Kevin A. Henry ^1* Greg Hussack ¹ Jan Gettemans ² Cory L Brooks ³

• ¹ National Research Council Canada (NRC), Ottawa, Canada
• ² Ghent University, Ghent, East Flanders, Belgium
• ³ California State University, Fresno, Fresno, California, United States

Since the first volume of this Research Topic was published in 2017 (1), the single-domain antibody (sdAb) field has evolved dramatically. The first sdAb-based drug, the anti-von Willebrand factor caplacizumab (Cablivi), was approved for the treatment of acquired thrombotic thrombocytopenic purpura by the EMA and FDA in 2018 and 2019, respectively (2). The emergence of SARS-CoV-2 and response to the resulting COVID-19 pandemic firmly established the antiviral neutralization potency of sdAbs, especially well-designed multiparatopic molecules (3). The fields of cell therapy and chimeric antigen receptor (CAR) design have exploded, leading to six FDAapproved CAR-T cell therapies including ciltacabtagene autoleucel (Carvykti), a BCMAtargeted tandem sdAb-based product for the treatment of relapsed or refractory multiple myeloma (4). Recent approvals of ozoralizumab (Nanozora) in Japan (5), a trimeric sdAb targeting TNF and serum albumin for the treatment of rheumatoid arthritis, and envafolimab in China (6), a PD-L1-specific sdAb fused to IgG1 Fc for various advanced solid tumours, highlight growing momentum in the field. Clearly, sdAbs are no mere biological curiosities or niche research objects but an entirely distinct class of binding molecules that are now coming into their own. Some of the themes of the first volume also extend to the second. The advantages of sdAbs over conventional antibodies and their fragments in a variety of applications are clearly illustrated in the 12 original research articles and 2 reviews of this collection, which together provide a snapshot of trends and recent developments in the field. In particular, many of the articles in the second volume investigated uses of sdAbs for non-invasive imaging and as diagnostics, often to detect SARS-CoV-2. One original research article addressed the fundamental properties of sdAbs. In the largest study of this type conducted to date, Gordon et al. (7) compared the structures of 345 sdAb:antigen complexes and 892 conventional antibody:antigen complexes with the goal of understanding potentially distinct mechanisms of antigen recognition by sdAbs. In agreement with prior studies, the results of this analysis show that the paratopes of sdAbs are smaller than those of conventional antibodies; however, neither differences in paratope amino acid composition nor differences in the size (defined as the number of residues), amino acid composition or accessibility of epitopes targeted by sdAbs were evident. The explanation for this apparent contradiction is that within smaller sdAb paratopes, a longer complementarity-determining region 3 (CDR3) loop contributes a greater number of interactions per residue and framework residues are more likely to play a role in binding. One original research article investigated a new approach for camelid sdAb discovery. While many groups have integrated high-throughput sequencing of antibody repertoires into existing discovery pipelines in which antigen reactivity of individual clones is evaluated in vitro, Matsuda et al. ( 8) developed a predictive algorithm to identify antigen-specific sdAbs without in vitro screening by longitudinal sequencing and phylogenetic analysis of the peripheral repertoire. The basis for identifying antigenspecific sdAbs is the accumulation of somatic hypermutations and high turnover rates within clonal families during the process of affinity maturation. While preliminary characterization of antigen-specific sdAbs recovered using this strategy showed variable binding data across assays, and concurrent immune responses mounted against non-immunizing antigens including pathogens would be expected to confound predictions, the encouraging overall results indicate it may one day be possible to accurately identify antigen-specific sdAbs following immunization via sequencing of the peripheral blood repertoire.Two original research articles examined the ability of sdAbs, or even smaller antibody-derived fragments, to extend the serum persistence of biologics via binding to serum albumin. Harmsen et al. ( 9) isolated and characterized sdAbs from the repertoire of a llama immunized with dog and horse serum albumin. Unlike previous efforts in this regard, the sdAbs bound the albumins of various animal species including horse, dog, cat and swine -but did not recognize those of human or mouse -and extended the half-life of a tetanus toxin-specific sdAb in pigs and horses. These sdAbs would be useful for therapeutic studies of molecules with intrinsic short half-lives in these animals. One original research article tackled the challenging problem of developing antibodies that are able to specifically recognize particular conformational states of proteins. Zupancic et al. (20) identified llama sdAbs from yeast-displayed libraries using MACS-and FACS-based selection that preferentially recognize aggregated (fibrillar) tau over soluble monomeric tau. These sdAbs were able to recognize tau aggregates in brain samples from transgenic mice as well as patients with tauopathies, and may have diagnostic or therapeutic applications in neurodegenerative diseases. Regulatory approval of four sdAb-based drugs (three biologics and one CAR-T cell) has substantially altered perceptions and attitudes towards these molecules in the medical and scientific communities. With mainstream acceptance has come increased visibility and interest. However, efforts and investment continue to center on discovery and biotechnological applications of sdAbs, and much work still remains to understand the basic immunobiology of these unique molecules as well as how to generate, engineer, characterize and manufacture them most effectively.The editors would again like to thank all contributors for the many excellent submissions to this Research Topic, as well as the reviewers and the Frontiers in Immunology editorial office.