The causative agent of coronavirus induced disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. Here, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of human COVID-19 autopsies revealed SARS-CoV-2 infected cells including olfactory neuronal cells facing the nasal cavity positive for NRP1. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.
The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19)is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . Understanding how SARS-CoV-2 enters and spreads within human organs is crucial for developing strategies to prevent viral dissemination. For many viruses, tissue tropism is determined by the availability of virus receptors on the surface of host cells 2 . Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as a host receptor, yet, their tropisms differ 3-5 . Here, we found that the cellular receptor neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was inhibited by a monoclonal blocking antibody against the extracellular b1b2 domain of NRP1. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial cells and in the epithelial cells facing the nasal cavity. Neuropathological analysis of human COVID-19 autopsies revealed SARS-CoV-2 infected NRP1-positive cells in the olfactory epithelium and bulb. In the olfactory bulb infection was detected particularly within NRP1-positive endothelial cells of small capillaries and medium-sized vessels. Studies in mice demonstrated, after intranasal application, NRP1-mediated transport of virus-sized particles into the central nervous system. Thus, NRP1 could explain the enhanced tropism and spreading of SARS-CoV-2.
Tumor-associated macrophages (TAMs) expressing the multi-ligand endocytic receptor mannose receptor (CD206/MRC1) contribute to tumor immunosuppression, angiogenesis, metastasis, and relapse. Here, we describe a peptide that selectively targets MRC1-expressing TAMs (MEMs). We performed in vivo peptide phage display screens in mice bearing 4T1 metastatic breast tumors to identify peptides that target peritoneal macrophages. Deep sequencing of the peptide-encoding inserts in the selected phage pool revealed enrichment of the peptide CSPGAKVRC (codenamed “UNO”). Intravenously injected FAM-labeled UNO (FAM-UNO) homed to tumor and sentinel lymph node MEMs in different cancer models: 4T1 and MCF-7 breast carcinoma, B16F10 melanoma, WT-GBM glioma and MKN45-P gastric carcinoma. Fluorescence anisotropy assay showed that FAM-UNO interacts with recombinant CD206 when subjected to reducing conditions. Interestingly, the GSPGAK motif is present in all CD206-binding collagens. FAM-UNO was able to transport drug-loaded nanoparticles into MEMs, whereas particles without the peptide were not taken up by MEMs. In ex vivo organ imaging, FAM-UNO showed significantly higher accumulation in sentinel lymph nodes than a control peptide. This study suggests applications for UNO peptide in diagnostic imaging and therapeutic targeting of MEMs in solid tumors.
Antiangiogenic and vascular disrupting compounds have shown promise in cancer therapy, but tend to be only partially effective. We previously reported a potent theranostic nanosystem that was highly effective in glioblastoma and breast cancer mouse models, retarding tumor growth and producing some cures [Agemy et al. 2011,2013]. The nanosystem consists of iron oxide NPs (“nanoworms”) coated with a composite peptide with tumor-homing and pro-apoptotic domains. The homing component targets tumor vessels by binding to p32/gC1qR at the surface or tumor endothelial cells. We sought to further improve the efficacy nanosystem by searching for an optimally effective homing peptide that would also incorporate a tumor-penetrating function. To this effect, we tested a panel of candidate p32 binding peptides with a sequence motif that conveys tumor-penetrating activity (CendR motif). We identified a peptide designated as Linear TT1 (Lin TT1) (sequence: AKRGARSTA) as most effective in causing tumor homing and penetration of the nanosystem. This peptide had the lowest affinity for p32 among the peptides tested. The low affinity may have moderated the avidity effect from the multivalent presentation on nanoparticles (NPs), such that the NPs avoid getting trapped by the so called “binding-site barrier”, which can hinder tissue penetration of compounds with a high affinity for their receptors. Treatment of breast cancer mice with the LinTT1 nanosystem showed greatly improved efficacy compared to the original system. These results identify a promising treatment modality and underscore the value of tumor penetration effect in improving the efficacy tumor treatment.
