Rapid Internalization Research Articles

Abstract 2658: ZW251, a novel glypican-3-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload

Abstract Background: Glypican-3 (GPC3) is a cell-surface oncofetal glycoprotein frequently expressed in hepatocellular carcinoma (HCC) with minimal presence in normal adult tissues. ZW251 is an antibody-drug conjugate (ADC) targeting human GPC3, composed of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor, ZD06519, via a maleimide anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable linker. Materials and Methods: Extensive functional characterization was performed to assess the mechanism of action and therapeutic potential of the ZW251 ADC. Antibody binding to human and cynomolgus monkey GPC3 was assessed by surface plasmon resonance and flow cytometry. A screen of off-target binding and target specificity was conducted using a membrane proteome array. ZW251 antibody internalization in GPC3-expressing tumor cell lines was assessed by flow cytometry. In vitro ADC cytotoxicity against tumor monolayers and spheroids was assessed in a panel of HCC cell lines. Tumor cell co-culture assays were also performed to assess bystander-mediated cell killing by ZW251. The pharmacokinetic (PK) profile of the ZW251 antibody was assessed in Tg32 mice expressing human FcRn. Anti-tumor activity of ZW251 was investigated in a large panel of cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models representing a range of GPC3 expression. Results: The ZW251 antibody backbone demonstrated nanomolar binding affinity to both human and cynomolgus monkey GPC3, and strong binding to target-expressing cancer cell lines. Rapid internalization of ZW251 antibody was observed in GPC3-expressing HCC cell lines. ZW251 exhibited potent and target-specific cytotoxicity in a panel of HCC cells cultured either in monolayer or as 3D spheroids. ZW251 showed effective bystander-mediated killing of GPC3 negative cancer cells when in co-culture with GPC3 positive cancer cells. The ZW251 antibody demonstrated a favorable PK profile in Tg32 mice. A single administration of ZW251 resulted in robust tumor growth inhibition of a large panel of CDX and PDX models representing a range of GPC3-expression. Overall, these results support the potential of ZW251 as a novel therapeutic agent against GPC3-bearing cancers. Citation Format: Laurence Madera, Andrea Hernández Rojas, Raffaele Colombo, Alex Wu, Chayne L. Piscitelli, Dunja Urosev, Allysha Bissessur, Chi Wing Cheng, Renee Duan, Catrina Kim, Kevin Yin, Vincent Fung, Kaylee Wu, Winnie Cheung, Diego A. Alonzo, Mark E. Petersen, Stuart D. Barnscher, Jamie R. Rich. ZW251, a novel glypican-3-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2658.

The Construction of Polyphotocage Platform for Anticancer Photochemotherapy

AbstractPhotocages enable the precise activation of molecular function with light in many research fields, such as anticancer treatment, where remote spatiotemporal control over the release of an active drug is needed. However, the poor physiological stability and tumor accumulation of conventional small molecular photocages are significant obstacles to developing efficient therapy in vivo. In this study, a new concept of “polyphotocage” is proposed through photocage–polymer hybrid macromolecular engineering. Photoresponsive Ru complex photocage is designed and fused with PEGylated polycarbonates, resulting in the polyphotocage. Various anticancer drugs can be readily conjugated to the polyphotocage via coordination linkage, which can be cut off to release drugs by red light. The polyphotocages can self‐assemble into nanoparticles, which enhances the stability of the Ru photocage and demonstrates the efficient accumulation in different‐sized tumorswith a high signal‐to‐background ratio. Furthermore, rapid cellular internalization and mitochondrial anchoring capability allowed the polyphotocages to deliver drugs into the mitochondria, which induces mitochondrial dysfunction and cell death. These properties ensure the effective delivery of anticancer drugs to solid tumors and multiple tiny tumors, ultimately inhibiting tumor proliferation. This strategy of polyphotocages provides a new platform for the future design of drug‐delivery systems for cancer photochemotherapy.

