Crystal Structure of a Human Single Domain Antibody Dimer Formed through VH-VH Non-Covalent Interactions

Single-domain antibodies (sdAbs) derived from human VH are considered to be less soluble and prone to aggregate which makes it difficult to determine the crystal structures. In this study, we isolated and characterized two anti-human epidermal growth factor receptor-2 (HER2) sdAbs, Gr3 and Gr6, from a synthetic human VH phage display library. Size exclusion chromatography and surface plasmon resonance analyses demonstrated that Gr3 is a monomer, but that Gr6 is a strict dimer. To understand this different molecular behavior, we solved the crystal structure of Gr6 to 1.6 Å resolution. The crystal structure revealed that the homodimer assembly of Gr6 closely mimics the VH-VL heterodimer of immunoglobulin variable domains and the dimerization interface is dominated by hydrophobic interactions

A rational engineering strategy for designing protein a-binding camelid single-domain antibodies

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species

Differential tumor-targeting abilities of three single-domain antibody formats

The large molecular size of antibody drugs is considered one major factor preventing them from becoming more efficient therapeutics. Variable regions of heavy chain antibodies (HCAbs), or single-domain antibodies (sdAbs), are ideal building blocks for smaller antibodies due to their molecular size and enhanced stability. In the search for better antibody formats for in vivo imaging and/or therapy of cancer, three types of sdAb-based molecules directed against epidermal growth factor receptor (EGFR) were constructed, characterized and tested. Eleven sdAbs were isolated from a phage display library constructed from the sdAb repertoire of a llama immunized with a variant of EGFR. A pentameric sdAb, or pentabody, V2C-EG2 was constructed by fusing one of the sdAbs, EG2, to a pentamerization protein domain. A chimeric HCAb (cHCAb), EG2-hFc, was constructed by fusing EG2 to the fragment crystallizable (Fc) of human IgG1. Whereas EG2 and V2C-EG2 localized mainly in the kidneys after i.v. injection, EG2-hFc exhibited excellent tumor accumulation, and this was largely attributed to its long serum half life, which is comparable to that of IgGs. The moderate size (~80 kDa) and intact human Fc make HCAbs a unique antibody format which may outperform whole IgGs as imaging and therapeutic reagents.Peer reviewed: YesNRC publication: Ye

Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook

Tremendous effort has been expended over the past two and a half decades to understand many aspects of camelid heavy chain antibodies, from their biology, evolution, and immunogenetics to their potential applications in various fields of research and medicine. In this article, I present a historical perspective on the development of camelid single-domain antibodies (sdAbs or VHHs, also widely known as nanobodies) since their discovery and discuss the advantages and disadvantages of these unique molecules in various areas of research, industry, and medicine. Commercialization of camelid sdAbs exploded in 2001 with a flurry of patents issued to the Vrije Universiteit Brussel (VUB) and later taken on by the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB) and, after 2002, the VIB-founded spin-off company, Ablynx. While entrepreneurial spirit has certainly catalyzed the exploration of nanobodies as marketable products, IP restrictions may be partially responsible for the relatively long time span between the discovery of these biomolecules and their entry into the pharmaceutical market. It is now anticipated that the first VHH-based antibody drug, Caplacizumab, a bivalent anti-vWF antibody for treating rare blood clotting disorders, may be approved and commercialized in 2018 or shortly thereafter. This elusive first approval, along with the expiry of key patents, may substantially alter the scientific and biomedical landscape surrounding camelid sdAbs and pave the way for their emergence as mainstream biotherapeutics

Camelid Single-Domain Antibodies: Promises and Challenges as Lifesaving Treatments

Since the discovery of camelid heavy-chain antibodies in 1993, there has been tremendous excitement for these antibody domains (VHHs/sdAbs/nanobodies) as research tools, diagnostics, and therapeutics. Commercially, several patents were granted to pioneering research groups in Belgium and the Netherlands between 1996–2001. Ablynx was established in 2001 with the aim of exploring the therapeutic applications and development of nanobody drugs. Extensive efforts over two decades at Ablynx led to the first approved nanobody drug, caplacizumab (Cablivi) by the EMA and FDA (2018–2019) for the treatment of rare blood clotting disorders in adults with acquired thrombotic thrombocytopenic purpura (TPP). The relatively long development time between camelid sdAb discovery and their entry into the market reflects the novelty of the approach, together with intellectual property restrictions and freedom-to-operate issues. The approval of the first sdAb drug, together with the expiration of key patents, may open a new horizon for the emergence of camelid sdAbs as mainstream biotherapeutics in the years to come. It remains to be seen if nanobody-based drugs will be cheaper than traditional antibodies. In this review, I provide critical perspectives on camelid sdAbs and present the promises and challenges to their widespread adoption as diagnostic and therapeutic agents

Site-directed mutagenesis for improving biophysical properties of VH domains

Recombinant antibody fragments are significant therapeutic and diagnostic reagents. As such, their efficacy depends heavily on their affinities and biophysical properties. Thus, mutagenesis approaches have been extensively applied to recombinant antibodies to improve their affinity, stability, and solubility. Among the existing recombinant antibody variants, human VH domains stand out as the ones with the general need of solubility engineering at some point during their development; this solubility engineering step transforms VHs into nonaggregating, functional entities, rendering them useful as therapeutic and diagnostic reagents. Here, we present one of several approaches that have been employed to develop nonaggregating human VH domains. We apply an in vitro site-directed mutagenesis approach to an aggregating human VH domain by means of a splice overlap extension technique. The resultant mutant VHs are nonaggregating in contrast to the parent wild type VH and less prone to aggregation following thermal unfolding.Peer reviewed: YesNRC publication: Ye

Isolation of monoclonal antibody fragments from phage display libraries

Techniques developed over the past 20 years for the display of foreign peptides and proteins on the surfaces of filamentous bacteriophages have been a major driving force in the rapid development of recombinant antibody technology in recent years. With phage display of antibodies as one of its key components, recombinant antibody technology has led to the development of an increasing number of therapeutic monoclonal antibodies. Antibody gene libraries are fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting antibody libraries in fusion with the coat protein are propagated in Escherichia coli. Phage displaying monoclonal antibodies with specificities for target antigens are isolated from the libraries by a process called panning. The genes encoding the desired antibodies selected from the libraries are packaged within the phage particles, linking genotype and phenotype. Here, we describe the application of this technology to the construction of a phage-displayed single-domain antibody (sdAb) library based on the heavy chain antibody repertoire of a llama, the panning of the library against a peptide antigen and the expression, purification, and characterization of sdAbs isolated by panning.Peer reviewed: YesNRC publication: Ye

Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold

For the past 25 years, phage display technology has been an invaluable tool for studies of protein-protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage's potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage's large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.Peer reviewed: YesNRC publication: Ye

Isolation of camelid single-domain antibodies against native protins using recombinant multivalent peptide ligands

Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (i) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (ii) expression, purification and characterization of the multivalent peptide-VT ligands; (iii) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (iv) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield cusstom peptide-VT ligands that appear to maintain the antigenetic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.Peer reviewed: YesNRC publication: Ye