Biology subjects

Xiang, Y.

Publications and source records attributed to Xiang, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Super-immunity by broadly protective nanobodies to sarbecoviruses

Vaccine boosters and infection can facilitate the development of SARS-CoV-2 antibodies with improved potency and breadth. Here, we observed super-immunity in a camelid extensively immunized with the SARS-CoV-2 receptor-binding domain (RBD). We rapidly isolated a large repertoire of specific ultrahigh-affinity nanobodies that bind strongly to all known sarbecovirus clades using integrative proteomics. These pan-sarbecovirus nanobodies (psNbs) are highly effective against SARS-CoV and SARS-CoV-2 variants including the Omicron, with the best median neutralization potency at single-digit ng/ml. Structural determinations of 13 psNbs with the SARS-CoV-2 spike or RBD revealed five epitope classes, providing insights into the mechanisms and evolution of their broad activities. The highly evolved psNbs target small, flat, and flexible epitopes that contain over 75% of conserved RBD surface residues. Their potencies are strongly and negatively correlated with the distance of the epitopes to the receptor binding sites. A highly potent, inhalable and bispecific psNb (PiN-31) was developed. Our findings inform on the development of broadly protective vaccines and therapeutics. One sentence summarySuccessive immunization of SARS-CoV-2 RBD in a camelid enhanced the development of super-immunity and isolation and systematic characterization of a large repertoire of ultrahigh-affinity pan-sarbecovirus single-chain VHH antibodies to understand the evolution of this potent and broad immune response.

microbiology↗

bioRxiv2021

HybridDTA: Hybrid Data Fusion through Pairwise Training for Drug-Target Affinity Prediction

Candidate compounds with high binding affinities toward a target protein are likely to be developed as drugs. Deep neural networks (DNNs) have attracted increasing attention for drug-target affinity (DTA) estimation owning to their efficiency. However, the negative impact of batch effects caused by measure metrics, system technologies, and other assay information is seldom discussed when training a DNN model for DTA. Suffering from the data deviation caused by batch effects, the DNN models can only be trained on a small amount of "clean" data. Thus, it is challenging for them to provide precise and consistent estimations. We design a batch-sensitive training framework, namely BatchDTA, to train the DNN models. BatchDTA implicitly aligns multiple batches toward the same protein, alleviating the impact of the batch effects on the DNN models. Extensive experiments demonstrate that BatchDTA facilitates four mainstream DNN models to enhance the ability and robustness on multiple DTA datasets. The average concordance index (CI) of the DNN models achieves a relative improvement of 4.0%. BatchDTA can also be applied to the fused data collected from multiple sources to achieve further improvement.

bioinformatics↗

bioRxiv2021

Llamanade: an open-source computational pipeline for robust nanobody humanization

Nanobodies (Nbs) have recently emerged as a promising class of antibody fragments for biomedical and therapeutic applications. Despite having marked physicochemical properties, Nbs are derived from camelids and may require "humanization" to improve translational potentials for clinical trials. Here we have systematically analyzed the sequence and structural properties of Nbs based on NGS (next-generation sequencing) databases and high-resolution structures. Our analysis reveals substantial framework diversities and underscores the key differences between Nbs and human Immunoglobulin G (IgG) antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen-binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade, a user-friendly, open-source to facilitate rational humanization of Nbs. Using Nb sequence as input, Llamanade provides information on the sequence features, model structures, and optimizes solutions to humanize Nbs. The full analysis for a given Nb takes less than a minute on a local computer. To demonstrate the robustness of this tool, we applied it to successfully humanize a cohort of structurally diverse and highly potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a web server to support the development of a rapidly expanding repertoire of therapeutic Nbs into safe and effective trials. Author SummaryCamelid Nbs are characterized by small size, excellent pharmacological properties and high flexibility in bioengineering for therapeutic development. However, Nbs are "xeno" antibodies, which require "humanization" to improve their translational potential. Currently, there is a lack of systematic investigation of Nbs to rationally guide humanization. No dedicated software has been developed for this purpose. Here, we report the development of Llamanade, an open-source computational pipeline and the first dedicated software to facilitate rational humanization of Nbs. To subjectively evaluate Llamanade, we used it to humanize a cohort of structurally diverse and ultrapotent antiviral Nbs against SARS-CoV-2. Robust humanization by Llamanade significantly improved the humanness level of Nbs to closely resemble fully human IgGs. Importantly, these highly humanized antiviral Nbs remained excellent solubility and comparably high bioactivities to the non-humanized Nb precursors. We envision that Llamanade will help advance Nb research into therapeutic development.