Gastrointestinal and gynecological malignancies disseminate in the peritoneal cavity - a condition known as peritoneal carcinomatosis (PC). Intraperitoneal (IP) administration can be used to improve therapeutic index of anticancer drugs used for PC treatment. Activity of IP anticancer drugs can be further potentiated by encapsulation in nanocarriers and/or affinity targeting with tumor-specific affinity ligands, such as tumor homing peptides. Here we evaluated a novel tumor penetrating peptide, linTT1 (AKRGARSTA), as a PC targeting ligand for nanoparticles. We first demonstrated that the primary homing receptor for linTTl, p32 (or gClqR), is expressed on the cell surface of peritoneal carcinoma cell lines of gastric (MKN-45P), ovarian (SKOV-3), and colon (CT-26) origin, and that peritoneal tumors in mice and clinical peritoneal carcinoma explants express p32 protein accessible from the IP space. Iron oxide nanoworms (NWs) functionalized with the linTT1 peptide were taken up and routed to mitochondria in cultured PC cells. NWs functionalized with linTT1 peptide in tandem with a pro-apoptotic [D(KLAKLAK)2] peptide showed p32-dependent cytotoxicity in MKN-45P, SKOV-3, and CT- 26 cells. Upon IP administration in mice bearing MKN-45P, SKOV-3, and CT-26 tumors, linTT1-functionalized NWs showed robust homing and penetration into malignant lesions, whereas only a background accumulation was seen in control tissues. In tumors, the linTT1-NW accumulation was seen predominantly in CD31-positive blood vessels, in LYVE-1-positive lymphatic structures, and in CD11b-positive tumor macrophages. Experimental therapy of mice bearing peritoneal MKN-45P xenografts and CT-26 syngeneic tumors with IP linTT1-D(KLAKLAK)2-NWs resulted in significant reduction of weight of peritoneal tumors and significant decrease in the number of metastatic tumor nodules, whereas treatment with untargeted D(KLAKLAK)2-NWs had no effect. Our data show that targeting of p32 with linTTl tumor-penetrating peptide improves tumor selectivity and antitumor efficacy of IP pro-apoptotic NWs. P32-directed intraperitoneal targeting of other anticancer agents and nanoparticles using peptides and other affinity ligands may represent a general strategy to increase their therapeutic index.
Tumor-selective drug conjugates can potentially improve the prognosis for patients affected by glioblastoma (GBM) -the most common and malignant type of brain cancer with no effective cure.Here we evaluated a novel tumor penetrating peptide that targets cell surface p32, LinTT1 (AKRGARSTA), as a GBM targeting ligand for systemically-administered nanoparticles. LinTT1-functionalization increased tumor homing of iron oxide nanoworms (NWs) across a panel of five GBM models ranging from infiltratively-disseminating to angiogenic phenotypes. LinTT1-NWs homed to CD31-positive tumor blood vessels, including to transdifferentiated endothelial cells, and showed co-localization with tumor macrophages and lymphatic vessels. LinTT1 functionalization also resulted in increased GBM delivery of other types of systemically-administered nanoparticles: silver nanoparticles and albumin-paclitaxel nanoparticles. Finally, LinTT1-guided proapoptotic NWs exerted strong anti-glioma activity in two models of GBM, including doubling the lifespan of the mice in an aggressive orthotopic stem cell-like GBM that recapitulates the histological hallmarks of human GBM. Our study suggests that LinTT1 targeting strategy can be used to increase GBM uptake of systemic nanoparticles for improved imaging and therapy.
Extracellular matrix in solid tumors has emerged as a specific, stable, and abundant target for affinityguided delivery of anticancer drugs. Here we describe the homing peptide that interacts with the C-isoform of Tenascin-C (TNC-C) upregulated in malignant tissues. TNC-C binding PL3 peptide (amino acid sequence: AGRGRLVR) was identified by in vitro biopanning on recombinant tnc-c. Besides tnc-C, PL3 interacts via its C-end Rule (CendR) motif with cell-and tissue penetration receptor neuropilin-1 (NRP-1). Functionalization of iron oxide nanoworms (NWs) and metallic silver nanoparticles (AgNPs) with PL3 peptide increased tropism of systemic nanoparticles towards glioblastoma (GBM) and prostate carcinoma xenograft lesions in nude mice (eight and five-fold respectively). Treatment of gliomabearing mice with proapoptotic PL3-guided NWs improved the survival of the mice, whereas treatment with untargeted particles had no effect. PL3-coated nanoparticles were found to accumulate in TNC-C and NRP-1-positive areas in clinical tumor samples, suggesting a translational relevance. The systemic tumor-targeting properties and binding of PL3-NPs to the clinical tumor sections, suggest that the PL3 peptide may have applications as a targeting moiety for the selective delivery of imaging and therapeutic agents to solid tumors.
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