Multifunctional elastin-like polypeptide nanocarriers for efficient miRNA delivery in cancer therapy.

BACKGROUND: The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation. RESULTS: Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice. CONCLUSIONS: Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects.

IL4 receptor targeting enables nab-paclitaxel to enhance reprogramming of M2-type macrophages into M1-like phenotype via ROS-HMGB1-TLR4 axis and inhibition of tumor growth and metastasis.

Rationale: Nab-paclitaxel (Abx) is widely employed in malignant tumor therapy. In tumor cells and pro-tumoral M2-type macrophages, the IL4 receptor (IL4R) is upregulated. This study aimed to elucidate the selective delivery of Abx to M2-type macrophages by targeting IL4R and reprogramming them into an anti-tumoral M1-type. Methods: Abx was conjugated with the IL4R-binding IL4RPep-1 peptide using click chemistry (IL4R-Abx). Cellular internalization, macrophage reprogramming and signal pathways, and tumor growth and metastasis by IL4R-Abx were examined. Results: IL4R-Abx was internalized into M2 macrophages more efficiently compared to the unmodified Abx and control peptide-conjugated Abx (Ctrl-Abx), which was primarily inhibited using an anti-IL4R antibody and a receptor-mediated endocytosis inhibitor compared with a macropinocytosis inhibitor. IL4R-Abx reprogrammed the M2-type macrophages into M1-like phenotype and increased reactive oxygen species (ROS) levels and extracellular release of high mobility group box 1 (HMGB1) in M2 macrophages at higher levels than Abx and Ctrl-Abx. The conditioned medium of IL4R-Abx-treated M2 macrophages skewed M2 macrophages into the M1-like phenotype, in which an anti-HMGB1 antibody and a toll-like receptor 4 (TLR4) inhibitor induced a blockade. IL4R-Abx accumulated at tumors, heightened immune-stimulatory cells while reducing immune-suppressing cells, and hampered tumor growth and metastasis in mice more efficiently than Abx and Ctrl-Abx. Conclusions: These results indicate that IL4R-targeting allows enhancement of M2-macrophage shaping into M1-like phenotype by Abx through the ROS-HMGB1-TLR4 axis, improvement of antitumor immunity, and thereby inhibition of tumor growth and metastasis, presenting a new approach to cancer immunotherapy.

Immune Checkpoint-Blocking Nanocages Cross the Blood-Brain Barrier and Impede Brain Tumor Growth.

Glioblastoma (GBM) is the deadliest tumor of the central nervous system, with a median survival of less than 15 months. Despite many trials, immune checkpoint-blocking (ICB) therapies using monoclonal antibodies against the PD-1/PD-L1 axis have demonstrated only limited benefits for GBM patients. Currently, the main hurdles in brain tumor therapy include limited drug delivery across the blood-brain barrier (BBB) and the profoundly immune-suppressive microenvironment of GBM. Thus, there is an urgent need for new therapeutics that can cross the BBB and target brain tumors to modulate the immune microenvironment. To this end, we developed an ICB strategy based on the BBB-permeable, 24-subunit human ferritin heavy chain, modifying the ferritin surface with 24 copies of PD-L1-blocking peptides to create ferritin-based ICB nanocages. The PD-L1pep ferritin nanocages first demonstrated their tumor-targeting and antitumor activities in an allograft colon cancer model. Next, we found that these PD-L1pep ferritin nanocages efficiently penetrated the BBB and targeted brain tumors through specific interactions with PD-L1, significantly inhibiting tumor growth in an orthotopic intracranial tumor model. The addition of PD-L1pep ferritin nanocages to triple in vitro cocultures of T cells, GBM cells, and glial cells significantly inhibited PD-1/PD-L1 interactions and restored T-cell activity. Collectively, these findings indicate that ferritin nanocages displaying PD-L1-blocking peptides can overcome the primary hurdle of brain tumor therapy and are, therefore, promising candidates for treating GBM.