Protein adsorption determines pulmonary cell uptake of lipid-based nanoparticles

The inhalable administration of lipid nanoparticles is an effective strategy for localised delivery of therapeutics against various lung diseases. Of this, improved intracellular delivery of pharmaceuticals for infectious disease and cancer management is of high significance. However, the influence of lipid nanoparticle composition and structure on uptake in pulmonary cell lines, especially in the presence of biologically relevant media is poorly understood. Here, the uptake of lamellar (liposomes) versus non-lamellar (cubosomes) lipid nanoparticles in macrophages and lung epithelial cells was quantified and the influence of bronchoalveolar lavage fluid (BALF), containing native pulmonary protein and surfactant molecules is determined. Cubosome uptake in both macrophages and epithelial cells was strongly mediated by a high percentage of molecular function regulatory and binding proteins present within the protein corona. In contrast, the protein corona did not influence the uptake of liposomes in epithelial cells. In macrophages, the proteins mediated a rapid internalisation, followed by exocytosis of liposomes after 6 h incubation. These findings on the influence of biological fluid in regulating lipid nanoparticle uptake mechanisms may guide future development of optimal intracellular delivery systems for therapeutics via the pulmonary route.

Piperazine-modified dendrimer achieves efficient intracellular protein delivery via caveolar endocytosis bypassing the endo-lysosomal pathway.

Intracellular protein delivery has been a major challenge due to various physiological barriers including low proteolytic stability and poor membrane permeability of the biologics. Nanoparticles were widely proposed to deliver cargo proteins into cells by endocytosis, however, the materials and complexes with proteins are often entrapped in endosomes and subject to lysosome degradation. In this study, we report a piperazine modified dendrimer for stabilizing the complexes via a combination of electrostatic interaction and hydrophobic interactions. The complexes show rapid cell internalization and the loaded proteins are released into the cytosols as early as half an hour post incubation. Mechanism study suggests that the complexes are endocytosed into cells via caveolae-based pathways, which could be inhibited by inhibitors such as genistein, filipin III, brefeldin A and nystatin. The phenylpiperazine-modified polymer enables the delivery of cargo proteins with reserved bioactivity and show high permeability in three-dimensional cell spheroids. The results prove the beneficial roles of phenylpiperazine ligands in polymer-mediated cytosolic protein delivery systems. STATEMENT OF SIGNIFICANCE: We synthesized a list of piperazine and derivatives modified dendrimers as cytosolic protein delivery vectors via facile reactions. Phenylpiperazine modification enables the efficient protein binding through the combination of electrostatic, hydrogen bonding and hydrophobic interactions. Phenylpiperazine modified dendrimers were internalized into the cells via a caveolae-based endo/lysosome-independent path and could release the cargo proteins into the cytosols as early as half an hour post incubation. Phenylpiperazine modified dendrimers delivered cargo proteins with reserved bioactivity and showed high permeability in three-dimensional cell spheroids.

Enhancing Anti-Tumorigenic Efficacy of Eugenol in Human Colon Cancer Cells Using Enzyme-Responsive Nanoparticles

This study is focused on the selective delivery and release of the plant-based anticancer compound eugenol (EUG) in colorectal cancer cells (CRC). EUG is an apoptotic and anti-growth compound in diverse malignant tumors, including CRC. However, EUG's rapid metabolization, excretion, and side effects on normal cells at higher dosages are major limitations of its therapeutic potential. To address this problem, we developed a "smart" enzyme-responsive nanoparticle (eNP) loaded with EUG that exposes tumors to a high level of the drug while keeping its concentration low among healthy cells. We demonstrated that EUG induces apoptosis in CRC cells irrespective of their grades in a dose- and time-dependent manner. EUG significantly decreases cancer cell migration, invasion, and the population of colon cancer stem cells, which are key players in tumor metastasis and drug resistance. The "smart" eNPs-EUG show a high affinity to cancer cells with rapid internalization with no affinity toward normal colon epithelial cells. NPs-EUG enhanced the therapeutic efficacy of EUG measured by a cell viability assay and showed no toxicity effect on normal cells. The development of eNPs-EUG is a promising strategy for innovative anti-metastatic therapeutics.

CXCR7 as a novel therapeutic target for advanced prostate cancer.

