Stromal netrin 1 coordinates renal arteriogenesis and mural cell differentiation.

The kidney vasculature has a complex architecture that is essential for renal function. The molecular mechanisms that direct development of kidney blood vessels are poorly characterized. We identified a regionally restricted, stroma-derived signaling molecule, netrin 1 (Ntn1), as a regulator of renal vascular patterning in mice. Stromal progenitor (SP)-specific ablation of Ntn1 (Ntn1SPKO) resulted in smaller kidneys with fewer glomeruli, as well as profound defects of the renal artery and transient blood flow disruption. Notably, Ntn1 ablation resulted in loss of arterial vascular smooth muscle cell (vSMC) coverage and in ectopic SMC deposition at the kidney surface. This was accompanied by dramatic reduction of arterial tree branching that perdured postnatally. Transcriptomic analysis of Ntn1SPKO kidneys revealed dysregulation of vSMC differentiation, including downregulation of Klf4, which we find expressed in a subset of SPs. Stromal Klf4 deletion similarly resulted in decreased smooth muscle coverage and arterial branching without, however, the disruption of renal artery patterning and perfusion seen in Ntn1SPKO. These data suggest a stromal Ntn1-Klf4 axis that regulates stromal differentiation and reinforces stromal-derived smooth muscle as a key regulator of renal blood vessel formation.

Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol.

Risk, severity, and outcome of infection depend on the interplay of pathogen virulence and host susceptibility. Systematic identification of genetic susceptibility to infection is being undertaken through genome-wide association studies, but how to expeditiously move from genetic differences to functional mechanisms is unclear. Here, we use genetic association of molecular, cellular, and human disease traits and experimental validation to demonstrate that genetic variation affects expression of VAC14, a phosphoinositide-regulating protein, to influence susceptibility to Salmonella enterica serovar Typhi (S Typhi) infection. Decreased VAC14 expression increased plasma membrane cholesterol, facilitating Salmonella docking and invasion. This increased susceptibility at the cellular level manifests as increased susceptibility to typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering agent, ezetimibe, reduced susceptibility to S Typhi. Thus, coupling multiple genetic association studies with mechanistic dissection revealed how VAC14 regulates Salmonella invasion and typhoid fever susceptibility and may open doors to new prophylactic/therapeutic approaches.

Dynamic Glycosylation Governs the Vertebrate COPII Protein Trafficking Pathway.

The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.

Preclinical Study of a Fully Human Anti-PD-L1 Antibody as a Theranostic Agent for Cancer Immunotherapy.

Recently, inhibiting the PD-1/PD-L1 checkpoint pathway utilizing anti-PD-1 or anti-PD-L1 antibodies has achieved great clinical success in cancer treatment. However, anti-PD-1 immunotherapy cannot be applied to all cancer patients, no more than 25% showed a positive response. Immunohistochemistry (IHC) is the gold standard to determine the PD-L1 expression level in malignant lesions, but a noninvasive imaging-meditated strategy is urgently required for clinical diagnosis to cover the shortcomings of invasive techniques. MX001, which is an anti-PD-L1 antibody, was labeled with Cu-64 ( t1/2 = 12.7 h) and purified by PD-10 chromatography. Comprehensive studies including positron emission tomography (PET), ex vivo biodistribution, IHC, and immunotherapy have been performed in mice bearing MC38 (PD-L1 positive (+)) and 4T1 (PD-L1 negative (-)) xenografts. PET imaging of [18F]FDG was taken before and after therapy to monitor the therapeutic efficacy. [64Cu]Cu-NOTA-MX001 exhibited 2.3 ± 1.2, 5.6 ± 2.1, 5.6 ± 1.2, 6.1 ± 1.1, 6.1 ± 0.5, and 10.2 ± 1.7%ID/g uptake in MC38 xenografts at 0.5, 12, 24, 36, 48, and 62 h post-injection (p.i.), respectively. Meanwhile, the uptake in the liver and muscle at corresponding time points was 17.5 ± 2.2, 8.4 ± 2.4, 11.3 ± 3.2, 7.2 ± 2.1, 7.9.1 ± 3.5, and 3.8 ± 1.8%ID/g, and 1.2 ± 0.5, 1.3 ± 0.4, 1.5 ± 0.5, 0.7 ± 0.1, 0.6 ± 0.2, and 0.2 ± 0.1%ID/g, respectively. The uptake of [18F]FDG in MC38 and 4T1 xenografts at 1-h p.i. was 5.3 ± 0.4 and 6.4 ± 0.6%ID/g, while the uptake of [64Cu]Cu-NOTA-MX001 was 5.6 ± 0.3 and 1.3 ± 0.4%ID/g at 12-h p.i. IHC analysis confirmed that the MC38 tumor exhibited high PD-L1 expression, and the 4T1 tumor, liver, and muscle exhibited low PD-L1 expression. In addition, MC38 xenografts were suppressed by MX001 about 88% in the immunotherapy study. MX001 was successfully developed as a fully human anti-PD-L1 antibody with a high binding affinity in mouse, monkey, and human. The in vivo pharmacokinetics of MX001 was evaluated with PET imaging after being radiolabeled with Cu-64. The uptake of [64Cu]Cu-NOTA-MX001 was clearly correlated to the PD-L1 expression on various types of cancer. Subsequent immunotherapy studies demonstrated that MX001 could effectively suppress tumor growth with positive PD-L1 expression, but had poor antitumor efficacy on tumors which exhibited low PD-L1 expression. Together with the above results, MX001 has the potential to be further developed as an antibody theranostic agent for both PET imaging and immunotherapy of cancers in clinics.

