Single domain antibodies from camelids in the treatment of microbial infections

Abstract

Infectious diseases continue to pose significant global health challenges. In addition to the enduring burdens of ailments like malaria and HIV, the emergence of nosocomial outbreaks driven by antibiotic-resistant pathogens underscores the ongoing threats. Furthermore, recent infectious disease crises, exemplified by the Ebola and SARS-CoV-2 outbreaks, have intensified the pursuit of more effective and efficient diagnostic and therapeutic solutions. Among the promising options, antibodies have garnered significant attention due to their favorable structural characteristics and versatile applications. Notably, nanobodies (Nbs), the smallest functional single-domain antibodies of heavy-chain only antibodies produced by camelids, exhibit remarkable capabilities in stable antigen binding. They offer unique advantages such as ease of expression and modification and enhanced stability, as well as improved hydrophilicity compared to conventional antibody fragments (antigen-binding fragments (Fab) or single-chain variable fragments (scFv)) that can aggregate due to their low solubility. Nanobodies directly target antigen epitopes or can be engineered into multivalent Nbs and Nb-fusion proteins, expanding their therapeutic potential. This review is dedicated to charting the progress in Nb research, particularly those derived from camelids, and highlighting their diverse applications in treating infectious diseases, spanning both human and animal contexts.

Keywords: antibiotic resistance; antimicrobial resistance (AMR); antimicrobial therapy; antiviral therapies; infectious diseases; nanobodies (Nbs); novel therapy for infectious diseases; passive immune therapy.

Figure 1

Origin, structure and formats of nanobodies. (A) Graphical representation of conventional and heavy-chain-only antibodies. Conventional antibodies are found in mammals. They consist of two heavy (green) and two light (blue) chains, and their antigen-binding region (paratope) is encoded by the variable domains of both chains (VH and VL). In camelids (camels, dromedary, Ilama, and alpaca), next to the conventional antibodies, heavy-chain-only antibodies are also found. The antigen is recognized by the variable domain of the heavy chain (VHH). Nanobodies (Nbs) are VHHs derived from heavy-chain-only antibodies with a size of approximately 15 kDa. (B) Schematic representations of the nanobody architecture. Nanobodies comprise four framework regions (FR1–4) and three hypervariable regions (CDR1–3). The structural architecture of nanobodies includes 2 β-sheets, one with 4 β-strands (A, B, D, and E) and one with one β-strand (C, C’, C”, F, and G). CDR1, CDR2, and CDR3 are unstructured loops. (C) Nanobody-based engineered molecules to improve the antimicrobial potency or stability of anti-pathogenic Nbs. Due to their modular structure, Nbs can function as building blocks in multimeric constructs binding the same (multivalent) or different (multiparatopic) epitopes. Monovalent Nbs can be conjugated genetically to toxins to promote target-cytotoxicity or to membrane-permeating peptides to allow entry into target cells. To produce bivalent or bispecific recombinant antibodies and to mediate different effector functions, Nbs can be fused to the constant Fc domain of conventional IgG or IgA antibodies. Monomeric, dimeric (bivalent or biparatopic) or trimeric (trivalent or trispecific) Nbs can be obtained by linking the different monovalent Nbs head to tail using a linker peptide.