Using phage display for rational engineering of a higher affinity humanized 3' phosphohistidine-specific antibody.

Histidine phosphorylation (pHis) is a non-canonical post-translational modification (PTM) that is historically understudied due to a lack of robust reagents that are required for its investigation, such as high affinity pHis-specific antibodies. Engineering pHis-specific antibodies is very challenging due to the labile nature of the phosphoramidate (P-N) bond and the stringent requirements for selective recognition of the two isoforms, 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis). Here, we present a strategy for engineering of antibodies for detection of native 3-pHis targets.

Monoclonal antibodies that block Roundabout 1 and 2 signaling target pathological ocular neovascularization through myeloid cells.

Roundabout (ROBO) 1 and 2 are transmembrane receptors that bind secreted SLIT ligands through their extracellular domains (ECDs) and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. SLIT-ROBO signaling regulates pathological ocular neovascularization, which is a major cause of vision loss worldwide, but pharmacological tools to prevent SLIT-ROBO signaling are lacking. Here, we developed human monoclonal antibodies (mAbs) against the ROBO1 and ROBO2 ECDs.

Structural and biophysical characterisation of ubiquitin variants that inhibit the ubiquitin conjugating enzyme Ube2d2.

The ubiquitin-conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases, and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function relies on interactions with partner proteins, and the disruption of these is an effective way to modulate activity.

Antigen-binding fragments with improved crystal lattice packing and enhanced conformational flexibility at the elbow region as crystallization chaperones.

It has been shown previously that a set of three modifications-termed S1, Crystal Kappa, and elbow-act synergistically to improve the crystallizability of an antigen-binding fragment (Fab) framework. Here, we prepared a phage-displayed library and performed crystallization screenings to identify additional substitutions-located near the heavy-chain elbow region-which cooperate with the S1, Crystal Kappa, and elbow modifications to increase expression and improve crystallizability of the Fab framework even further. One substitution (K141Q) supports the signature Crystal Kappa-mediated Fab:Fab crystal lattice packing interaction.

Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling.

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception.

Structural Survey of Antigen Recognition by Synthetic Human Antibodies.

Synthetic antibody libraries have been used extensively to isolate and optimize antibodies. To generate these libraries, the immunological diversity and the antibody framework(s) that supports it outside of the binding regions are carefully designed/chosen to ensure favorable functional and biophysical properties. In particular, minimalist, single-framework synthetic libraries pioneered by our group have yielded a vast trove of antibodies to a broad array of antigens.

Exploiting spatiotemporal regulation of FZD5 during neural patterning for efficient ventral midbrain specification.

The Wnt/β-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level.

Structural and functional validation of a highly specific Smurf2 inhibitor.

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest.

Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework.

Single-cell transcriptomics identifies a WNT7A-FZD5 signaling axis that maintains fallopian tube stem cells in patient-derived organoids.

The study of fallopian tube (FT) function in health and disease has been hampered by limited knowledge of FT stem cells and lack of in vitro models of stem cell renewal and differentiation. Using optimized organoid culture conditions to address these limitations, we find that FT stem cell renewal is highly dependent on WNT/β-catenin signaling and engineer endogenous WNT/β-catenin signaling reporter organoids to biomark, isolate, and characterize these cells. Using functional approaches, as well as bulk and single-cell transcriptomics analyses, we show that an endogenous hormonally regulated WNT7A-FZD5 signaling axis is critical for stem cell renewal and that WNT/β-catenin pathway-activated cells form a distinct transcriptomic cluster of FT cells enriched in extracellular matrix (ECM) remodeling and integrin signaling pathways.

Construction of Synthetic Antibody Phage Display Libraries.

Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival natural antibodies and be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions.

Engineering SH2 Domains with Tailored Specificities and Affinities.

The Src Homology 2 (SH2) domain is an emerging biotechnology with applications in basic science, drug discovery, and even diagnostics. The SH2 domains rapid uptake into different areas of research is a direct result of the wealth of information generated on its biochemical, biological, and biophysical role in mammalian cell biology. Functionally, the SH2 domain binds and recognizes specific phosphotyrosine (pTyr) residues in the cell to mediate protein-protein interactions (PPIs) that govern signal transduction networks.

Activity-based profiling of cullin-RING E3 networks by conformation-specific probes.

The cullin-RING ubiquitin 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.

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.

Nebulization of pharmacological solutions with an innovative medical device based on microvaporization.

The currently available nebulization devices have a slow aerosol flow and produce vapor with large microdrops. Improved devices that achieve higher airflow and produce smaller microdrops are needed to improve the clinical care of patients. To address this critical need, we developed a novel system for the molecular vaporization of liquids.

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.

Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation.

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood.

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.