Beyond Natural Immune Repertoires: Synthetic Antibodies.

Synthetic antibody libraries, in which the antigen-binding sites are precisely designed, offer unparalleled precision in antibody engineering, exceeding the potential of natural immune repertoires and constituting a novel generation of research tools and therapeutics. Recent advances in artificial intelligence-driven technologies and their integration into synthetic antibody discovery campaigns hold the promise to further streamline and effectively develop antibodies. Here, we provide an overview of synthetic antibodies.

Generation and Selection of Synthetic Human Antibody Libraries via Phage Display.

Synthetic antibody libraries enable the development of antibodies that can recognize virtually any antigen, with affinity and specificity profiles that are superior to those of natural antibodies. By using highly stable and optimized frameworks, synthetic antibody libraries can be rapidly generated by precisely designing synthetic DNA, allowing absolute control over the position and chemical diversity introduced while expanding the sequence space for antigen recognition. Here, we describe a detailed protocol for the generation of highly diverse synthetic antibody phage display libraries based on a single framework, with diversity genetically incorporated by using finely designed mutagenic oligonucleotides.

Activity-based profiling of cullin-RING ligase networks by conformation-specific probes.

The cullin-RING E3 ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle.

High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression.

Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells.

Antigenic mapping reveals sites of vulnerability on α-HCoV spike protein.

Understanding the antigenic signatures of all human coronaviruses (HCoVs) Spike (S) proteins is imperative for pan-HCoV epitopes identification and broadly effective vaccine development. To depict the currently elusive antigenic signatures of α-HCoVs S proteins, we isolated a panel of antibodies against the HCoV-229E S protein and characterized their epitopes and neutralizing potential. We found that the N-terminal domain of HCoV-229E S protein is antigenically dominant wherein an antigenic supersite is present and appears conserved in HCoV-NL63, which holds potential to serve as a pan-α-HCoVs epitope.