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. Another substitution (E172G) improves the compatibility of the elbow modification with the Fab framework by alleviating some of the strain incurred by the shortened and bulkier elbow linker region. A third substitution (F170W) generates a split-Fab conformation, resulting in a powerful crystal lattice packing interaction comprising the biological interaction interface between the variable heavy and light chain domains. In sum, we have used K141Q, E172G, and F170W substitutions-which complement the S1, Crystal Kappa, and elbow modifications-to generate a set of highly crystallizable Fab frameworks that can be used as chaperones to enable facile elucidation of Fab:antigen complex structures by x-ray crystallography.

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. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

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

The Wnt/ß-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. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of ß-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/ß-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

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. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.

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/ß-catenin signaling and engineer endogenous WNT/ß-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/ß-catenin pathway-activated cells form a distinct transcriptomic cluster of FT cells enriched in extracellular matrix (ECM) remodeling and integrin signaling pathways. Overall, we provide a deep characterization of FT stem cells and their molecular requirements for self-renewal, paving the way for mechanistic work investigating the role of stem cells in FT health and disease.

Targeted alveolar regeneration with Frizzled-specific agonists.

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.

Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b.

The conclusive identity of Wnts regulating liver zonation (LZ) and regeneration (LR) remains unclear despite an undisputed role of ß-catenin. Using single-cell analysis, we identified a conserved Wnt2 and Wnt9b expression in endothelial cells (ECs) in zone 3. EC-elimination of Wnt2 and Wnt9b led to both loss of ß-catenin targets in zone 3, and re-appearance of zone 1 genes in zone 3, unraveling dynamicity in the LZ process. Impaired LR observed in the knockouts phenocopied models of defective hepatic Wnt signaling. Administration of a tetravalent antibody to activate Wnt signaling rescued LZ and LR in the knockouts and induced zone 3 gene expression and LR in controls. Administration of the agonist also promoted LR in acetaminophen overdose acute liver failure (ALF) fulfilling an unmet clinical need. Overall, we report an unequivocal role of EC-Wnt2 and Wnt9b in LZ and LR and show the role of Wnt activators as regenerative therapy for ALF.

A Descriptor Set for Quantitative Structure-property Relationship Prediction in Biologics.

There has been a remarkable increase in the number of biologics, especially monoclonal antibodies, in the market over the last decade. In addition to attaining the desired binding to their targets, a crucial aspect is the 'developability' of these drugs, which includes several desirable properties such as high solubility, low viscosity and aggregation, physico-chemical stability, low immunogenicity and low poly-specificity. The lack of any of these desirable properties can lead to significant hurdles in advancing them to the clinic and are often discovered only during late stages of drug development. Hence, in silico methods for early detection of these properties, particularly the ones that affect aggregation and solubility in the earlier stages can be highly beneficial. We have developed a computational framework based on a large and diverse set of protein specific descriptors that is ideal for making liability predictions using a QSPR (quantitative structure-property relationship) approach. This set offers a high degree of feature diversity that may coarsely be classified based on (1) sequence (2) structure and (3) surface patches. We assess the sensitivity and applicability of these descriptors in four dedicated case studies that are believed to be representative of biophysical characterizations commonly employed during the development process of a biologics drug candidate. In addition to data sets obtained from public sources, we have validated the descriptors on novel experimental data sets in order to address antibody developability and to generate prospective predictions on Adnectins. The results show that the descriptors are well suited to assist in the improvement of protein properties of systems that exhibit poor solubility or aggregation.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.

A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy.

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to ßcatenin-dependent formation of the blood-retina-barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger ßcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12-/- mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen-induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction.

A T cell redirection platform for co-targeting dual antigens on solid tumors.

In order to direct T cells to specific features of solid cancer cells, we engineered a bispecific antibody format, named Dual Antigen T cell Engager (DATE), by fusing a single-chain variable fragment targeting CD3 to a tumor-targeting antigen-binding fragment. In this format, multiple novel paratopes against different tumor antigens were able to recruit T-cell cytotoxicity to tumor cells in vitro and in an in vivo pancreatic ductal adenocarcinoma xenograft model. Since unique surface antigens in solid tumors are limited, in order to enhance selectivity, we further engineered "double-DATEs" targeting two tumor antigens simultaneously. The double-DATE contains an additional autonomous variable heavy-chain domain, which binds a second tumor antigen without itself eliciting a cytotoxic response. This novel modality provides a strategy to enhance the selectivity of immune redirection through binary targeting of native tumor antigens. The modularity and use of a common, stable human framework for all components enables a pipeline approach to rapidly develop a broad repertoire of tailored DATEs and double-DATEs with favorable biophysical properties and high potencies and selectivities.

Inhibition of Cancer Cell Adhesion, Migration and Proliferation by a Bispecific Antibody that Targets two Distinct Epitopes on αv Integrins.

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFß) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFß. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvß1, αvß3 and αvß5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.

A phage-displayed single-chain Fab library optimized for rapid production of single-chain IgGs.

Phage-displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full-length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate-limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage-displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage-displayed synthetic single-chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single-chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage-derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell-surface receptors, and scIgGs behave the same as conventional IgGs.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.

Discovery of a chemical probe for PRDM9.

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.

Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice.

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.

Blockade of TGF-ß signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models.

Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. EOC is often diagnosed at late stages, with peritoneal metastases and ascites production. Current surgery and platinum-based chemotherapy regimes fail to prevent recurrence in most patients. High levels of Transforming growth factor-ß (TGF-ß) within ascites has been linked to poor prognosis. TGF-ß signaling promotes epithelial-mesenchymal transition (EMT) in EOC tumor cells, and immune suppression within the tumor microenvironment, with both contributing to chemotherapy resistance and metastasis. The goal of this study was to develop specific synthetic inhibitory antibodies to the Type II TGF-ß receptor (TGFBR2), and test these antibodies in EOC cell and tumor models. Following screening of a phage-displayed synthetic antigen-binding fragment (Fab) library with the extracellular domain of TGFBR2, we identified a lead inhibitory Fab that suppressed TGF-ß signaling in mouse and human EOC cell lines. Affinity maturation of the lead inhibitory Fab resulted in several derivative Fabs with increased affinity for TGFBR2 and efficacy as suppressors of TGF-ß signaling, EMT and EOC cell invasion. In EOC xenograft and syngeneic tumor models, blockade of TGFBR2 with our lead antibodies led to improved chemotherapy response. This correlated with reversal of EMT and immune exclusion in these tumor models with TGFBR2 blockade. Together, these results describe new inhibitors of the TGF-ß pathway that improve antitumor immunity, and response to chemotherapy in preclinical EOC models.

A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties.

Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells.

Targeting human SET1/MLL family of proteins.

The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein-protein interactions within the MLL1 complex.

A Suite of Biochemical Assays for Screening RNA Methyltransferase BCDIN3D.

BCDIN3D is an RNA-methyltransferase that O-methylates the 5' phosphate of RNA and regulates microRNA maturation. To discover small-molecule inhibitors of BCDIN3D, a suite of biochemical assays was developed. A radiometric methyltransferase assay and fluorescence polarization-based S-adenosylmethionine and RNA displacement assays are described. In addition, differential scanning fluorimetry and surface plasmon resonance were used to characterize binding. These assays provide a comprehensive package for the development of small-molecule modulators of BCDIN3D activity.