Synthesis of Bispecific Conjugates by ADP-Ribosyl Cyclases
Adv. Sci.
Armed with two distinct targeting moieties, artificially created bispecific agents can uniquely modulate various processes and events implicated in human health and diseases. Despite recent approvals of multiple bispecific therapeutics, generation of homogeneous dual-targeting constructs with desired pharmacological properties remains technically challenging. Here, we report a strategy for synthesis of bispecific agents by utilizing CD38, a member of the ADP-ribosyl cyclase family, and its covalent inhibitor. A model ADP-ribosyl cyclase-enabled bispecific conjugate (ARC-BsC) against human T-cell CD3 and prostate-specific membrane antigen (PSMA) is generated through site-specific conjugation of an anti-CD3 antibody-CD38 fusion with the CD38 covalent inhibitor derivatized by a PSMA small-molecule ligand. The resulting ARC-BsC can redirect and activate cytotoxic T cells toward killing PSMA-expressing tumor cells, eliciting highly potent and selective anti-cancer immunity in vitro and in vivo. This proof-of-concept work demonstrates ARC-BsC as a potentially general approach for the development of bispecific therapeutics with diverse applications.
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A versatile platform for chemical engineering of exosomes empowered by ADP-ribosyl cyclases
Nat. Commun.
Cell-secreted exosomes have been emerging as an increasingly attractive form of nanomaterials for biomedical research. Various approaches have been established to genetically modify exosomes with proteins of interest for new and/or improved functions. However, equipping exosomes with diverse non-protein biomolecules remains largely dependent on random chemical conjugation or membrane insertion, hindering the application potential of exosomes. Herein, we develop a technology for site-specific functionalization of exosome with different synthetic groups by exploiting surface-expressed CD38, an ADP-ribosyl cyclase, and its covalent inhibitor derived from nicotinamide adenine dinucleotide (NAD+). The designed ADP-ribosyl cyclase-enabled exosomes (ARC Exos) carrying conjugated fluorescent imaging probes, small-molecule ligands, cytotoxic payloads, and bone-targeting agents are demonstrated with in vitro and/or in vivo activities and specificities. This ARC Exos-based platform provides a general approach with great versatility for chemically reprogramming exosomes.
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Preclinical assessment of genetically modified exosomes for colorectal cancer immunotherapy
J. Control. Release.
Colorectal cancer (CRC) claims nearly one million lives every year worldwide. Current treatments for CRC suffer from limited efficacy and developed resistance. Exosomes are cell-derived membranous vesicles, characterized by unique properties for drug discovery. We previously designed genetically engineered multifunctional immune-modulating exosomes (GEMINI-Exos) with surface-displayed antibodies against T-cell CD3 and cancer-associated epidermal growth factor receptor (EGFR) antigens as well as programmed death 1 receptors and OX40 ligands. Here, GEMINI-Exos were evaluated in cellular and animal models of CRC. By recruiting and activating cytotoxic T cells toward attacking EGFR-positive CRC cells and subsequently engaging with distinct checkpoint pathways, GEMINI-Exos elicit robust cellular immunity to CRC tumors in vitro and in vivo. This work presents GEMINI-Exos as an immunotherapeutic candidate with promising preclinical activity for CRC.
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Genetically Reprogrammed Exosomes for Immunotherapy of Acute Myeloid Leukemia
Mol. Ther.
Current treatments for acute myeloid leukemia (AML) remain challenging and are characterized by poor clinical outcomes. Exosomes, cell-derived membranous vesicles, have been emerging as a new modality of therapy. Here, we designed and generated genetically reprogrammed exosomes with surface-displayed antibodies and immunoregulatory proteins, namely programmed immune-engaging exosomes (PRIME Exos). By simultaneously targeting T cells and AML cells expressing C-type lectin-like molecule-1 (CLL-1), PRIME Exos can elicit tumor-specific immune responses and sustain cellular immunity against AML by modulating programmed death 1 (PD-1)- and CD27-mediated immune checkpoint pathways. In preclinical models of AML, PRIME Exos have shown promising efficacy and safety for suppressing leukemia expansion. This study developed a new exosome-based approach for AML immunotherapy.