Chemokines and their cognate receptors comprise an intricate signaling network that becomes high-jacked by cancer cells for uncontrollable tumor growth and dissemination. ACKR3 (Atypical Chemokine Receptor 3), traditionally called CXCR7, is up-regulated in many cancers, including advanced prostate cancer, and represents promising targets for therapeutic intervention. Unlike typical G protein-coupled receptors such as CXCR4, CXCR7, once bound by its cognate ligand CXCL12, initiates the recruitment of β-arrestin instead of G proteins, and results in rapid internalization and degradation of CXCL12, functioning as a scavenger receptor. However, recent evidence suggests that CXCR7 may be more than a scavenger or auxiliary receptor of CXCR4 and that it may play essential roles in regulating cancer progression, some of which are independent of CXCR4 and its ligands, such as CXCL12. Constitutively active CXCR7 binds to β-arrestin. This protein complex internalizes to form a scaffold for assembling and activating various cytoplasmic kinases necessary for cell survival and tumor growth. Here we review and discuss the up-to-date knowledge on CXCR7 regulation and function and how this new understanding guides the development of CXCR7 inhibitors, focusing on prostate cancer.

Feasibility of Co-Targeting HER3 and EpCAM Using Seribantumab and DARPin-Toxin Fusion in a Pancreatic Cancer Xenograft Model.

Pancreatic cancer (PC) is one of the most aggressive malignancies. A combination of targeted therapies could increase the therapeutic efficacy in tumors with heterogeneous target expression. Overexpression of the human epidermal growth factor receptor type 3 (HER3) and the epithelial cell adhesion molecule (EpCAM) in up to 40% and 30% of PCs, respectively, is associated with poor prognosis and highlights the relevance of these targets. Designed ankyrin repeat protein (DARPin) Ec1 fused with the low immunogenic bacterial toxin LoPE provides specific and potent cytotoxicity against EpCAM-expressing cancer cells. Here, we investigated whether the co-targeting of HER3 using the monoclonal antibody seribantumab (MM-121) and of EpCAM using Ec1-LoPE would improve the therapeutic efficacy in comparison to the individual agents. Radiolabeled 99mTc(CO)3-Ec1-LoPE showed specific binding with rapid internalization in EpCAM-expressing PC cells. MM-121 did not interfere with the binding of Ec1-LoPE to EpCAM. Evaluation of cytotoxicity indicated synergism between Ec1-LoPE and MM-121 in vitro. An experimental therapy study using Ec1-LoPE and MM-121 in mice bearing EpCAM- and HER3-expressing BxPC3 xenografts demonstrated the feasibility of the therapy. Further development of the co-targeting approach using HER3 and EpCAM could therefore be justified.

A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection.

Most of the cholesterol in the plasma membranes (PMs) of animal cells is sequestered through interactions with phospholipids and transmembrane domains of proteins. However, as cholesterol concentration rises above the PM's sequestration capacity, a new pool of cholesterol, called accessible cholesterol, emerges. The transport of accessible cholesterol between the PM and the endoplasmic reticulum (ER) is critical to maintain cholesterol homeostasis. This pathway has also been implicated in the suppression of both bacterial and viral pathogens by immunomodulatory oxysterols. Here, we describe a mechanism of depletion of accessible cholesterol from PMs by the oxysterol 25-hydroxycholesterol (25HC). We show that 25HC-mediated activation of acyl coenzyme A: cholesterol acyltransferase (ACAT) in the ER creates an imbalance in the equilibrium distribution of accessible cholesterol between the ER and PM. This imbalance triggers the rapid internalization of accessible cholesterol from the PM, and this depletion is sustained for long periods of time through 25HC-mediated suppression of SREBPs and continued activation of ACAT. In support of a physiological role for this mechanism, 25HC failed to suppress Zika virus and human coronavirus infection in ACAT-deficient cells, and Listeria monocytogenes infection in ACAT-deficient cells and mice. We propose that selective depletion of accessible PM cholesterol triggered by ACAT activation and sustained through SREBP suppression underpins the immunological activities of 25HC and a functionally related class of oxysterols.

A CO2-Responsive Imidazole-Functionalized Fluorescent Material Mediates Cancer Chemotherapy.

