The elimination half-lives range from 1.6?days (Nb100) to 7.6?days (Nb75), which correspond to approximately (S)-(-)-5-Fluorowillardiine 771-fold improvement compared to the control Nb (Figures 4B and 4C; Data S2). (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected (S)-(-)-5-Fluorowillardiine NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs Duraleukin and demonstrated Duraleukin’s high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs. half-life make HSA an excellent (S)-(-)-5-Fluorowillardiine trojan horse for drug delivery. Different albumin-based approaches have been developed including direct fusion to HSA and development of albumin nanoparticles as well as albumin-binding domains (ABDs) derived from gram-positive bacteria to improve the pharmacokinetics of target therapeutics (Larsen et?al., 2016; Merlot et?al., 2014; Sleep et?al., 2013). Despite these advances, however, successful preclinical and clinical applications of these technologies remain limited (Kontermann, 2016). Development of robust, highly specific and cost-effective new HSA binders, ideally with inherent low immunogenicity, high bioavailability, and versatility in modulating pharmacokinetic properties based on specific medical needs remains highly desirable. Nbs are robust and small antigen-binding fragments (15?kDa) that (S)-(-)-5-Fluorowillardiine are derived from camelid’s heavy-chain-only antibodies (HcAbs) (Hamers-Casterman et?al., 1993). They are characterized by marked physiochemical properties including high solubility and stability (Xiang et?al., 2020a, 2021). Nbs can be rapidly produced in microbes such as and yeast cells at low costs. Because of the small size and robustness, Nbs have fast tissue distributions and high bioavailability for therapeutic and diagnostic applications (Jov?evska and Muyldermans, 2020; Kijanka et?al., 2015). Monomeric Nbs can be easily bioengineered into multivalent forms to improve binding affinities and bioactivities (Fridy et?al., 2014; Harmsen and De Haard, 2007; Xiang et?al., 2020a, 2021). Importantly, camelid Nbs have high sequence and structural similarity with human IgG heavy chains (Kijanka et?al., 2015; Vincke et?al., 2009). To date, over 35 clinical trials have been conducted on more than 1,000 patients and healthy volunteers, revealing high biosafety of Nbs in humans. In addition, the first Nb drug (caplacizumab) has recently been approved by the FDA (Scully et?al., 2019). Stable Nbs are flexible for administration including direct inhalation therapy by Nb aerosols, making their use against respiratory viruses very appealing (Cunningham et?al., 2021; Larios Mora et?al., 2018; Liang et al., 2020; Nambulli et?al., 2021; Patton and Byron, 2007; Van Heeke et?al., 2017). Recently, we have developed a robust proteomic technology for large-scale identification and affinity classification of drug-quality Nbs (cells and have excellent stability and bioactivity. EPLG6 Using a melanoma mouse model, we demonstrated the high preclinical efficacy of Duraleukin for cancer treatment. This versatile and well-characterized Nb resource is shared with the research community to help advance biomedical discoveries into therapeutic development. Results Characterization of a repertoire of high-quality NbHSA We hypothesize that the half-lives of NbHSAs are related to their biophysical (such as binding kinetics, solubility, stability, etc.) and structural properties (Ovacik and Lin, 2018). To maximize these properties, we included a collection of 90 NbHSAs with highly diverse complementarity-determining region (CDR) 3 sequences, which form hypervariable loop structures (fingerprints) for specific antigen binding. As shown in Figure?1B, the CDR3s of these highly selected Nbs vary substantially in amino acid composition, sequence length, isoelectric point (pI), and hydrophobicity. To produce recombinant Nbs, their amino acid.
The elimination half-lives range from 1