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Nicotinamide Riboside and CD38: Covalent Inhibition and Live-Cell Labeling
JACS Au
Nicotinamide adenine dinucleotide (NAD+) is required for a myriad of metabolic, signaling, and post-translational events in cells. Its levels in tissues and organs are closely associated with health conditions. The homeostasis of NAD+ is regulated by biosynthetic pathways and consuming enzymes. As a membrane-bound protein with robust NAD+ hydrolase activity, cluster of differentiation 38 (CD38) is a major degrader of NAD+. Deficiency or inhibition of CD38 enhances NAD+ levels in vivo, resulting in various therapeutic benefits. As a metabolic precursor of NAD+, nicotinamide mononucleotide can be rapidly hydrolyzed by CD38, whereas nicotinamide riboside (NR) lacks CD38 substrate activity. Given their structural similarities, we explored the inhibition potential of NR. To our surprise, NR exhibits marked inhibitory activity against CD38 by forming a stable ribosyl-ester bond with the glutamate residue 226 at the active site. Inspired by this discovery, we designed and synthesized a clickable NR featuring an azido substitution at the 5'-OH position. This cell-permeable NR analogue enables covalent labeling and imaging of both extracellular and intracellular CD38 in live cells. Our work discovers an unrecognized molecular function of NR and generates a covalent probe for health-related CD38. These findings offer new insights into the role of NR in modulating NAD+ metabolism and CD38-mediated signaling as well as an innovative tool for in-depth studies of CD38 in physiology and pathophysiology.
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Eliciting Anti-Cancer Immunity by Genetically Engineered Multifunctional Exosomes
Mol. Ther.
Exosomes are cell-derived nanovesicles involved in regulating intercellular communications. In contrast to conventional nanomedicines, exosomes are characterized by unique advantages for therapeutic development. Despite their major successes in drug delivery, the full potential of exosomes for immunotherapy remains untapped. Herein we designed genetically engineered exosomes featured with surfaced-displayed antibody targeting groups and immunomodulatory proteins. Through genetic fusions with exosomal membrane proteins, Expi293F cell-derived exosomes were armed with monoclonal antibodies specific for human T-cell CD3 and epidermal growth factor receptor (EGFR) as well as immune checkpoint modulators, programmed death 1 (PD-1) and OX40 ligand (OX40L). The resulting genetically engineered multifunctional immune-modulating exosomes (GEMINI-Exos) can not only redirect and activate T cells toward killing EGFR-positive triple negative breast cancer (TNBC) cells but also elicit robust anti-cancer immunity, giving rise to highly potent inhibition against established TNBC tumors in mice. GEMINI-Exos represent candidate agents for immunotherapy and may offer a general strategy for generating exosome-based immunotherapeutics with desired functions and properties.
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Discovery of an NAD+ Analogue with Enhanced Specificity for PARP1
Chem. Sci.
Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key player for regulating numerous cellular processes and events through enzymatic attachments of target proteins with ADP-ribose units donated by nicotinamide adenine dinucleotide (NAD+). Human PARP1 is involved in the pathogenesis and progression of many diseases. PARP1 inhibitors have received approvals for cancer treatment. Despite these successes, our understanding about PARP1 remains limited, partially due to the presence of various ADP-ribosylation reactions catalyzed by other PARPs and their overlapped cellular functions. Here we report a synthetic NAD+ featuring an adenosyl 3'-azido substitution. Acting as an ADP-ribose donor with high activity and specificity for human PARP1, this compound enables labelling and profiling of possible protein substrates of endogenous PARP1. It provides a unique and valuable tool for studying PARP1 in biology and pathology and may shed light on the development of PARP isoform-specific modulators.
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Expanding the Toolbox of Exosome-Based Modulators of Cell Functions
Biomaterials
Exosomes are cell-derived extracellular vesicles and play important roles in mediating intercellular communications. Due to their unique advantages in transporting a variety of biomolecules, exosomes have been emerging as a new class of nanocarriers with great potential for therapeutic applications. Despite advancements in loading chemotherapeutics and interfering RNAs into exosomes, active incorporation of protein molecules into exosomes remains challenging owing to their distinctive physicochemical properties and/or a lack of knowledge of cargo sorting during exosome biogenesis. Here we report the generation of a novel type of engineered exosomes with actively incorporated membrane proteins or soluble protein cargos, named genetically infused functionally tailored exosomes (GIFTed-Exos). Through genetic fusion with exosome-associated tetraspanin CD9, transmembrane protein CD70 and glucocorticoid-induced tumor necrosis factor receptor family-related ligand (GITRL) could be displayed on exosome surface, resulting in GIFTed-Exos with excellent T-cell co-stimulatory activities. By genetically linking to a CD9-photocleavable protein fusion, fluorescent protein mCherry, apoptosis-inducing protein apoptin, and antioxidant enzyme catalase could be effectively packed into exosomes for light-controlled release. The generated GIFTed-Exos display notable in vitro and in vivo activities for delivering distinct types of protein cargos to target cells. As a possibly general approach, GIFTed-Exos provide new opportunities to create exosomes with new functions and properties for biomedical research.
