Cost-effective, efficacious therapeutics are urgently needed against the COVID-19 pandemic. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration and determined a structure of one of the most potent in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. We constructed multivalent Nb constructs that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization.

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The outbreak of COVID-19 has severely impacted global health and economy. Cost-effective therapeutics are urgently needed. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike protein receptor-binding domain (RBD). We discovered multiple elite Nbs of picomolar to femtomolar affinities that inhibit viral infection at as low as sub-ng/ml concentration, more potent than some of the best human neutralizing antibodies. We then determined a crystal structure of such an elite neutralizing Nb in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. Structural characterization facilitated the bioengineering of novel multivalent Nb constructs into multi-epitope cocktails that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. Our Nbs can be rapidly produced in bulk from microbes and resist heat, lyophilization, and aerosolization. These highly promising agents are readily translated into efficient, economic, and convenient therapeutics to help end this once-in-a-century health crisis.

Current single-cell RNA-seq approaches are hindered by preamplification bias, loss of strand of origin information, and the inability to observe small-RNA and mRNA dual transcriptomes. Here, we introduce a single-cell holo-transcriptome sequencing (Holo-Seq) that overcomes all three hurdles. Holo-Seq has the same quantitative accuracy and uniform coverage with a complete strand of origin information as bulk RNA-seq. Most importantly, Holo-Seq can simultaneously observe small RNAs and mRNAs in a single cell. Furthermore, we acquire small RNA and mRNA dual transcriptomes of 32 human hepatocellular carcinoma single cells, which display the genome-wide super-enhancer activity and hepatic neoplasm kinetics of these cells.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1553-7) contains supplementary material, which is available to authorized users.

Summary Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (Nb HSA ) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of Nb HSA s, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected Nb HSA s in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of Nb HSA s were drastically prolonged by 771-fold. Nb HSA s have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive Nb HSA -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.