Japanese medaka (Oryzias latipes) Nectin4 plays an important role against red spotted grouper nervous necrosis virus infection.

Nectins are adhesion molecules that play a crucial role in the organization of epithelial and endothelial junctions and function as receptors for the entry of herpes simplex virus. However, the role of Nectin4 remains poorly understood in fish. In this study, nectin4 gene was cloned from medaka (OlNectin4). OlNectin4 was located on chromosome 18 and contained 11 exons, with a total genome length of 25754 bp, coding sequences of 1689 bp, coding 562 amino acids and a molecular weight of 65.5 kDa. OlNectin4 contained four regions, including an Immunoglobulin region, an Immunoglobulin C-2 Type region, a Transmembrane region and a Coiled coil region. OlNectin4 shared 47.18 % and 25.00 % identity to Paralichthys olivaceus and Mus musculus, respectively. In adult medaka, the transcript of nectin4 was predominantly detected in gill. During red spotted grouper nervous necrosis virus (RGNNV) infection, overexpression of OlNectin4 in GE cells significantly increased viral gene transcriptions. Meanwhile, Two mutants named OlNectin4△4 (+4 bp) and OlNectin4△7 (-7 bp) medaka were established using CRISPR-Cas9 system. Nectin4-KO medaka had higher mortality than WT after infected with RGNNV. Moreover, the expression of RGNNV RNA2 gene in different tissues of the Nectin4-KO were higher than WT medaka after challenged with RGNNV. The brain and eye of Nectin4-KO medaka which RGNNV mainly enriched, exhibited significantly higher expression of interferon signaling genes than in WT. Taken together, the OlNectin4 plays a complex role against RGNNV infection by inducing interferon responses for viral clearance.

Nectin-4-targeted immunoSPECT/CT imaging and photothermal therapy of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is more prone to distant metastasis and visceral recurrence in comparison to other breast cancer subtypes, and is related to dismal prognosis. Nevertheless, TNBC has an undesirable response to targeted therapies. Therefore, to tackle the huge challenges in the diagnosis and treatment of TNBC, Nectin-4 was selected as a theranostic target because it was recently found to be highly expressed in TNBC. We developed anti-Nectin-4 monoclonal antibody (mAbNectin-4)-based theranostic pair, 99mTc-HYNIC-mAbNectin-4 and mAbNectin-4-ICG. 99mTc-HYNIC-mAbNectin-4 was applied to conduct immuno-single photon emission computed tomography (SPECT) for TNBC diagnosis and classification, and mAbNectin-4-ICG to mediate photothermal therapy (PTT) for relieving TNBC tumor growth. METHODS: Nectin-4 expression levels of breast cancer cells (MDA-MB-468: TNBC cells; and MCF-7, non-TNBC cells) were proved by western blot, flow cytometry, and immunofluorescence imagning. Cell uptake assays, SPECT imaging, and biodistribution were performed to evaluate Nectin-4 targeting of 99mTc-HYNIC-mAbNectin-4. A photothermal agent (PTA) mAbNectin-4-ICG was generated and characterized. In vitro photothermal therapy (PTT) mediated by mAbNectin-4-ICG was conducted under an 808 nm laser. Fluorescence (FL) imaging was performed for mAbNectin-4-ICG mapping in vivo. In vivo PTT treatment effects on TNBC tumors and corresponding systematic toxicity were evaluated. RESULTS: Nectin-4 is overexpressed in MDA-MB-468 TNBC cells, which could specifically uptake 99mTc-HYNIC-mAbNectin-4 with high targeting in vitro. The corresponding immunoSPECT imaging demonstrated exceptional performance in TNBC diagnosis and molecular classification. mAbNectin-4-ICG exhibited favourable biocompatibility, photothermal effects, and Nectin-4 targeting. FL imaging mapped biodistribution of mAbNectin-4-ICG with excellent tumor-targeting and retention in vivo. Moreover, mAbNectin-4-ICG-mediated PTT provided advanced TNBC tumor destruction efficiency with low systematic toxicity. CONCLUSION: mAbNectin-4-based radioimmunoimaging provides visualization tools for the stratification and diagnosis for TNBC, and the corresponding mAbNectin-4-mediated PTT shows a powerful anti-tumor effect. Our findings demonstrate that this Nectin-4 targeting strategy offers a simple theranostic platform for TNBC.

Nectin-1 Is an Entry Mediator for Varicella-Zoster Virus Infection of Human Neurons.

