INDI-integrated nanobody database for immunoinformatics.

Nanobodies, a subclass of antibodies found in camelids, are versatile molecular binding scaffolds composed of a single polypeptide chain. The small size of nanobodies bestows multiple therapeutic advantages (stability, tumor penetration) with the first therapeutic approval in 2018 cementing the clinical viability of this format. Structured data and sequence information of nanobodies will enable the accelerated clinical development of nanobody-based therapeutics. Though the nanobody sequence and structure data are deposited in the public domain at an accelerating pace, the heterogeneity of sources and lack of standardization hampers reliable harvesting of nanobody information. We address this issue by creating the Integrated Database of Nanobodies for Immunoinformatics (INDI, http://naturalantibody.com/nanobodies). INDI collates nanobodies from all the major public outlets of biological sequences: patents, GenBank, next-generation sequencing repositories, structures and scientific publications. We equip INDI with powerful nanobody-specific sequence and text search facilitating access to >11 million nanobody sequences. INDI should facilitate development of novel nanobody-specific computational protocols helping to deliver on the therapeutic promise of this drug format.

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

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.

Single domain antibody multimers confer protection against rabies infection.

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90-95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.

Overview and discovery of IgNARs and generation of VNARs.

Immunoglobulin new antigen receptors (IgNARs) from sharks are a distinct class of immune receptors, consisting of homodimers with no associated light chains. Antigen binding is encapsulated within single VNAR immunoglobulin domains of 13-14 kDa in size. This small size and single domain format means that they exhibit considerable stability and are readily produced in heterologous protein expression systems. In this chapter, I describe the history and discovery of IgNARs, the development of VNAR biotechnology, and highlight important factors in VNAR protein production.