We present a breakthrough in the synthesis and development of functional gas-responsive materials as highly potent anticancer agents suitable for applications in cancer treatment. Herein, we successfully synthesised a stimuli-responsive multifunctional material (I-R6G) consisting of a carbon dioxide (CO2)-sensitive imidazole moiety and spirolactam-containing conjugated rhodamine 6G (R6G) molecule. The resulting I-R6G is highly hydrophobic and non- or weakly fluorescent. Simple CO2 bubbling treatment induces hydrophobic I-R6G to completely dissolve in water and subsequently form self-assembled nanoparticles, which exhibit unique optical absorption and fluorescence behaviours in water and extremely low haemolytic ability against sheep red blood cells. Reversibility testing indicated that I-R6G undergoes reversible CO2/nitrogen (N2)-dependent stimulation in water, as its structural and physical properties can be reversibly and stably switched by alternating cycles of CO2 and N2 bubbling. Importantly, in vitro cellular assays clearly demonstrated that the CO2-protonated imidazole moiety promotes rapid internalisation of CO2-treated I-R6G into cancer cells, which subsequently induces massive levels of necrotic cell death. In contrast, CO2-treated I-R6G was not internalised and did not affect the viability of normal cells. Therefore, this newly created system may provide an innovative and efficient route to remarkably improve the selectivity, safety and efficacy of cancer treatment.

Fusogenic liposome-coated nanoparticles for rapid internalization into donor corneal endothelial tissue to enable prophylaxis before transplantation.

Cold stress (hypothermia) during storage and cytokine stress due to acute allograft rejection adversely affect the donor corneal endothelium in the short term. Pharmacological pre-treatment (before transplantation) of the donor corneal endothelium or cells (propagated in vitro for cell injection therapy) with microtubule stabilizers, cold stress protectants, and other molecules is an attractive strategy to tackle damage caused by hypothermia and cytokine stress. These molecules can be delivered intracellularly to the donor corneal endothelium or cells at controlled rates for desired periods and with one-time administration using nanoparticles. However, the death-to-preservation time of donor corneas of more than 4 to 6 h significantly decreases endothelial cell density and increases the risk of microbial contamination. Therefore, we have developed fusogenic liposome-coated nanoparticles for rapid internalization of nanoparticles into cultured corneal endothelial cells and ex vivo corneal endothelial tissue. Here, we have shown that the fusogenic liposome-coated nanoparticles have the intrinsic ability to efficiently and rapidly internalize into cultured corneal endothelial cells and ex vivo corneal tissue within 3 h by possibly fusing with the cell membrane and bypassing the endocytic pathway. Lactate dehydrogenase assay showed that the internalized fusogenic liposome-coated nanoparticles did not cause cytotoxicity in endothelial cells associated with the ex vivo cornea for at least up to 2 days. Thus, fusogenic liposome-coated nanoparticles have great potential as a platform for engineering cells and endothelial tissue of donor corneas to facilitate prophylactic drug delivery during storage and after transplantation.

Zwitterionic rhodamine-CPT prodrug nanoparticles with GSH/H2O2 responsiveness for cancer theranostics.

Rationale: Fluorescently traceable prodrugs, which can monitor their biodistribution in vivo and track the kinetics of drug delivery in living cells, are promising for constructing theranostic medicines. However, due to their charge and hydrophobicity, most of the fluorescently traceable prodrugs exhibit high protein binding and non-specific tissue retention affecting in vivo distribution and toxicity, with high background signals. Methods: Herein, the zwitterionic rhodamine (RhB) and camptothecin (CPT) were bridged with a disulfide bond to construct a tumorous heterogeneity-activatable prodrug (RhB-SS-CPT). The interaction of zwitterionic RhB-SS-CPT with proteins was detected by UV and fluorescence spectroscopy, and further demonstrated by molecular docking studies. Then, intracellular tracking and cytotoxicity of RhB-SS-CPT were determined in tumor and normal cells. Finally, the in vivo biodistribution, pharmacokinetics, and anticancer efficacy of RhB-SS-CPT were evaluated in a mouse animal model. Results: The tumorous heterogeneity-activatable RhB-SS-CPT prodrug can self-assemble into stable nanoparticles in water based on its amphiphilic structure. Particularly, the zwitterionic prodrug nanoparticles reduce the non-specific binding to generate a low background signal for better identification of cancerous lesions, achieve rapid internalization into cancer cells, selectively release bioactive CPT as a cytotoxic agent in response to high levels of GSH and H2O2, and exhibit high fluorescence that contributes to the visual chemotherapy modality. In addition, the RhB-SS-CPT prodrug nanoparticles show longer circulation time and better antitumor activity than free CPT in vivo. Interestingly, the zwitterionic nature allows RhB-SS-CPT to be excreted through the renal route, with fewer side effects. Conclusions: Zwitterionic features and responsive linkers are important considerations for constructing potent prodrugs, which provide some useful insights to design the next-generation of theranostic prodrugs for cancer.