Varicella-zoster virus (VZV) maintains lifelong latency in neurons following initial infection and can subsequently be reactivated to result in herpes zoster or severe neurological manifestations such as encephalitis. Mechanisms of VZV neuropathogenesis have been challenging to study due to the strict human tropism of the virus. Although neuronal entry mediators of other herpesviruses, including herpes simplex virus, have been identified, little is known regarding how VZV enters neurons. Here, we utilize a human stem cell-based neuronal model to characterize cellular factors that mediate entry. Through transcriptional profiling of infected cells, we identify the cell adhesion molecule nectin-1 as a candidate mediator of VZV entry. Nectin-1 is highly expressed in the cell bodies and axons of neurons. Either knockdown of endogenous nectin-1 or incubation with soluble forms of nectin-1 produced in mammalian cells results in a marked decrease in infectivity of neurons. Notably, while addition of soluble nectin-1 during viral infection inhibits infectivity, addition after infection has no effect on infectivity. Ectopic expression of human nectin-1 in a cell line resistant to productive VZV infection confers susceptibility to infection. In summary, we have identified nectin-1 as a neuronal entry mediator of VZV. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox, gains access to neurons during primary infection where it resides lifelong, and can later be reactivated. Reactivation is associated with shingles and postherpetic neuralgia, as well as with severe neurologic complications, including vasculitis and encephalitis. Although the varicella vaccine substantially decreases morbidity and mortality associated with primary infection, the vaccine cannot prevent the development of neuronal latency, and vaccinated populations are still at risk for reactivation. Furthermore, immunocompromised individuals are at higher risk for VZV reactivation and associated complications. Little is known regarding how VZV enters neurons. Here, we identify nectin-1 as an entry mediator of VZV in human neurons. Identification of nectin-1 as a neuronal VZV entry mediator could lead to improved treatments and preventative measures to reduce VZV related morbidity and mortality.

A monoclonal antibody neutralizes pesudorabies virus by blocking gD binding to the receptor nectin-1.

Pseudorabies virus (PRV) was isolated from some human cases recently and the infected patients manifested respiratory dysfunction and acute neurological symptoms. However, no effective drug or vaccine, preventing the progression of PRV infection, is available. Nectin-1 was the only reported receptor for PRV cell entry both swine and human origin, representing an excellent target to block PRV infection, and especially its transmission from pigs to humans. A PRV-gD specific mAbs (10B6) was isolated from hybridomas and its neutralizing activities in vitro and in vivo were determined. 10B6 exhibited effective neutralizing activities in vitro with IC50 = 2.514 µg/ml and 4.297 µg/ml in the presence and absence of complement. And in vivo, 10B6 provided 100% protection against PRV lethal challenge with a dose of 15 mg/kg. Further, 10B6 could bind to a conserved epitope, 316QPAEPFP322, locating in gD pro-fusion domain, and finally blocks the binding of PRV-gD to nectin-1. Moreover, 10B6 showed an effective inhibition on PRV cell-attachment in a cell type-independent manner and could also block the virus spreading among cells. 10B6 exhibited effectively neutralizing activities to Chinese PRV variant strain in vitro and in vivo by blocking gD binding to nectin-1, implied both prophylactic and therapeutic interventions against PRV infections.

Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus.

The measles virus (MV) is a major cause of childhood morbidity and mortality worldwide. We previously established a mouse monoclonal antibody, 2F4, which shows high neutralizing titers against eight different genotypes of MV. However, the molecular basis for the neutralizing activity of the 2F4 antibody remains incompletely understood. Here, we have evaluated the binding characteristics of a Fab fragment of the 2F4 antibody. Using the MV infectious assay, we demonstrated that 2F4 Fab inhibits viral entry via either of two cellular receptors, SLAM and Nectin4. Surface plasmon resonance (SPR) analysis of recombinant proteins indicated that 2F4 Fab interacts with MV hemagglutinin (MV-H) with a KD value at the nm level. Furthermore, we designed a single-chain Fv fragment of 2F4 antibody as another potential biopharmaceutical to target measles. The stable 2F4 scFv was successfully prepared by the refolding method and shown to interact with MV-H at the µm level. Like 2F4 Fab, scFv inhibited receptor binding and viral entry. This indicates that 2F4 mAb uses the receptor-binding site and/or a neighboring region as an epitope with high affinity. These results provide insight into the neutralizing activity and potential therapeutic use of antibody fragments for MV infection.

