Single‐domain antibodies (sdAbs), the autonomous variable domains of camelid and shark heavy‐chain antibodies, have many desirable properties as components of biologic drugs. However, their sequences may increase the risk of immunogenicity and antidrug antibody (ADA) development in humans, and thus, sdAbs are routinely humanized during development. Here, we review and summarize the available evidence regarding the factors governing immunogenicity of sdAbs and our current state of knowledge of strategies to mitigate immunogenicity risks by humanization. While several sdAb properties, including high homology of camelid V_HHs with human IGHV3 gene products, favor low immunogenicity in humans, epitopes absent in the human repertoire including the exposed V_H:V_L interface may be intrinsically immunogenic. While most clinical trials have demonstrated minimal sdAb immunogenicity, two notable exceptions (the tetrameric DR5‐specific V_HH TAS266 and the TNFR1‐specific V_H GSK1995057) illustrate that special caution must be taken in identifying preexisting ADAs against highly potent sdAbs. Nonhuman sequence alone does not adequately explain sdAb immunogenicity, as some camelid V_HHs are nonimmunogenic while some fully human V_Hs elicit ADAs. The presence of preexisting ADAs directed against the exposed C‐termini of some sdAbs in a significant proportion of individuals awaits a molecular explanation. Whether sdAb humanization reduces or promotes immunogenicity remains unclear: reduction of nonhuman sequence content at the expense of introducing low‐level aggregation in humanized variants may be counterproductive. Further work will establish thresholds for V_HH and V_NAR humanization to maximize human sequence content while avoiding loss of binding affinity and/or immunogenicity resulting from aggregation or decreased stability.

Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional V_H:V_L antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.

For the past twenty-five 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 various spheres impose obstacles to its widespread adoption and use.

Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies (sdAbs or V_HHs) directed against human EGFR. The source of these V_HHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The V_HHs targeted multiple sites on the receptor's surface with high affinity (K_D range: 1–40 nM), including one epitope overlapping that of cetuximab, several epitopes conserved in the cynomolgus EGFR orthologue, and at least one epitope conserved in the mouse EGFR orthologue. Interestingly, despite their generation against human EGFR expressed from cDNA by llama cells in vivo (presumably in native conformation), the V_HHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc, one of the V_HH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC₅₀: ∼30 nM). Thus, DNA immunization elicited high-affinity, functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.