Preclinical characterization and phase 1 results of ADG106 in patients with advanced solid tumors and non-Hodgkin's lymphoma.

ADG106, a ligand-blocking agonistic antibody targeting CD137 (4-1BB), exhibits promising results in preclinical studies, demonstrating tumor suppression in various animal models and showing a balanced profile between safety and efficacy. This phase 1 study enrolls 62 patients with advanced malignancies, revealing favorable tolerability up to the 5.0 mg/kg dose level. Dose-limiting toxicity occurs in only one patient (6.3%) at 10.0 mg/kg, resulting in grade 4 neutropenia. The most frequent treatment-related adverse events include leukopenia (22.6%), neutropenia (22.6%), elevated alanine aminotransferase (22.6%), rash (21.0%), itching (17.7%), and elevated aspartate aminotransferase (17.7%). The overall disease control rates are 47.1% for advanced solid tumors and 54.5% for non-Hodgkin's lymphoma. Circulating biomarkers suggest target engagement by ADG106 and immune modulation of circulating T, B, and natural killer cells and cytokines interferon γ and interleukin-6, which may affect the probability of clinical efficacy. ADG106 has a manageable safety profile and preliminary anti-tumor efficacy in patients with advanced cancers (this study was registered at ClinicalTrials.gov: NCT03802955).

Targeting CD137 (4-1BB) towards improved safety and efficacy for cancer immunotherapy.

T cells play a critical role in antitumor immunity, where T cell activation is regulated by both inhibitory and costimulatory receptor signaling that fine-tune T cell activity during different stages of T cell immune responses. Currently, cancer immunotherapy by targeting inhibitory receptors such as CTLA-4 and PD-1/L1, and their combination by antagonist antibodies, has been well established. However, developing agonist antibodies that target costimulatory receptors such as CD28 and CD137/4-1BB has faced considerable challenges, including highly publicized adverse events. Intracellular costimulatory domains of CD28 and/or CD137/4-1BB are essential for the clinical benefits of FDA-approved chimeric antigen receptor T cell (CAR-T) therapies. The major challenge is how to decouple efficacy from toxicity by systemic immune activation. This review focuses on anti-CD137 agonist monoclonal antibodies with different IgG isotypes in clinical development. It discusses CD137 biology in the context of anti-CD137 agonist drug discovery, including the binding epitope selected for anti-CD137 agonist antibody in competition or not with CD137 ligand (CD137L), the IgG isotype of antibodies selected with an impact on crosslinking by Fc gamma receptors, and the conditional activation of anti-CD137 antibodies for safe and potent engagement with CD137 in the tumor microenvironment (TME). We discuss and compare the potential mechanisms/effects of different CD137 targeting strategies and agents under development and how rational combinations could enhance antitumor activities without amplifying the toxicity of these agonist antibodies.

Directing Traffic: Regulation of COPI Transport by Post-translational Modifications.

