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Frontiers in immunology2024; 15; 1346328; doi: 10.3389/fimmu.2024.1346328

Serum immunoglobulin or albumin binding single-domain antibodies that enable tailored half-life extension of biologics in multiple animal species.

Abstract: Single-domain antibody fragments (sdAbs) can be isolated from heavy-chain-only antibodies that occur in camelids or the heavy chain of conventional antibodies, that also occur in camelids. Therapeutic application of sdAbs is often complicated by their low serum half-life. Fusion to sdAb that bind to long-lived serum proteins albumin or IgG can prolong serum half-life of fusion partners. Such studies mostly focused on human application. For half-life prolongation in multiple animal species novel species cross-reacting sdAb are needed. We here describe the isolation from immunized llamas of sdAbs G6 and G13 that bound IgG of 9-10 species analysed, including horse, dog, cat, and swine, as well as sdAb A12 that bound horse, dog, swine and cat albumin. A12 bound albumin with 13 to 271 nM affinity dependent on the species. G13 affinity was difficult to determine by biolayer interferometry due to low and heterogeneous signals. G13 and G6 compete for the same binding domain on Fab fragments. Furthermore, they both lack the hallmark residues typical of camelid sdAbs derived from heavy-chain antibodies and had sequence characteristics typical of human sdAbs with high solubility and stability. This suggests they are derived from conventional llama antibodies. They most likely bind IgG through pairing with VL domains at the VH-VL interface rather than a paratope involving complementarity determining regions. None of the isolated sdAb interfered with FcRn binding to albumin or IgG, and thus do not prevent endosomal albumin/IgG-sdAb complex recycling. Fusions of albumin-binding sdAb A12 to several tetanus neurotoxin (TeNT) binding sdAbs prolonged the terminal serum half-life in piglets to about 4 days, comparable to authentic swine albumin. However, G13 conferred a much lower half-life of 0.84 days. Similarly, in horse, G13 prolonged half-life to only 1.2 days whereas A12 fused to two TeNT binding domains (T6T16A12) had a half-life of 21 days. The high half-life of T6T16A12, which earlier proved to be a highly potent TeNT antitoxin, further supports its therapeutic value. Furthermore, we have identified several additional sdAbs that enable tailored half-life extension of biologicals in multiple animal species.
Copyright © 2024 Harmsen, Ackerschott and de Smit.
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Publication Date: 2024-01-30 PubMed ID: 38352869PubMed Central: PMC10862077DOI: 10.3389/fimmu.2024.1346328Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study reports the development of single-domain antibodies (sdAbs) targeting serum IgG and albumin across multiple animal species to extend the half-life of therapeutic biologics.
  • The researchers isolated sdAbs from llamas that bind serum proteins in various animals, enabling prolonged circulation time of biologic drugs by leveraging these natural serum proteins.

Background on Single-Domain Antibodies (sdAbs)

  • sdAbs are small antibody fragments derived either from:
    • Heavy-chain-only antibodies found naturally in camelids (like llamas), or
    • The heavy chain variable region (VH) of conventional antibodies in camelids.
  • sdAbs have therapeutic potential but suffer from short serum half-life, limiting their effectiveness.
  • One strategy to improve therapeutic persistence is fusion of sdAbs to proteins that naturally have long serum half-lives, such as albumin or IgG.

Purpose of the Study

  • Most prior research focused on human applications and human serum proteins.
  • This study aims to identify sdAbs that can cross-react with albumin and IgG in multiple animal species, enabling half-life extension of biologics in veterinary or preclinical models.