Green synthesis of gardenia seeds-based carbon dots for bacterial imaging and antioxidant activity in aqueous and oil samples.

In this work, luminescent carbon dots with gardenia seeds as carbon precursors (GCDs) were synthesized using a one-step mild pyrolysis process and were then used as probes for imaging of bacterial (Escherichia coli). The GCDs showed a strong emission at 430 nm when excited at 370 nm. The relative fluorescence quantum yield of GCDs was found to be 1.13% in an aqueous medium. Rapid internalization of the GCDs by bacteria was confirmed by three colors (blue, green, and yellow) images that were obtained using confocal fluorescence microscopy. In addition, GCDs were noted to exhibit potent scavenging activities against DPPH˙, ˙OH, and ˙O2- free radicals. GCDs were also assayed as antioxidants in an oil sample by volumetric determination of the peroxide value. Thus, GCDs exhibited good antioxidant properties both in aqueous and oil media. In addition, a free fatty acid quantification kit in the presence of GCDs showed enhanced fluorescence detection of palmitic acid with a remarkably good limit of detection of 0.08 μM, which is lower than that in the absence of GCDs (0.76 μM). The proposed fluorescence method was then successfully used to determine the concentration of palmitic acid spiked in milk powder samples, with spiked recoveries of 82.6-109.6% and relative standard deviations of 0.9-4.6%.

Effects of small molecule-induced dimerization on the programmed death ligand 1 protein life cycle

The programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) checkpoint blockade is central to Immuno-Oncology based therapies, and alternatives to antibody blockers of this interaction are an active area of research due to antibody related toxicities. Recently, small molecule compounds that induce PD-L1 dimerization and occlusion of PD-1 binding site have been identified and developed for clinical trials. This mechanism invokes an oligomeric state of PD-L1 not observed in cells previously, as PD-L1 is generally believed to function as a monomer. Therefore, understanding the cellular lifecycle of the induced PD-L1 dimer is of keen interest. Our report describes a moderate but consistent increase in the PD-L1 rate of degradation observed upon protein dimerization as compared to the monomer counterpart. This subtle change, while not resolved by measuring total PD-L1 cellular levels by western blotting, triggered investigations of the overall protein distribution across various cellular compartments. We show that PD-L1 dimerization does not lead to rapid internalization of neither transfected nor endogenously expressed protein forms. Instead, evidence is presented that dimerization results in retention of PD-L1 intracellularly, which concomitantly correlates with its reduction on the cell surface. Therefore, the obtained data for the first time points to the ability of small molecules to induce dimerization of the newly synthesized PD-L1 in addition to the protein already present on the plasma membrane. Overall, this work serves to improve our understanding of this important target on a molecular level in order to guide advances in drug development.

Live‐cell imaging of internalised oligomeric tau reveals differences between neuronal cell types

