharma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Characterization of the CR3022 and its variants. (A) Coomassie brilliant blue staining of non‐reduced and reduced protein gels depicting monoclonal antibodies (mAbs). (B, C) ELISA analysis illustrating the binding affinity of mAbs and intravenous immunoglobulin (IVIG) to the SARS‐CoV‐2 spike RBD protein (B) and FcRn protein (C). Each data point represents the mean ± standard deviation (n = 3).
Figure 2
Figure 2
Basal‐to‐apical transport of CR3022 and its variants across WD‐HAE cell culture. (A) The mAb concentration in the apical wash over time at basal doses of 40 and 400 µg. (B) The cumulative amount of mAbs transported to the apical side over time at basal doses of 40 and 400 µg. (C) Cumulative transcytosed fraction based on the applied dose to the basal side over time. Each point represents the mean ± standard deviation (n = 3 for each condition/ID, total of three donor IDs).
Figure 3
Figure 3
(A) BulkSeq of cultures of Large Airway Epithelium (LAE) and Small Airway Epithelium (SAE). (B) Immunohistochemical detection of FcRn proteins in WD‐HAE cultures and human trachea, compared to isotype staining control. (C–E) Effect of IVIG on the basal‐to‐apical transport of CR3022 through WD‐HAE cell culture. (C) The mAb concentration in the apical wash over time at basal doses of 400 μg, with or without co‐application of IVIG (4 mg). (D) The cumulative amount of mAb transported to the apical side over time at basal doses of 400 μg with or without IVIG (4 mg). (E) Cumulative transcytosed fraction, calculated based on the applied dose to the basal side, over time with or without IVIG. Each point represents the mean ± standard deviation (n = 3 for each condition/ID, a total of three donor IDs). *p < 0.01: indicates statistical significance compared to CR3022 alone, using Bonferroni‐corrected t‐test. “ns” stands for “Not significant”.
Figure 4
Figure 4
Apical‐to‐basal transport of CR3022 and its variants across WD‐HAE cell culture. (A) The mAb concentration in the basal media over time at apical doses of 0.2 and 2 µg. (B) The cumulative amount of mAbs transported to the basal side over time at apical doses of 0.2 and 2 µg. (C) Cumulative transcytosed fraction based on the applied dose to the apical side over time. Each point represents the mean ± standard deviation (n = 3 for each condition/ID, a total of three donor IDs).
Figure 5
Figure 5
Comparative analysis of apical (A) and basal (B) concentrations of CR3022 and its variants after 96 h of apical (0.2 or 2 µg) or basal (40 or 400 µg) application on WD‐HAE cell culture. (C) The ratio of mAb concentrations in the apical/basal compartment. Each point shows an average between three donor IDs. Each point represents the mean ± standard deviation (n = 3 for each condition/ID, a total of three donor IDs). *p < 0.01 indicates statistical significance compared to the basal dose of 40 µg. #p < 0.01 indicates statistical significance compared to the basal dose of 400 µg. Statistical comparison was based on one‐way ANOVA.
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