An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, natural antibodies isolated from convalescent patients are vulnerable to SARS-CoV-2 Spike mutations. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb dimer, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern or interest. Deep-mutational scanning experiments identify the potential binding epitopes of the monomeric Nb1 and Nb2 on the RBD and demonstrate that bivalent Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that Nb1-Nb2 broadly neutralizes SARS-CoV-2, including variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to the biparatopic Nb (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0*10E-12 M) and neutralizing activity (IC50 = 0.0017 nM). Together, we developed a biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

A Heterodimeric Antibody Fragment for Passive Immunotherapy Against Norovirus Infection

Human noroviruses cause an estimated 685 million infections and 200 000 deaths annually worldwide. Although vaccines against GII.4 and GI.1 genotypes are under development, no information is available regarding vaccines or monoclonal antibodies to other noroviral genotypes. Here, we developed 2 variable-domain llama heavy-chain antibody fragment (VHHs) clones, 7C6 and 1E4, against GII.4 and GII.17 human noroviruses, respectively. Although 7C6 cross-reacted with virus-like particles (VLPs) of GII.17, GII.6, GII.3, and GII.4, it neutralized only GII.4 norovirus. In contrast, 1E4 reacted with and neutralized only GII.17 VLPs. Both VHHs blocked VLP binding to human induced pluripotent stem cell-derived intestinal epithelial cells and carbohydrate attachment factors. Using these 2 VHHs, we produced a heterodimeric VHH fragment that neutralized both GII.4 and GII.17 noroviruses. Because VHH fragments are heat- and acid-stable recombinant monoclonal antibodies, the heterodimer likely will be useful for oral immunotherapy and prophylaxis against GII.4 and GII.17 noroviruses in young, elderly, or immunocompromised persons.

PCR-based approach for site-specific conjugation of long double-stranded DNA to a single-domain VHH antibody

Site-specific conjugation of double-stranded DNA using antibodies enables the development of unique applications for antibody–drug conjugates utilizing recent advances in nucleic acid medicines. Here, we describe a novel method to conjugate a camelid-derived single-domain VHH (variable domain of a heavy chain antibody) antibody with arbitrarily sized double-stranded DNA by PCR. Cysteine in anti-human epidermal growth factor receptor (EGFR) VHH was replaced by alanine, and an unpaired cysteine was introduced at the carboxyl terminus. These modifications enabled site-specific labelling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product—single-stranded DNA conjugated at the carboxyl terminus of VHH—retained its affinity for EGFR. To investigate whether this VHH–single-stranded DNA conjugate might be used as a forward primer, we subjected it to PCR, producing 100–500 bp DNA. We confirmed the amplification of the VHH–double-stranded DNA conjugate by examining its mobility on acrylamide gel; retention of the binding affinity of the conjugate for EGFR was identified by immuno-PCR.

The (Fab)ulous Destiny of Idarucizumab: Highlighting Its Interference with Urine Protein Immunofixation

Idarucizumab is a humanized antigen binding fragment (Fab) of a recombinant anti-dabigatran monoclonal antibody (IgG1-kappa) that allows rapid and sustained reversal of dabigatran-induced anticoagulation in case of bleeding or urgent surgery. Herein, we report a very unusual case of dabigatran reversal by idarucizumab in a 79-year-old woman with acute kidney failure admitted to a hospital in a context of hemoptysis. Three repeated injections were necessary because of massive dabigatran overdose and high rebounds of dabigatran plasma concentration. Idarucizumab was found on urine immunofixation up to 6 days after the last injection where it reacted with anti-kappa light chain antibody, but not with anti-gamma heavy chain antibody. Physicians should be aware of the increased half-life of idarucizumab in this context of acute kidney impairment and of its interference with urine immunofixation because it could lead to false-positive results and misdiagnosis of a paraprotein.