Single domain antibody: Development and application in biotechnology and biopharma

Abstract

Heavy-chain antibodies (HCAbs) are a unique type of antibodies devoid of light chains, and comprised of two heavy chains-only that recognize their cognate antigen by virtue of a single variable domain also referred to as VHH, single domain antibody (sdAb), or nanobody (Nb). These functional HCAbs, serendipitous discovered about three decades ago, are exclusively found in camelids, comprising dromedaries, camels, llamas, and vicugnas. Nanobodies have become an essential tool in biomedical research and medicine, both in diagnostics and therapeutics due to their beneficial properties: small size, high stability, strong antigen-binding affinity, low immunogenicity, low production cost, and straightforward engineering into more potent affinity reagents. The occurrence of HCAbs in camelids remains intriguing. It is believed to be an evolutionary adaptation, equipping camelids with a robust adaptive immune defense suitable to respond to the pressure from a pathogenic invasion necessitating a more profound antigen recognition and neutralization. This evolutionary innovation led to a simplified HCAb structure, possibly supported by genetic mutations and drift, allowing adaptive mutation and diversification in the heavy chain variable gene and constant gene regions. Beyond understanding their origins, the application of nanobodies has significantly advanced over the past 30 years. Alongside expanding laboratory research, there has been a rapid increase in patent application for nanobodies. The introduction of commercial nanobody drugs such as Cablivi, Nanozora, Envafolimab, and Carvykti has boosted confidence among in their potential. This review explores the evolutionary history of HCAbs, their ontogeny, and applications in biotechnology and pharmaceuticals, focusing on approved and ongoing medical research pipelines.

Keywords: VHH; biotechnological tools; diagnosis; heavy chain antibodies; nanobody; single‐domain antibody; therapeutics.

FIGURE 1

Adaptive evolution of HCAbs and nanobody structures. (A) Schematic representation of typical antibody and HCAb (left); Structural organization of intact IgG, VH (PDB: 5DK3), and VHH (PDB: 7DJX) (right). Characteristic amino acid substitutions are shown in cartoon pattern and the CDR3 region is shown in surface view (bottom right). (B) Structural alignment of VHH region from Camelid (Modena, PDB:8H3Y; Lilac, PDB:5IMM; Blue, PDB:7DJX) and VH region from Homo sapiens (Yellow, PDB:5DK3; Pink, PDB:1MCO) and Mus musculus (Green, PDB:1IGY). The right showed the sequence alignment of CDR3 regions. (C) Structural alignment of two different nanobodies, containing an additional non‐canonical disulfide bond. (Orange, PDB:1MEL). (D) Amino acid sequences of long hinge regions from rearranged llama IgG transcript (Modified from Kevin et.al⁴⁷).

FIGURE 2

Search results of the LENS.ORG patent database by applying the terms “nanobody” (https://www.lens.org/, accessed on 19 July 2024). (A) Annual patent trends related to nanobody. (B) Patent documents by Jurisdiction and trend over 5 years. (C) Top applicants from North America and Europe.

FIGURE 3

(A) Schematic illustration of regular nanobody production operations using a phage display library. It involves immunizing an alpaca with the target antigen, followed by blood sampling to isolate peripheral blood lymphocytes (PBLs). mRNA is extracted from PBLs and reverse‐transcribed into cDNA. VHH genes are amplified via PCR and inserted into a phage display vector to create a library. Phages displaying unique VHHs undergo selection for antigen binding through panning. Selected phage clones are screened, and top candidates are subcloned into expression vectors. The VHHs are produced in host cells like E. coli and purified using affinity chromatography. Created with BioRender.com (B) Structural diagram of nanobody binding to the antigen (PDB: 8H3Y) and summary of the important applications and related targets of Nbs in the realm of medicine, especially on cancer, neurological disorders, infectious diseases and diagnostic tools. Created with BioRender.com (C) Timeline of approved therapeutic nanobodies. The names of the products are colored orange, followed by its target. The marketing authorization number is on the dotted line.