The coat protein complex I (COPI) is an essential, highly conserved pathway that traffics proteins and lipids between the endoplasmic reticulum (ER) and the Golgi. Many aspects of the COPI machinery are well understood at the structural, biochemical and genetic levels. However, we know much less about how cells dynamically modulate COPI trafficking in response to changing signals, metabolic state, stress or other stimuli. Recently, post-translational modifications (PTMs) have emerged as one common theme in the regulation of the COPI pathway. Here, we review a range of modifications and mechanisms that govern COPI activity in interphase cells and suggest potential future directions to address as-yet unanswered questions.

Mapping Interactions of Microbial Metabolites with Human G-Protein-Coupled Receptors.

Despite evidence linking the human microbiome to health and disease, how the microbiota affects human physiology remains largely unknown. Microbiota-encoded metabolites are expected to play an integral role in human health. Therefore, assigning function to these metabolites is critical to understanding these complex interactions and developing microbiota-inspired therapies. Here, we use large-scale functional screening of molecules produced by individual members of a simplified human microbiota to identify bacterial metabolites that agonize G-protein-coupled receptors (GPCRs). Multiple metabolites, including phenylpropanoic acid, cadaverine, 9-10-methylenehexadecanoic acid, and 12-methyltetradecanoic acid, were found to interact with GPCRs associated with diverse functions within the nervous and immune systems, among others. Collectively, these metabolite-receptor pairs indicate that diverse aspects of human health are potentially modulated by structurally simple metabolites arising from primary bacterial metabolism.

A Novel Glycoproteomics Workflow Reveals Dynamic O-GlcNAcylation of COPγ1 as a Candidate Regulator of Protein Trafficking.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is an abundant and essential intracellular form of protein glycosylation in animals and plants. In humans, dysregulation of O-GlcNAcylation occurs in a wide range of diseases, including cancer, diabetes, and neurodegeneration. Since its discovery more than 30 years ago, great strides have been made in understanding central aspects of O-GlcNAc signaling, including identifying thousands of its substrates and characterizing the enzymes that govern it. However, while many O-GlcNAcylated proteins have been reported, only a small subset of these change their glycosylation status in response to a typical stimulus or stress. Identifying the functionally important O-GlcNAcylation changes in any given signaling context remains a significant challenge in the field. To address this need, we leveraged chemical biology and quantitative mass spectrometry methods to create a new glycoproteomics workflow for profiling stimulus-dependent changes in O-GlcNAcylated proteins. In proof-of-principle experiments, we used this new workflow to interrogate changes in O-GlcNAc substrates in mammalian protein trafficking pathways. Interestingly, our results revealed dynamic O-GlcNAcylation of COPγ1, an essential component of the coat protein I (COPI) complex that mediates Golgi protein trafficking. Moreover, we detected 11 O-GlcNAc moieties on COPγ1 and found that this modification is reduced by a model secretory stress that halts COPI trafficking. Our results suggest that O-GlcNAcylation may regulate the mammalian COPI system, analogous to its previously reported roles in other protein trafficking pathways. More broadly, our glycoproteomics workflow is applicable to myriad systems and stimuli, empowering future studies of O-GlcNAc in a host of biological contexts.

Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H) and V(L) for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H) frameworks and V(H)-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display.

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

Adapter-directed display: a modular design for shuttling display on phage surfaces.

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.

Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters.

A novel adapter-directed yeast display system with modular features was developed. This display system consists of two modules, a display vector and a helper vector, and is capable of displaying proteins of interest on the surface of Saccharomyces cerevisiae through the interaction of two small adapters that are expressed from the display and helper vectors. In this report, an anti-VEGF scFv antibody gene was cloned into the display vector and introduced alone into yeast S. cerevisiae cells. This led to the expression and secretion of a scFv antibody that was fused in-frame with the coiled-coil adapter GR1. For display purposes, a helper vector was constructed to express the second coiled-coil adapter GR2 that was fused with the outer wall protein Cwp2, and this was genetically integrated into the yeast genome. Co-expression of the scFv-GR1 and GR2-Cwp2 fusions in the yeast cells resulted in the functional display of anti-VEGF scFv antibodies on the yeast cell surfaces through pairwise interaction between the GR1 and GR2 adapters. Visualization of the co-localization of GR1 and GR2 on the cell surfaces confirmed the adapter-directed display mechanism. When the adapter-directed phage and yeast display modules are combined, it is possible to expand the adapter-directed display to a novel cross-species display that can shuttle between phage and yeast systems.