Methods and Key Findings

  • Llamas were immunized to produce sdAbs against serum proteins.
  • Several sdAbs were isolated, including:
    • G6 and G13: binding IgG from 9–10 species such as horse, dog, cat, swine.
    • A12: binding albumin from horse, dog, swine, and cat.
  • Binding affinities:
    • A12 bound albumin with affinity ranging from 13 to 271 nM depending on the species.
    • G13’s affinity could not be precisely measured by biolayer interferometry due to low and inconsistent signal but competes with G6 for binding on the Fab fragment of IgG.
  • sdAbs G13 and G6 exhibit sequence features typical of human sdAbs (high solubility and stability), lacking hallmark camelid heavy-chain antibody residues.
  • This suggests these sdAbs derive from conventional llama antibodies binding IgG through interaction at the VH-VL interface rather than traditional antigen binding regions.
  • None of the sdAbs interfered with FcRn-mediated recycling, preserving normal half-life extension mechanisms of albumin/IgG and their sdAb fusions.

Functional Testing of Half-Life Extension

  • sdAbs fused to tetanus neurotoxin (TeNT) binding sdAbs to test in vivo serum half-life extension in animals.
  • Results in piglets:
    • A12 fusion extended the terminal serum half-life to about 4 days, similar to native swine albumin.
    • G13 fusion extended half-life only to about 0.84 days.
  • Results in horses:
    • G13 extended half-life to about 1.2 days.
    • A12 fused to two TeNT binding domains (T6T16A12) extended half-life dramatically to 21 days.
  • The long half-life of T6T16A12 supports its potential as a potent long-acting therapeutic antitoxin for tetanus.

Conclusions and Impact

  • The study identified novel sdAbs that bind albumin or IgG across multiple animal species allowing tailored half-life extension of biologics.
  • This is particularly advantageous for veterinary therapeutics and preclinical testing in species other than humans.
  • The findings highlight a promising approach to improve pharmacokinetics of sdAb-based biologics without hindering natural protein recycling processes mediated by FcRn.
  • The technology can be broadly applied to enhance therapeutic efficacy and dosing convenience for biologics in diverse animal models.

Cite This Article

APA
Harmsen MM, Ackerschott B, de Smit H. (2024). Serum immunoglobulin or albumin binding single-domain antibodies that enable tailored half-life extension of biologics in multiple animal species. Front Immunol, 15, 1346328. https://doi.org/10.3389/fimmu.2024.1346328

Publication

Frontiers in immunology
ISSN: 1664-3224
NlmUniqueID: 101560960
Country: Switzerland
Language: English
Volume: 15
Pages: 1346328
PII: 1346328

Researcher Affiliations

Harmsen, Michiel M
  • Wageningen Bioveterinary Research, Wageningen University & Research, Lelystad, Netherlands.
Ackerschott, Bart
  • Research and Development, Smivet B.V., Wijchen, Netherlands.
de Smit, Hans
  • Research and Development, Smivet B.V., Wijchen, Netherlands.

MeSH Terms

  • Animals
  • Humans
  • Dogs
  • Horses
  • Swine
  • Single-Domain Antibodies
  • Biological Products
  • Immunoglobulin Heavy Chains
  • Albumins
  • Antitoxins
  • Immunoglobulin G

Conflict of Interest Statement

BA and HS were employed by Smivet B.V. while performing the work described here. A patent application was filed describing part of this work. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that the study was funded by Smivet B.V.. The funder (HS) had the following involvement in the study: conceptualization, study design, supervision, validation, decision to publish, and preparation of the manuscript.

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Citations

This article has been cited 2 times.
  1. Zhu J, Zhang Y, Wang X, Peng L, Ma X, Qiu Z, Kang Z, Zheng F, Zhang X, Song M, Du J, Shi Y, Yu L, Gu C, Shi J. Engineered long-acting Irisin-albumin binding domain fusion protein for enhanced anti-inflammatory efficacy in lipopolysaccharide-induced systemic inflammation.. Commun Biol 2025 Nov 17;8(1):1592.
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  2. Zettl I, Bauernfeind C, Kollárová J, Flicker S. Single-Domain Antibodies-Novel Tools to Study and Treat Allergies.. Int J Mol Sci 2024 Jul 11;25(14).
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