Enteric Pharmacokinetics of Monomeric and Multimeric Camelid Nanobody Single-domain Antibodies
Michelle DebatesHillary Dance, Jacqueline M. Tremblay, Kimberly Gaspie, Raymond K. Kudej, Vladimir Vigdorovich, Noah Sather, Justyna J. Jaskiewicz, Saul Tzipori, Charles B. Shoemaker
Abstract
Single-domain antibodies (sdAbs) derived from Camelidae heavy-chain-only antibodies (also called nanobodies or VHHs) have advantages over conventional antibodies in terms of their small size and stability to pH and temperature extremes, their ability to express well in microbial hosts, and to be functionally multimerized for enhanced properties. For these reasons, VHHs are showing promise as enteric disease therapeutics, yet little is known as to their pharmacokinetics (PK) within the digestive tract. To improve understanding of enteric VHH PK, we investigated the functional and structural stability of monomeric and multimeric camelid VHH-agents following in vitro incubation with intestinal extracts (chyme) from rabbits and pigs or fecal extracts from human sources, and in vivo in rabbits. The results showed that unstructured domains such as epitopic tags and flexible spacers composed of different amino acid sequences were rapidly degraded by enteric proteases while the functional core VHHs were much more stable to these treatments. Individual VHHs were widely variable in their functional stability to GI tract proteases. Some VHH-based agents which neutralize enteric Shiga toxin Stx2 displayed a functional stability to chyme incubations comparable to that of Stx2-neutralizing IgG and IgA mAbs, thus indicating that selected nanobodies can approach the functional stability of conventional immunoglobulins.
Introduction
Enteric diseases substantially impact health in both human and veterinary medicine. While modern sanitation methods largely control enteric disease in the developed world, food-borne pathogens remain a major cause of disease and death in the developing world, particularly among children. Vaccines and drugs such as antibiotics and anthelmintics are available to prevent and/or treat many enteric diseases but are underutilized in the developing world due to factors such as cost, appearance of drug-resistant pathogens and inadequate medical infrastructure. Antibody therapeutics could also provide enteric disease benefits but are currently costlier and more challenging to distribute and administer than vaccines and small molecule drugs.
Materials and methods
Ethics statement
All animal studies were performed within the guidelines established by the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) under Protocols No. G2017-18, No. G2017-55 and No. G2017-119. Human infant fecal solids were obtained in 2018 from a deidentified donor under informed parental consent in accordance with Tufts Health Sciences Campus Institutional Review Board guidelines and in compliance with certification of exempt IRB status No. 9893.
Enteric extract preparations and incubations
Variable segments of the rabbit or porcine GI tract representing sections of duodenum, jejunum, ileum, cecum, colon and rectum were removed by dissection and the contents were squeezed out into 50 ml tubes and diluted approximately 1:4 (v:v) in PBS. Human infant fecal solids were similarly diluted approximately 1:4 (v:v) in PBS. Tubes were then vigorously vortexed and centrifuged (∼10,000 xg). The resulting chyme or fecal extract supernatants were stored in aliquots at -80°C as 1x chyme or 1x fecal extract.
Results
Human fecal and porcine intestinal extracts appear to preferentially cleave the spacers separating VHH multimers
Early western blot studies incubating intestinal and fecal extracts with VHH multimers had suggested that the proteins were preferentially cleaved within the spacer regions separating the VHH monomer components. A particularly clear example occurred when we incubated a heterohexameric agent consisting of VHHs linked by conventional (GGGGS)N spacers [13] with dilute extracts (final 1:50 dilution) obtained from human feces or porcine intestine. As shown in Fig 1, western blots that recognize the epitopic E-tags which flank the six linked VHHs produced a similar partial digestion pattern when incubated with either human or porcine enteric proteases. The size of the products, multiples of ∼15 kDa, suggested that both extracts cleaved the heteromultimer within the spacers separating the 15 kDa VHH components.
Discussion
Single-domain antibodies, such as VHHs, are particularly promising as components of enteric therapeutics because they can be produced at low cost in microbes and delivered orally or expressed in patients by probiotics or, possibly by engineering their own microbiome. Such products have already been reported to show some therapeutic benefits in numerous animal models of enteric diseases [2–11]. Because the GI tract is a hostile environment for protein therapeutics due to its role in proteolytic food digestion and its rapid luminal flow leading to elimination, optimal exploitation of VHH-based agents as enteric therapeutics will require a good understanding of the factors that influence the functional stability of these agents while resident within this environment. We report here on some initial enteric PK studies with VHH-based agents.
Acknowledgments
We gratefully acknowledge the excellent assistance of Jean Mukherjee and Alexa Foss for immunizing and caring for the rabbits. We thank David Lee-Paritz for assistance with the rabbit gut loop work and Sue Chapman for technical assistance. We also thank Michael Berne at the Tufts University Core Facility (TUCF) for the Edman degradation and amino acid sequence analysis.
Citation: Debatis M, Danz H, Tremblay JM, Gaspie K, Kudej RK, Vigdorovich V, et al. (2023) Enteric pharmacokinetics of monomeric and multimeric camelid nanobody single-domain antibodies. PLoS ONE 18(11): e0291937. https://doi.org/10.1371/journal.pone.0291937
Editor: Sebastian D. Fugmann, Chang Gung University, TAIWAN
Received: June 7, 2023; Accepted: September 10, 2023; Published: November 27, 2023
Copyright: © 2023 Debatis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The datasets generated during and/or analyzed during the current study are available in this article and its Supporting Information files.
Funding: This research was funded by the Bill and Melinda Gates Foundation (https://www.gatesfoundation.org) award #OPP1172434 granted to CBS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: sdAb, single-domain antibody; VHH, heavy-chain-only antibody VH domain; VNA, VHH-based neutralizing agent; PK, pharmacokinetics; GFP, green fluorescent protein; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; CHO, Chinese hamster ovary; EK, enterokinase; Stx2, Shiga toxin 2; Hc, heavy chain; Lc, light chain; VL, variable light chain domain; VH, variable heavy chain domain; pIgR, polymeric immunoglobulin receptor; TcdB, Clostridium difficile toxin B
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0291937#abstract0