bioinformatics↗

bioRxiv2021

A concurrent canonical and modified miRNAome pan-cancer study on TCGA and TARGET cohorts leads to an enhanced resolution in cancer

MiRNA Epitranscriptomics has placed a new layer of complexity in the cancer field. Despite the fast-growing interest in miRNA editing and shifted miRNA isoforms, a simultaneous study of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modifications, including A-to-I RNA editing and shifted miRNA isoforms, in >13K adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments datasets. We investigated the differences among canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint. The combination of canonical miRNAs/miRNA isoforms boosted the quality of clustering results, outlining unique cohorts clinical-pathological features. We described modified miRNAs showing opposite dysregulation with respect to their canonical counterparts in cancer, potentially impacting their targetome and function. The abundance of expressed miRNA isoforms directly impacted the activation/deactivation of critical carcinogenesis pathways. Finally, we experimentally validated unique targeting for a shifted and edited miRNA isoform. Our findings outlined once more the importance of going beyond the well-established paradigm of one-mature-miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression.

cancer biology↗

bioRxiv2021

Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity

Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error-prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampicin tolerance. However, the mechanisms by which these clinically-derived mutations in gatA impact GatCAB function was unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains, but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.

molecular biology↗

bioRxiv2021

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 {micro}M. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 {micro}M, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 {micro}M). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

pharmacology and toxicology↗

bioRxiv2021

Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes

There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.

biophysics↗

bioRxiv2021

Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses

Globally there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals weight loss post-infection and decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology and prevents viral pneumonia. Combined with the marked stability and low production cost, this novel therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.

microbiology↗

bioRxiv2021

Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

immunology↗

bioRxiv2021

Robust RNA editing via recruitment of endogenous ADARs using circular guide RNAs

Akin to short-hairpin RNAs and antisense oligonucleotides which efficaciously recruit endogenous cellular machinery such as Argonaute and RNase H to enable targeted RNA knockdown, simple long antisense guide RNAs (1) can recruit endogenous adenosine deaminases acting on RNA (ADARs) to enable programmable A-to-I RNA editing, without requiring co-delivery of any exogenous proteins. This approach is highly specific, however the efficiency is typically lower than observed with enzyme overexpression. Conjecturing this was due in part to the short half-life and residence times of guide RNAs, here we engineer highly stable circular ADAR recruiting guide RNAs (cadRNAs), which can be delivered not only by genetically encoding on DNA vectors, but also via transfection of RNA molecules transcribed in vitro. Using these cadRNAs, we observed robust RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, vastly improved efficiency and durability of RNA editing, and high transcriptome-wide specificity. High transcript-level specificity was achieved by further engineering to reduce bystander editing. Additionally, in vivo delivery of cadRNAs via adeno-associated viruses (AAVs) enabled robust 38% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Taken together, cadRNAs enable efficacious programmable RNA editing with application across diverse protein modulation and gene therapeutic settings.

bioengineering↗

bioRxiv2020

An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 M and EC50 of 1.27 M, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the -methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

pharmacology and toxicology↗

bioRxiv2020

Scalable production of tissue-like vascularised liver organoids from human PSCs.

