Rational engineering strategies to improve antibody fragment pharmacokinetics and bioavailability
Kelly, Vince Wesley
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Permalink
https://hdl.handle.net/2142/129476
Description
Title
Rational engineering strategies to improve antibody fragment pharmacokinetics and bioavailability
Author(s)
Kelly, Vince Wesley
Issue Date
2024-04-15
Director of Research (if dissertation) or Advisor (if thesis)
Sirk, Shannon J
Doctoral Committee Chair(s)
Sirk, Shannon J
Committee Member(s)
Underhill, Gregory H
Smith, Andrew M
Stadtmueller, Beth M
Department of Study
Bioengineering
Discipline
Bioengineering
Degree Granting Institution
University of Illinois Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Pharmacokinetics
protein engineering
therapeutics
bioavailability
Abstract
Therapeutic antibodies have become one of the most widely used classes of biotherapeutics due to their unique antigen specificity and their ability to be engineered against diverse disease targets. There is significant interest in utilizing truncated antibody fragments, including single-domain antibodies (sdAb), as therapeutics, as their small size affords favorable properties such as increased tumor penetration as well as the ability to utilize lower-cost prokaryotic production methods. However, they suffer from poor pharmacokinetics in vivo. This poor pharmacokinetic profile can be attributed to multiple factors, including an inability to bind the neonatal Fc receptor (FcRn) – the receptor responsible for the long half-lives of natural IgG and serum albumin. A common approach to circumvent these limitations is to enable engagement with the half-life extending neonatal Fc receptor (FcRn). This is usually achieved via fusion with a large Fc domain, which negates the benefits of the antibody fragment’s small size. In this work, we show that modifying antibody fragments with short FcRn-binding peptide domains that mimic engagement of IgG and albumin with FcRn enables binding and FcRn-mediated recycling and trans-membrane transcytosis in cell-based assays, and alters pharmacokinetics in vivo. SdAb also fall below the cutoff for first-pass clearance through the renal filtration system (50-60 kDa), leading to rapid blood clearance, which limits the efficacy of these potentially powerful therapeutics. Strong net negative charge has also been shown to decrease fractional kidney filtration in vivo. Here, we demonstrate that rational swapping of surface-exposed cationic residues with anionic restudies, in combination with fusion to a homotrimeric peptide domain, significantly extends half-life and delays renal filtration in vivo. Further, we combine these modifications with FcRn-binding peptide fusions to further increase half-life and facilitate trans-membrane transport in a mouse model. These rational modifications may improve the suitability of nanobodies for use as therapeutics, particularly in combination with alternative delivery systems such as enteric capsules or engineered commensal bacteria.
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