AbstractBackgroundIt has recently been established that pathological forms of aggregated tau spread from affected to recipient cells in the brain, facilitating disease progression. Here we have applied live‐cell imaging to visualise internalised tau in recipient neuronal cells.MethodRecombinant human 2N4R tau was aggregated using heparin and dithiothreitol, sonicated, and labelled with the fluorescent dye, Cy5. Differentiated SH‐SY5Y human neuroblastoma cells (dSH‐SY5Y), mouse primary cortical neurons (PCNs) and human induced pluripotent stem cell‐derived neurons (iPSCs) were exposed to Cy5‐tau for up to 7 days. Confocal microscopy was used to visualise internalised Cy5‐tau in live‐cells and paraformaldehyde‐fixed cells. BODIPY 493/503 and Lysotracker dyes were used to label neutral lipids and lysosomes, respectively.ResultLive‐cell imaging showed rapid (within five minutes) internalisation of Cy5‐tau by all cell types, appearing as small speckles (<0.1µm diameter), with larger masses (>0.5 µm diameter) forming over time. In contrast, only small speckles of Cy5‐tau were detectable inside fixed cells. The size of Cy5‐tau accumulations in PCNs, but not in dSH‐SY5Y or iPSCs, increased further over time, reaching up to 6 µm diameter after 7 days. Lysotracker labelling of live cells indicated significant colocalisation with Cy5‐tau in all three cell types. BODIPY also partially colocalised with both Lysotracker and Cy5‐tau in PCN and iPSCs, but not in dSH. Furthermore, some large (>5 µm diameter) Cy5‐tau spheres in PCNs were Lysotracker‐negative, indicating that incorporation of Cy5‐tau may deacidify lysosomes.ConclusionLive‐cell imaging of neuronal cells has revealed previously unreported internalised tau structures that are not detectable in fixed cells. The sizes of tau spheres that form inside cells exposed to oligomeric tau differ between neuronal cell types, which is a consideration for selecting the most appropriate disease model. The colocalisation between Cy5‐tau and specific organelle labelling indicated tau intracellular trafficking pathway.

Gallic Acid-Triethylene Glycol Aptadendrimers Synthesis, Biophysical Characterization and Cellular Evaluation.

Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid-triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.

Less is More: Preorganization Leads to Better Tumor Retention and Therapeutic Efficacy

AbstractProtein preorganization is ubiquitous in nature and enables subtle structural rearrangements and precise function executions, making it an attractive approach for generating functional peptide materials. Here, a phosphorylation‐dependent pre‐organization approach to optimize the therapeutic outcomes of a self‐assembling peptide targeting programmed cell death ligand 1 (PD‐L1) is reported. Upon incorporating the imaging probe cyanine 5.5 (Cy5.5) and therapeutic drug 10‐hydroxycamptothecin (HCPT), three phosphorylated precursors (1P, 2P, and 3P) containing identical peptide sequences with different tyrosine phosphorylation sites are constructed. The presence of a phosphate group can stabilize the self‐assembling peptide in a well‐defined, preorganized state. Moreover, variations in the phosphorylation sites lead to different preorganized secondary structures, dephosphorylation rates, and PD‐L1 binding affinities. Responding to alkaline phosphatase dephosphorylation, subtle structural transformation to more ordered states, an increase in binding affinity against PD‐L1, and rapid cellular internalization are observed. Compared with the unphosphorylated control (0P), the preorganization of 1P, 2P, and 3P can optimize tumor retention and resultant therapeutic performance, of which 3P maximizes the final therapeutic outcomes, leading to a 70.3% tumor inhibition rate. This study highlights the great potential to control the subtle structural transformation and therapeutic effects of self‐assembling peptides by preparing their preorganized entities.

Preclinical Evaluation of 68Ga- and 177Lu-Labeled Integrin αvβ6-Targeting Radiotheranostic Peptides.