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A Bifunctional NAD+ for Profiling Poly-ADP-Ribosylation-Dependent Interacting Proteins
ACS Chem. Biol.
Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It participates in a variety of biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD+) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD+ enables photo-cross-linking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD+ provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.
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A Poly-ADP-Ribose Polymer-Based Antibody-Drug Conjugate
Chem. Sci.
Protein poly-ADP-ribosylation (PARylation) plays vital roles in many aspects of physiology and pathophysiology. This posttranslational modification is catalyzed by poly-ADP-ribose polymerases (PARPs) through additions of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to protein residues, forming linear or branched poly-ADP-ribose (PAR) polymers. In this study, we explored a new concept of utilizing functionalized PAR polymers for targeted drug delivery. This was achieved by rapid and efficient generation of auto-PARylated PARP1 with 3′-azido ADP-riboses and subsequent conjugations of anti-human epidermal growth factor receptor 2 (HER2) antibodies and monomethyl auristatin F (MMAF) payloads. This designed PARylated PARP1-antibody-MMAF conjugate could potently kill HER2-expressing cancer cells in high specificity. This proof-of-principle work demonstrates the feasibility of production of PAR polymer-based antibody-drug conjugate and its application in targeted delivery. The PAR polymer-based conjugates may lead to new types of therapeutics with potentially improved physicochemical and pharmacological properties.
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Synthesis of Site-Specific Antibody-Drug Conjugates by ADP-Ribosyl Cyclases
Sci. Adv.
Most of the current antibody-drug conjugates (ADCs) in clinic are heterogeneous mixtures. To produce homogeneous ADCs, established procedures often require multiple steps or long reaction times. The introduced mutations or foreign sequences may cause high immunogenicity. Here, we explore a new concept of transforming CD38 enzymatic activity into a facile approach for generating site-specific ADCs. This was achieved through coupling bifunctional antibody-CD38 fusion proteins with designer dinucleotide-based covalent inhibitors with stably attached payloads. The resulting adenosine diphosphate–ribosyl cyclase–enabled ADC (ARC-ADC) with a drug-to-antibody ratio of 2 could be rapidly generated through single-step conjugation. The generated ARC-ADC targeting human epidermal growth factor receptor 2 (HER2) displays excellent stability and potency against HER2-positive breast cancer both in vitro and in vivo. This proof-of-concept study demonstrates a new strategy for production of site-specific ADCs. It may provide a general approach for the development of a novel class of ADCs with potentially enhanced properties.
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A Ribose-Functionalized NAD+ with Unexpected High Activity and Selectivity for Protein Poly-ADP-Ribosylation
Nat. Commun.
Nicotinamide adenine dinucleotide (NAD+)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD+ analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3′-OH of nicotinamide riboside enables enzymatic synthesis of an NAD+ analogue with high efficiency and yields. Notably, the generated 3′-azido NAD+ exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3′-azido NAD+ in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3′-azido NAD+ provides an important tool for studying cellular PARylation.
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Reprogramming Exosomes as Nanoscale Controllers of Cellular Immunity
J. Am. Chem. Soc.
Exosomes are naturally occurring membranous vesicles secreted by various types of cells. Given their unique and important biological and pharmacological properties, exosomes have been emerging as a promising form of nanomedicine acting via efficient delivery of endogenous and exogenous therapeutics. Here we explore a new concept of utilizing endogenously derived exosomes as artificial controllers of cellular immunity to redirect and activate cytotoxic T cells toward cancer cells for killing. This was achieved through genetically displaying two distinct types of antibodies on exosomal surface. The resulting synthetic multivalent antibodies retargeted exosomes (SMART-Exos), which express monoclonal antibodies specific for T-cell CD3 and cancer cell-associated epidermal growth factor receptor (EGFR), were shown to not only induce cross-linking of T cells and EGFR-expressing breast cancer cells but also elicit potent antitumor immunity both in vitro and in vivo. This proof-of-concept study demonstrates a novel application of exosomes in cancer immunotherapy and may provide a general and versatile approach for the development of a new class of cell-free therapy.
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