A pipeline for high-throughput discovery of drug-quality nanobodies

We have recently 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. In collaboration with Dina Schneidman's group, we have made some really interesting discoveries about how mammalian circulating antibodies recognize an antigen. Currently, there are several directions that we are taking to move this technology forward. For example, we are interested in developing novel AI-based algorithms to facilitate nanobody discovery by proteomics. We are also exploring ways to humanize nanobodies. Finally, we are developing tools to enable high-throughput screening and production of therapeutic biomolecules including but not limited to nanobodies.

Ultrapotent and multi-epitope neutralizing nanobodies against SARS-CoV-2 and its variants of concern (VOCs)

Cost-effective, efficacious therapeutics are urgently needed to combat the COVID-19 pandemic. In this study, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (RBD). We discovered Nbs with picomolar to femtomolar affinities that inhibit viral infection at concentrations below the nanograms-per-milliliter level, and we determined a structure of one of the most potent Nbs in complex with the RBD. Structural proteomics and integrative modeling revealed multiple distinct and nonoverlapping epitopes and indicated an array of potential neutralization mechanisms. We bioengineered multivalent Nb constructs that achieved ultrahigh neutralization potency (half-maximal inhibitory concentration 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.

Pittsburgh inhalable Nanobody-21 (PiN-21): novel inhalation therapy for COVID-19

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 after infection, 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 innovative therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.

How do potent neutralizing nanobodies resist the circulating variants of SARS-CoV-2?

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 8 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.

A robust toolbox for tailored drug delivery and the story behind Duraleukin 

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by poor pharmacokinetics. Here we report the development of a repertoire of robust, high-affinity and multi-epitope nanobodies (Nbs) that target human serum albumin (NbHSA) to enable half-life extension of small therapeutic cargos. We developed a quantitative proteomic assay that facilitates specific and paralleled pharmacokinetic analysis of Nbs and determined the half-lives of a cohort of representative NbHSA in an Alb-/- FcRn-humanized mouse model. Interestingly, we found that NbHSAs span a wide range of pharmacokinetic profiles. The half-lives of NbHSAs  are positively correlated with their binding affinity to albumin at the endosomal pH. Compared to control Nbs that were rapidly cleared from the serum, the half-lives of NbHSAs  were drastically prolonged by up to 771-fold. To demonstrate the utility of NbHSAs, we developed stable NbHSA-cytokine fusion constructs “Duraleukin” and confirmed the excellent preclinical efficacy of a Duraluekin for cancer treatment in a mouse melanoma model. This high-quality and versatile Nb resource will help advance biomedical research into therapeutic development. 

AI-based computational tools for drug disocvery (coming soon)

To be updated...

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

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.

Development of novel therapeutics for human diseases

To be updated