The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood–brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.

Prolonged serum half‐life is required for the efficacy of most protein therapeutics. One strategy for half‐life extension is to exploit the long circulating half‐life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single‐domain antibodies (V _H Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half‐lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional V _H H (M75) that is naturally pH‐sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton‐linked binding of M75 to HSA, with differing binding enthalpies observed for full‐length HSA and an HSA domain 1‐domain 2 fusion protein in which surface‐exposed His residues were substituted with Ala. M75 conferred moderate half‐life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn‐mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized V _H Hs maintained in vivo half‐life extension capabilities. These V _H Hs represent a new set of tools for extending protein therapeutic half‐life and one (M75) demonstrates a unique pH‐sensitive binding interaction that can be exploited to achieve modest in vivo half‐life.

Antibodies that cross-react with multiple isoforms or homologue of a given protein are often desirable, especially in therapeutic applications. Here, we report the identification of several unique, clonally unrelated, single-domain antibodies (sdAbs) that bind to multiple serum albumin orthologues (human, rhesus, rat and mouse) with low-to-medium nanomolar affinity from a llama immunized only with human serum albumin. Using single-round panning of a phage-displayed sdAb library against serum albumins and next-generation DNA sequencing, we were able to predict patterns of sdAb reactivity to the albumins of different species with ∼90% accuracy. We expect this strategy to be generally applicable for identifying cross-reactive sdAbs, particularly when these exist at low frequency and/or are poorly enriched by panning. Moreover, the sdAbs identified here are of potential interest for serum half-life extension of biologics.

Therapeutic agents targeting bacterial virulence factors are gaining interest as non-antibiotic alternatives for the treatment of infectious diseases. Clostridium difficile is a Gram-positive pathogen that produces two primary virulence factors, enterotoxins A and B (TcdA and TcdB), which are responsible for Clostridium difficile-associated disease (CDAD) and are targets for CDAD therapy. Antibodies specific for TcdA and TcdB have been shown to effectively treat CDAD and prevent disease relapse in animal models and in humans. This review summarizes the various toxin-specific antibody formats and strategies under development, and discusses future directions for CDAD immunotherapy, including the use of engineered antibody fragments with robust biophysical properties for systemic and oral delivery.

An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C. difficile immunotherapeutics, we previously isolated several single-domain antibodies (V_HHs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing V_HHs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 V_HHs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as K_D = 70 pM, none of the V_HHs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with V_HH-V_HH dimers and to a much greater extent with V_HH-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several V_HH-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other V_HH-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the V_HHs described here are effective at toxin B neutralization when formatted as bivalent V_HH-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site.

Approximately one fifth of all malignancies worldwide are etiologically associated with a persistent viral or bacterial infection. Thus, there is a particular interest in therapeutic molecules which use components of a natural immune response to specifically inhibit oncogenic microbial proteins, as it is anticipated they will elicit fewer off-target effects than conventional treatments. This concept has been explored in the context of human papillomavirus 16 (HPV16)-related cancers, through the development of monoclonal antibodies and fragments thereof against the viral E6 oncoprotein. Challenges related to the biology of E6 as well as the functional properties of the antibodies themselves appear to have precluded their clinical translation. Here, we addressed these issues by exploring the utility of the variable domains of camelid heavy-chain-only antibodies (denoted as VHHs). Through construction and panning of two llama, immune VHH phage display libraries, a pool of potential VHHs was isolated. The interactions of these with recombinant E6 were further characterized using an enzyme-linked immunosorbent assay (ELISA), Western blotting under denaturing and native conditions, and surface plasmon resonance. Three VHHs were identified that bound recombinant E6 with nanomolar affinities. Our results lead the way for subsequent studies into the ability of these novel molecules to inhibit HPV16-infected cells in vitro and in vivo.