The CC' loop of IgV domains of the immune checkpoint receptors, plays a key role in receptor:ligand affinity modulation.

Antibodies targeting negative regulators of immune checkpoints have shown unprecedented and durable response against variety of malignancies. While the concept of blocking the negative regulators of the immune checkpoints using mAbs appears to be an outstanding approach, their limited effect and several drawbacks, calls for the rational design of next generation of therapeutics. Soluble isoforms of the negative regulators of immune checkpoint pathways are expressed naturally and regulate immune responses. This suggests, affinity-modified versions of these self-molecules could be effective lead molecules for immunotherapy. To obtain better insights on the hotspot regions for modification, we have analysed structures of 18 immune receptor:ligand complexes containing the IgV domain. Interestingly, this analysis reveals that the CC' loop of IgV domain, a loop which is distinct from CDRs of antibodies, plays a pivotal role in affinity modulation, which was previously not highlighted. It is noteworthy that a ~5-residue long CC' loop in a ~120 residue protein makes significant number of hydrophobic and polar interactions with its cognate ligand. The post-interaction movement of CC' loop to accommodate the incoming ligands, seems to provide additional affinity to the interactions. In silico replacement of the CC' loop of TIGIT with that of Nectin-2 and PVR followed by protein docking trials suggests a key role of the CC' loop in affinity modulation in the TIGIT/Nectin pathway. The CC' loop appears to be a hotspot for the affinity modification without affecting the specificity to their cognate receptors.

Sea Urchin Extracellular Proteins Design a Complex Protein Corona on Titanium Dioxide Nanoparticle Surface Influencing Immune Cell Behavior.

Extensive exploitation of titanium dioxide nanoparticles (TiO2NPs) augments rapid release into the marine environment. When in contact with the body fluids of marine invertebrates, TiO2NPs undergo a transformation and adhere various organic molecules that shape a complex protein corona prior to contacting cells and tissues. To elucidate the potential extracellular signals that may be involved in the particle recognition by immune cells of the sea urchin Paracentrotus lividus, we investigated the behavior of TiO2NPs in contact with extracellular proteins in vitro. Our findings indicate that TiO2NPs are able to interact with sea urchin proteins in both cell-free and cell-conditioned media. The two-dimensional proteome analysis of the protein corona bound to TiO2NP revealed that negatively charged proteins bound preferentially to the particles. The main constituents shaping the sea urchin cell-conditioned TiO2NP protein corona were proteins involved in cellular adhesion (Pl-toposome, Pl-galectin-8, Pl-nectin) and cytoskeletal organization (actin and tubulin). Immune cells (phagocytes) aggregated TiO2NPs on the outer cell surface and within well-organized vesicles without eliciting harmful effects on the biological activities of the cells. Cells showed an active metabolism, no oxidative stress or caspase activation. These results provide a new level of understanding of the extracellular proteins involved in the immune-TiO2NP recognition and interaction in vitro, confirming that primary immune cell cultures from P. lividus can be an optional model for swift and efficient immune-toxicological investigations.

Functional characterization of seven-band grouper immunoglobulin like cell adhesion molecule, Nectin4 as a cellular receptor for nervous necrosis virus.

Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1 cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.

Identification of targets for prostate cancer immunotherapy.

BACKGROUND: We performed profiling of the immune microenvironment of castration-resistant (CRPC) and castration-sensitive (CSPC) prostate cancer (PC) in order to identify novel targets for immunotherapy. METHODS: PD-L1 and CD3/CD8 immunohistochemistry, PD-L1/2 fluorescent in situ hybridization, tumor mutation burden, microsatellite instability, and RNA-seq of 395 immune-related genes were performed in 19 CRPC and CSPC. Targeted genomic sequencing and fusion analysis were performed in 17 of these specimens. RESULTS: CD276, PVR, and NECTIN2 were highly expressed in PC. Comparison of CRPC versus CSPC and primary versus metastatic tissue revealed the differential expression of immunostimulatory, immunosuppressive, and epithelial-to-mesenchymal transition (EMT)-related genes. Unsupervised clustering of differentially expressed genes yielded two final clusters best segregated by CRPC and CSPC status. CONCLUSION: CD276 and the alternative checkpoint inhibition PVR/NECTIN2/CD226/TIGIT pathway emerged as relevant to PC checkpoint inhibition target development.

