A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels.

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

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. Here, we review their systematic and iterative development to provide insights into the design principles that make them a powerful tool for drug discovery. In addition, the ongoing accumulation of crystal structures of antigen-binding fragment (Fab)-antigen complexes generated with synthetic antibodies enables a deepening understanding of the structural determinants of antigen recognition and usage of immunoglobulin sequence diversity, which can assist in developing new strategies for antibody and library optimization. Toward this, we also survey here the structural landscape of a comprehensive and unbiased set of 50 distinct complexes derived from these libraries and compare it to a similar set of natural antibodies with the goal of better understanding how each achieves molecular recognition and whether opportunities exist for iterative improvement of synthetic libraries. From this survey, we conclude that despite the minimalist strategies used for design of these synthetic antibody libraries, the overall structural interaction landscapes are highly similar to natural repertoires. We also found, however, some key differences that can help guide the iterative design of new synthetic libraries via the introduction of positionally tailored diversity.

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways.

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.

Structural surfaceomics reveals an AML-specific conformation of integrin ß2 as a CAR T cellular therapy target.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

A Conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function.

The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of "negative" interactions in determining the specificity of a domain.

Monoclonal antibodies binding data for SARS-CoV-2 proteins.

SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and therapeutic applications. The present data provide the biochemical characterization of synthetically developed monoclonal antibodies for the SARS-CoV-2 proteins. The antibodies from phage-displayed antibody libraries were selected with the SARS-CoV-2 proteins immobilized in microwell plates. The clones which bind to the antigen in Fab-phage ELISA were selected, and a two-point competitive phage ELISA was performed. Antibodies binding kinetic of IgGs for SARS-CoV2 proteins further carried with B.L.I. Systematic analysis of binding with different control proteins and purified SARS-CoV-2 ensured the robustness of the antibodies.

Development of Monoclonal Antibodies to Detect for SARS-CoV-2 Proteins.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.

Systematic Engineering of Optimized Autonomous Heavy-Chain Variable Domains.

Autonomous heavy-chain variable (VH) domains are the smallest functional antibody fragments, and they possess unique features, including small size and convex paratopes, which provide enhanced targeting of concave epitopes that are difficult to access with larger conventional antibodies. However, human VH domains have evolved to fold and function with a light chain partner, and alone, they typically suffer from low stability and high aggregation propensity. Development of autonomous human VH domains, in which aggregation propensity is reduced without compromising antigen recognition, has proven challenging. Here, we used an autonomous human VH domain as a scaffold to construct phage-displayed synthetic libraries in which aspartate was systematically incorporated at different paratope positions. In selections, the library yielded many anti-EphA1 receptor VH domains, which were characterized in detail. Structural analyses of a parental anti-EphA1 VH domain and an improved variant provided insights into the effects of aspartate and other substitutions on preventing aggregation while retaining function. Our naïve libraries and in vitro selection procedures offer a systematic approach to generating highly functional autonomous human VH domains that resist aggregation and could be used for basic research and biomedical applications.

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.

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.

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.

A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases.

Skp1-Cul1-F-box (SCF) E3 ligases constitute the largest and best-characterized family of the multisubunit E3 ligases with important cellular functions and numerous disease links. The specificity of an SCF E3 ligase is established by one of the 69 human F-box proteins that are recruited to Cul1 through the Skp1 adaptor. We previously reported generation of ubiquitin variants (UbVs) targeting Fbw7 and Fbw11, which inhibit ligase activity by binding at the F-box-Skp1 interface to competitively displace Cul1. In the present study, we employed an optimized engineering strategy to generate specific binding UbVs against 17 additional Skp1-F-box complexes. We validated our design strategy and uncovered the structural basis of binding specificity by crystallographic analyses of representative UbVs bound to Skp1-Fbl10 and Skp1-Fbl11. Our study highlights the power of combining phage display with structure-based design to develop UbVs targeting specific protein surfaces.

Cotargeting Ephrin Receptor Tyrosine Kinases A2 and A3 in Cancer Stem Cells Reduces Growth of Recurrent Glioblastoma.

Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023-37. ©2018 AACR.

Specific targeting of the deubiquitinase and E3 ligase families with engineered ubiquitin variants.

The ubiquitin proteasome system (UPS) has garnered much attention due to its potential for the development of therapeutics. Following a successful clinical application of general proteasome inhibitors much effort has been devoted to targeting individual UPS components including E3 enzymes and deubiquitinases that control specificity of ubiquitination. Our group has developed a novel approach for targeting the UPS proteins using engineered ubiquitin variants (Ubvs). These drug-like proteins can serve as valuable tools to study biological function of UPS components and assist in the development of small molecules for clinical use. In this review, we summarize studies of Ubvs targeting members of three major families, including deubiquitinases, HECT E3 ligases, and CRL E3 ligases. In particular, we focus on Ubv binding mechanisms, structural studies, and effects on enzyme function. Furthermore, new insights gained from the Ubvs are discussed in the context of small molecule studies.