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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.

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 β2 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.

Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of ideal target antigens that are both absolutely tumor specific and homogeneously expressed. We show that multi-antigen prime-and-kill recognition circuits provide flexibility and precision to overcome these challenges in the context of glioblastoma. A synNotch receptor that recognizes a specific priming antigen, such as the heterogeneous but tumor-specific glioblastoma neoantigen epidermal growth factor receptor splice variant III (EGFRvIII) or the central nervous system (CNS) tissue-specific antigen myelin oligodendrocyte glycoprotein (MOG), can be used to locally induce expression of a CAR. This enables thorough but controlled tumor cell killing by targeting antigens that are homogeneous but not absolutely tumor specific. Moreover, synNotch-regulated CAR expression averts tonic signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state. In immunodeficient mice bearing intracerebral patient-derived xenografts (PDXs) with heterogeneous expression of EGFRvIII, a single intravenous infusion of EGFRvIII synNotch-CAR T cells demonstrated higher antitumor efficacy and T cell durability than conventional constitutively expressed CAR T cells, without off-tumor killing. T cells transduced with a synNotch-CAR circuit primed by the CNS-specific antigen MOG also exhibited precise and potent control of intracerebral PDX without evidence of priming outside of the brain. In summary, by using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity, completeness, and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.

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. One antibody that inhibited in vitro SLIT2 signaling through ROBO1 and ROBO2 (anti-ROBO1/2) also reduced ocular neovascularization in oxygen-induced retinopathy (OIR) and laser-induced corneal neovascularization (CNV) mouse models in vivo. Single-cell RNA sequencing of OIR retinas revealed that antibody treatment affected several cell types relevant to physiological and pathological angiogenesis, including endothelial cells, pericytes, and a heterogeneous population of myeloid cells. mAb treatment improved blood-retina barrier integrity and prevented pathological pericyte activation in OIR. SLIT-ROBO signaling inhibition prevented pathological activation of myeloid cells and increased neuroprotective myeloid populations normally seen in the developing retina. Microglia/infiltrating macrophage–specific ablation of Robo1 and Robo2 or knockout of the downstream effector phosphatidylinositol 3-kinase (Pik3cg) encoding PI3Kγ in both OIR and CNV models phenocopied anti-ROBO1/2 treatment, further demonstrating the key role of myeloid cells as drivers of ocular neovascular diseases. ROBO1/2 blocking antibodies may thus provide a promising strategy to combat inflammation in blinding eye diseases. Editor's summary: Ocular neovascularization underlies blindness in several pathological conditions. Current treatments focus on inhibiting vascular endothelial growth factor A (VEGFA) signaling, but targeting other pathways in diverse cell types could improve therapies. Geraldo et al. developed human monoclonal antibodies (mAbs) targeting Roundabout (ROBO) receptors that regulate cell migration and proliferation to target ocular neovascularization. mAb treatment in an oxygen-induced retinopathy mouse model and in laser-induced corneal neovascularization reduced neovascular area and increased myeloid cell populations associated with retinal development. Combining mAb treatment with standard anti-VEGFA treatment further improved pathology. Genetically deleting Robo1 and 2 in microglia and infiltrating macrophages mimicked the mAb treatment. These results demonstrate how inhibiting ROBO signaling in myeloid cells could be a strategy to treat ocular neovascularization diseases. —Brandon Berry [ABSTRACT FROM AUTHOR]

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.

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. [ABSTRACT FROM AUTHOR]

Magnetotactic bacteria are aquatic organisms that produce subcellular magnetic particles in order to orient in the earth's geomagnetic field. MamE, a predicted HtrA protease required to produce magnetite crystals in the magnetotactic bacterium Magnetospirillum magneticum AMB-1, was recently shown to promote the proteolytic processing of itself and two other biomineralization factors in vivo Here, we have analyzed the in vivo processing patterns of three proteolytic targets and used this information to reconstitute proteolysis with a purified form of MamE. MamE cleaves a custom peptide substrate with positive cooperativity, and its autoproteolysis can be stimulated with exogenous substrates or peptides that bind to either of its PDZ domains. A misregulated form of the protease that circumvents specific genetic requirements for proteolysis causes biomineralization defects, showing that proper regulation of its activity is required during magnetite biosynthesis in vivo Our results represent the first reconstitution of the proteolytic activity of MamE and show that its behavior is consistent with the previously proposed checkpoint model for biomineralization.

Cells reuse signaling proteins in multiple pathways, raising the potential for improper cross talk. Scaffold proteins are thought to insulate against such miscommunication by sequestering proteins into distinct physical complexes. We show that the scaffold protein Ste5, which organizes the yeast mating mitogen-activated protein kinase (MAPK) pathway, does not use sequestration to prevent misactivation of the mating response. Instead, Ste5 appears to use a conformation mechanism: Under basal conditions, an intramolecular interaction of the pleckstrin homology (PH) domain with the von Willebrand type A (VWA) domain blocks the ability to coactivate the mating-specific MAPK Fus3. Pheromone-induced membrane binding of Ste5 triggers release of this autoinhibition. Thus, in addition to serving as a conduit guiding kinase communication, Ste5 directly receives input information to decide if and when signal can be transmitted to mating output.

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. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross‐reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein‐based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2–E3 protein interaction interface. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]