A lack of physiological parity between 2D cell culture and in vivo, has paved the way towards more organotypic models. Organoids exist for a number of tissues, including the liver. However, current approaches to generate hepatic organoids suffer drawbacks, including a reliance on extracellular matrices (ECM), the requirement to pattern in 2D culture, costly growth factors and a lack of cellular diversity, structure and organisation. Current hepatic organoid models are generally simplistic, composed of hepatocytes or cholangiocytes, which renders them less physiologically relevant when compared to native tissue. Here we aim to address these drawbacks. To address this, we have developed an approach that does not require 2D patterning, is ECM independent combined with small molecules to mimic embryonic liver development that produces massive quantities of liver like organoids. Using single-cell RNA sequencing and immunofluorescence we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, innervation and a population of resident macrophage - the Kupffer cells. The organoids exhibit key liver functions including drug metabolism, serum protein production, coagulation factor production, bilirubin uptake and urea synthesis. The organoids can be transplanted and maintained in mice producing human albumin long term. The organoids exhibit a complex cellular repertoire reflective of the organ, have de novo vascularization and innervation, enhanced function and maturity. This is a pre-requisite for a myriad of applications from cellular therapy, tissue engineering, drug toxicity assessment, disease modeling, to basic developmental biology.

cell biology↗

bioRxiv2020

STAT3 inhibits Myocardin induced cardiac hypertrophy

BackgroundIn order to explore the molecular mechanism of cardiomyocyte-dependent myocardial gene expression and cardiomyocyte differentiation in cardiac hypertrophy, and to provide new insights for cardiac hypertrophy. MethodsCardiac myocytes were isolated from day 1-3 Sprague-Dawley rat pups. Real time quantitative PCR, western blot and immunocytochemistry Assay were used to detect the expression and localization of related genes. CO-IP was used to detect direct protein interactions between Myocardin and STAT3. Luciferase reporter assay and chromatin immunoprecipitation were used to detect the binding of Myocardin to the promoter of a downstream target gene. Microinjection of zebrafish embryos was used to examine the effects of STAT3 and Myocardin interactions on cardiac development in vivo ResultsThe N-terminus of STAT3 directly binds to the basic domain of myocardin and inhibits the transcriptional activity of Myocardin-mediated cardiac-specific genes ANF and -actinin, thereby inhibiting their expression, and further inhibit myocardin-mediated cardiac hypertrophy in vivo. ConclusionsIn summary, our report states that signal transduction and transcriptional activation factor 3 (STAT3) are inhibitors of the major cardiac hypertrophic transcription factor Myocardiin, which is required for cardiomyocyte differentiation. The STAT3-cardiacin interaction identified nuclear hormone receptor-mediated and cardiac-specific gene-regulated convergence sites and suggested a possible mechanism for cardioprotective effects.

cell biology↗

bioRxiv2020

HiCAR: a robust and sensitive multi-omic co-assay for simultaneous measurement of transcriptome, chromatin accessibility, and cis-regulatory chromatin contacts

The long-range interactions of cis-regulatory elements (cREs) play a central role in regulating the spatial-temporal gene expression program of multi-cellular organism. cREs are characterized by the presence of accessible (or open) chromatin, which can be identified at genome-wide scale with assays such as ATAC-seq, DHS-seq, and FAIRE-seq. However, it remains technically challenging to comprehensively identify the long-range physical interactions that occur between cREs, especially in a cost effective manner using low-input samples. Here, we report HiCAR (High-throughput Chromosome conformation capture on Accessible DNA with mRNA-seq co-assay), a method that enables simultaneous assessment of cis-regulatory chromatin interactions and chromatin accessibility, as well as evaluation of the transcriptome, which represents the functional output of chromatin structure and accessibility. Unlike immunoprecipitation-based methods such as HiChIP, PLAC-seq, and ChIA-PET, HiCAR does not require target-specific antibodies and thus can comprehensively capture the cis-regulatory chromatin contacts anchored at accessible regulatory DNA regions and associated with diverse epigenetic modifications and transcription factor binding. Compared to Trac-looping, another method designed to capture interactions between accessible chromatin regions, HiCAR produced a 17-fold greater yield of informative long-range cis- reads at a similar sequencing depth and required 1,000-fold fewer cells as input. Applying HiCAR to H1 human embryonic stem cells (hESCs) revealed 46,792 cis-regulatory chromatin interactions at 5kb resolution. Interestingly, we found that epigenetically poised, bivalent, and repressed cREs exhibit comparable spatial interaction activity to those transcriptionally activated cREs. Using machine learning approaches, we predicated 22 epigenome features that are potentially important for the spatial interaction activity of cREs in H1 hESC. Lastly, we also identified long-range cis-regulatory chromatin interactions in GM12878 and mouse embryonic stem cells with HiCAR. Our results demonstrate that HiCAR is a robust and cost-effective multi-omics assay, which is broadly applicable for simultaneous analysis of genome architecture, chromatin accessibility, and the transcriptome using low-input samples.