Interaction of PVR/PVRL2 with TIGIT/DNAM-1 as a novel immune checkpoint axis and therapeutic target in cancer.

Avoiding immune surveillance and inducing a tumor-promoting inflammatory milieu found entry into the new generation of the hallmarks of cancer. Cancer cells hijack immune mechanisms which physiologically protect the body from the development of autoimmune diseases and excessive tissue damage during inflammation by downregulating immune responses. This is frequently achieved by upregulation of immune checkpoints. Therefore, the blocking of immune checkpoint ligand-receptor interactions can reinstall the immune systems capability to fight cancer cells as shown for CTLA4 and PD-1 inhibitors in a clinical setting. Newly described checkpoint antigens are currently under investigation in cancer immunotherapy. Preclinical data emphasize the immune checkpoint axis TIGIT-PVR/PVRL2 as very promising target. This axis includes additional receptors such as DNAM-1, CD96, and CD112R. In this review, we discuss the recent findings of the relevance of this complex receptor ligand system in hematologic and solid cancers. Emphasis is also laid on the discussion of potential combinations with other immunotherapeutic approaches.

Fc engineering of anti-Nectin-2 antibody improved thrombocytopenic adverse event in monkey.

Nectin-2 is a transmembrane glycoprotein which is involved in the process of Ca2+-independent cell-cell adhesion. In our previous study, we have demonstrated that Nectin-2 is over-expressed in breast and ovarian cancer tissues by using gene expression analysis and immunohistochemistry. Furthermore, we discovered multiple anti-Nectin-2 fully human monoclonal antibodies which inhibited tumor growth in in vivo subcutaneous xenograft models with antibody-dependent cellular cytotoxicity (ADCC) as the principal mechanism of action. In this report, we assessed the toxicity of Y-443, a fully human IgG1/kappa anti-Nectin-2 monoclonal antibody exhibiting strong in vitro ADCC and in vivo anti-tumor activity in cynomolgus monkeys (Macaca fascicularis (Cynos)). Unexpectedly, upon administration, Y-443 induced strong thrombocytopenia through Nectin-2 expressed on Cyno platelets, presumably followed by phagocytosis in the mononuclear phagocytic system. To mitigate the adverse safety profile, we mutated the Fc region of Y-443 to reduce the Fc binding activity to Fcγ receptor I, which is the primary receptor for phagocytosis on macrophages. Moreover, we further engineered the Fc through defucosylation to maintain ADCC activity. The resultant Fc engineered antibody, termed Y-634, demonstrated diminished thrombocytopenia in Cyno toxicological studies and maintained anti-tumor activity in a mouse xenograft model. These findings suggest that Y-634 may have a therapeutic potential for the treatment of Nectin-2 positive cancers, and moreover, Fc engineering is a potential mitigation strategy to ameliorate safety liabilities in antibody induced thrombocytopenia while maintaining antibody potency.

Anti-CD155 and anti-CD112 monoclonal antibodies conjugated to a fluorescent mesoporous silica nanosensor encapsulating rhodamine 6G and fluorescein for sensitive detection of liver cancer cells.

A novel method for sensitive detection of liver cancer cells using anti-CD155 and anti-CD112 monoclonal antibodies conjugated to ultrabright fluorescent mesoporous silica nanoparticles (FMSNs) encapsulating Rhodamine 6G and fluorescein was developed. The diameter of the obtained nanoparticles was 90 nm, and the quantum yield was 69%. Because the emission of fluorescein has a high degree of overlap with the excitation of Rhodamine 6G, and these two dyes were sufficiently close to each other on the nanoparticles, fluorescence resonance energy transfer can occur between these two dyes. This transfer not only maintains the original feature of the nanochannels and the skeletal network of the silica weakening the inner filtering of the dye, but also makes the excitation peak of the nanoparticles wider and increases the useful load amount of the dye. Because the wider Stokes shifts weaken the interference of excitation, the detection sensitivity is enhanced at the same time. The NaIO4 oxidation method does not use a cross-linker but rather uses covalent immobilization of the monoclonal antibodies on the FMSNs. This method can maintain the activity of the monoclonal antibodies more easily than the glutaraldehyde method. These advantages ensure that the nanosensor has high sensitivity and specificity for detecting liver cancer SMMC-7721 and HHCC cells. The in vivo imaging experiment also ensured that the biosensor can target tumor tissue in mice.