The integrin αvβ6, an epithelium-specific cell surface receptor, is overexpressed on numerous malignancies, including the highly lethal pancreatic ductal adenocarcinomas. Here, we developed and tested a novel αvβ6-targeting peptide, DOTA-5G (1) radiolabeled with 68Ga, for PET/CT imaging and 177Lu for treatment. With the goal to develop a radiotheranostic, further modifications were made for increased circulation time, renal recycling, and tumor uptake, yielding DOTA-albumin-binding moiety-5G (2). Methods: Peptides 1 and 2 were synthesized on solid phase, and their affinity for αvβ6 was assessed by enzyme-linked immunosorbent assay. The peptides were radiolabeled with 68Ga and 177Lu. In vitro cell binding, internalization, and efflux of 68Ga-1 and 177Lu-2 were evaluated in αvβ6-positive BxPC-3 human pancreatic cancer cells. PET/CT imaging of 68Ga-1 and 68Ga-2 was performed on female nu/nu mice bearing subcutaneous BxPC-3 tumors. Biodistribution was performed for 68Ga-1 (1 and 2 h after injection), 68Ga-2 (2 and 4 h after injection), and 177Lu-1 and 177Lu-2 (1, 24, 48, and 72 h after injection). The 177Lu-2 biodistribution data were extrapolated for human dosimetry data estimates using OLINDA/EXM 1.1. Therapeutic efficacy of 177Lu-2 was evaluated in mice bearing BxPC-3 tumors. Results: Peptides 1 and 2 demonstrated high affinity (<55 nM) for αvβ6 by enzyme-linked immunosorbent assay. 68Ga-1, 68Ga-2, 177Lu-1, and 177Lu-2 were synthesized in high radiochemical purity. Rapid invitro binding and internalization of 68Ga-1 and 177Lu-2 were observed in BxPC-3 cells. PET/CT imaging and biodistribution studies demonstrated uptake in BxPC-3 tumors. Introduction of the albumin-binding moiety in 177Lu-2 resulted in a 5-fold increase in tumor uptake and retention over time. Based on the extended dosimetry data, the dose-limiting organ for 177Lu-2 is the kidney. Treatment with 177Lu-2 prolonged median survival by 1.5- to 2-fold versus controls. Conclusion: 68Ga-1 and 177Lu-2 demonstrated high affinity for the integrin αvβ6 both invitro and invivo, were rapidly internalized into BxPC-3 cells, and were stable in mouse and human serum. Both radiotracers showed favorable pharmacokinetics in preclinical studies, with predominantly renal excretion and good tumor-to-normal-tissue ratios. Favorable human dosimetry data suggest the potential of 177Lu-2 as a treatment for pancreatic ductal adenocarcinoma.

Ca2+ transport via TRPV6 is regulated by rapid internalization of the channel

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca2+-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca2+]i with a half time in the range of 0.5–1 h. Upon wash-out of the toxin, the [Ca2+]i rose to a steady-state level within 2–3 h. Consistently, the synchronized forward trafficking of TRPV6VL-eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6VL-bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca2+ transport.

Rapid macropinocytic transfer of α-synuclein to lysosomes.

The nervous system spread of alpha-synuclein fibrils is thought to cause Parkinson's disease (PD) and other synucleinopathies; however, the mechanisms underlying internalization and cellular spread are enigmatic. Here, we use confocal and superresolution microscopy, subcellular fractionation, and electron microscopy (EM) of immunogold-labeled α-synuclein preformed fibrils (PFFs) to demonstrate that this form of the protein undergoes rapid internalization and is targeted directly to lysosomes in as little as 2min. Uptake of PFFs is disrupted by macropinocytic inhibitors and circumvents classical endosomal pathways. Immunogold-labeled PFFs are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, in multivesicular bodies and in lysosomes. While most fibrils remain in lysosomes, a portion is transferred to neighboring naive cells along with markers of exosomes. These data indicate that PFFs use a unique internalization mechanism as a component of cell-to-cell propagation.

EGFR Activation Impairs Antiviral Activity of Interferon Signaling in Brain Microvascular Endothelial Cells During Japanese Encephalitis Virus Infection

The establishment of Japanese encephalitis virus (JEV) infection in brain microvascular endothelial cells (BMECs) is thought to be a critical step to induce viral encephalitis with compromised blood–brain barrier (BBB), and the mechanisms involved in this process are not completely understood. In this study, we found that epidermal growth factor receptor (EGFR) is related to JEV escape from interferon-related host innate immunity based on a STRING analysis of JEV-infected primary human brain microvascular endothelial cells (hBMECs) and mouse brain. At the early phase of the infection processes, JEV induced the phosphorylation of EGFR. In JEV-infected hBMECs, a rapid internalization of EGFR that co-localizes with the endosomal marker EEA1 occurred. Using specific inhibitors to block EGFR, reduced production of viral particles was observed. Similar results were also found in an EGFR-KO hBMEC cell line. Even though the process of viral infection in attachment and entry was not noticeably influenced, the induction of IFNs in EGFR-KO hBMECs was significantly increased, which may account for the decreased viral production. Further investigation demonstrated that EGFR downstream cascade ERK, but not STAT3, was involved in the antiviral effect of IFNs, and a lowered viral yield was observed by utilizing the specific inhibitor of ERK. Taken together, the results revealed that JEV induces EGFR activation, leading to a suppression of interferon signaling and promotion of viral replication, which could provide a potential target for future therapies for the JEV infection.