genomics↗

bioRxiv2020

Regulation of cGAS activity through RNA-mediated phase separation

Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA (dsDNA) sensor that functions in the innate immune system. Upon binding dsDNA in the cytoplasm, cGAS and dsDNA form phase-separated aggregates in which cGAS catalyzes synthesis of 23-cyclic GMP-AMP that subsequently triggers a STING-dependent, type I IFN response. Here, we showed that cytoplasmic RNAs, especially tRNAs, regulate cGAS activity. We discovered that RNAs did not activate cGAS but rather promoted phase separation in vitro. In cells, cGAS colocalized with RNAs and formed phase-separated granules even in the absence of cytoplasmic dsDNA. An Opti-prep gradient analysis of cell lysates showed that the endogenous cGAS was associated with cytoplasmic RNAs in an aggregative form. Further in vitro assays showed that RNAs compete for binding of cGAS with dsDNA and inhibit cGAS activity when the dsDNA concentration is high and promote the formation of phase separations and enhance cGAS activity when the dsDNA concentration is low. Thus, cytoplasmic RNAs regulate cGAS activity by interfering with formation of cGAS-containing aggregates.

biochemistry↗

bioRxiv2020

Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5 guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5-GA-3 motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

synthetic biology↗

bioRxiv2020

Integrative proteomics reveals exceptional diversity and versatility of mammalian humoral immunity

The humoral immune response is essential for the survival of mammals. However, we still lack a systematic understanding of the specific serologic antibody repertoire in response to an antigen. We developed a proteomic strategy to survey, at an unprecedented scale, the landscapes of antigen-engaged, serum-circulating repertoires of camelid heavy-chain antibodies (hcAbs). The sensitivity and robustness of this technology were validated using three antigens spanning orders of magnitude in immune response; thousands of divergent, high-affinity hcAb families were confidently identified and quantified. Using high-throughput structural modeling, cross-linking mass spectrometry, mutagenesis, and deep learning, we mapped and analyzed the epitopes of > 100,000 antigen-antibody complexes. Our results revealed a surprising diversity of high-affinity hcAbs for specific antigen binding on a variety of dominant epitopes. hcAbs perfect both shape and charge complementarity to target challenging antigens specifically; they can rapidly evolve to recognize a conserved, promiscuous cellular protein interaction interface, unraveling the convergent force that drives protein-protein interactions.

bioengineering↗

bioRxiv2020

A robust and versatile nanobody platform for drug delivery

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by the short half-lives. Here we report the development of a repertoire of diverse, high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. By integrating biophysics, and hybrid structural biology, we have systematically characterized the NbHSA for albumin binding, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate, multiplex Nb pharmacokinetic analysis. Using a humanized albumin mouse model, we found that the NbHSA has outstanding pharmacokinetics; the most stable NbHSA has a 771-fold T1/2 improvement compared with a control Nb. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed a highly stable NbHSA-hIL-2 cytokine conjugate "Duraleukin" and confirmed its improved anticancer properties than hIL-2 alone. We envision that this high-quality Nb resource will advance research into novel biotherapeutics.

bioengineering↗