IRF3 Activation in Mast Cells Promotes FcεRI-Mediated Allergic Inflammation.

(1) Background: This study aims to elucidate a novel non-transcriptional action of IRF3 in addition to its role as a transcription factor in mast cell activation and associated allergic inflammation; (2) Methods: For in vitro experiments, mouse bone-marrow-derived mast cells (mBMMCs) and a rat basophilic leukemia cell line (RBL-2H3) were used for investigating the underlying mechanism of IRF3 in mast-cell-mediated allergic inflammation. For in vivo experiments, wild-type and Irf3 knockout mice were used for evaluating IgE-mediated local and systemic anaphylaxis; (3) Results: Passive cutaneous anaphylaxis (PCA)-induced tissues showed highly increased IRF3 activity. In addition, the activation of IRF3 was observed in DNP-HSA-treated mast cells. Phosphorylated IRF3 by DNP-HSA was spatially co-localized with tryptase according to the mast cell activation process, and FcεRI-mediated signaling pathways directly regulated that activity. The alteration of IRF3 affected the production of granule contents in the mast cells and the anaphylaxis responses, including PCA- and ovalbumin-induced active systemic anaphylaxis. Furthermore, IRF3 influenced the post-translational processing of histidine decarboxylase (HDC), which is required for granule maturation; and (4) Conclusion: Through this study, we demonstrated the novel function of IRF3 as an important factor inducing mast cell activation and as an upstream molecule for HDC activity.

Peptides as multifunctional players in cancer therapy.

Peptides exhibit lower affinity and a shorter half-life in the body than antibodies. Conversely, peptides demonstrate higher efficiency in tissue penetration and cell internalization than antibodies. Regardless of the pros and cons of peptides, they have been used as tumor-homing ligands for delivering carriers (such as nanoparticles, extracellular vesicles, and cells) and cargoes (such as cytotoxic peptides and radioisotopes) to tumors. Additionally, tumor-homing peptides have been conjugated with cargoes such as small-molecule or chemotherapeutic drugs via linkers to synthesize peptide-drug conjugates. In addition, peptides selectively bind to cell surface receptors and proteins, such as immune checkpoints, receptor kinases, and hormone receptors, subsequently blocking their biological activity or serving as hormone analogs. Furthermore, peptides internalized into cells bind to intracellular proteins and interfere with protein-protein interactions. Thus, peptides demonstrate great application potential as multifunctional players in cancer therapy.

Lupeol alleviates atopic dermatitis-like skin inflammation in 2,4-dinitrochlorobenzene/Dermatophagoides farinae extract-induced mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects from children to adults widely, presenting symptoms such as pruritus, erythema, scaling, and dryness. Lupeol, a pentacyclic triterpenoid, has anti-inflammatory and antimicrobial activities. Based on these properties, the therapeutic effects of lupeol on skin disorders have been actively studied. In the present study, we aimed to determine the effectiveness of lupeol on AD. METHODS: We utilized tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated keratinocytes and 2, 4-dinitrochlorobenzene/Dermatophagoides farinae extract (DFE)-induced AD mice to confirm the action. RESULTS: Lupeol inhibited TNF-α/IFN-γ-stimulated keratinocytes activation by reducing the expressions of pro-inflammatory cytokines and chemokines which are mediated by the activation of signaling molecules such as signal transducer and activator of transcription 1, mitogen-activated protein kinases (p38 and ERK), and nuclear factor-κB. Oral administration of lupeol suppressed epidermal and dermal thickening and immune cell infiltration in ear tissue. Immunoglobulin (Ig) E (total and DFE-specific) and IgG2a levels in serum were also reduced by lupeol. The gene expression and protein secretion of T helper (Th) 2 cytokines, Th1 cytokines, and pro-inflammatory cytokine in ear tissue were decreased by lupeol. CONCLUSIONS: These results suggest that lupeol has inhibitory effects on AD-related responses. Therefore, lupeol could be a promising therapeutic agent for AD.

Epigenetic therapy reprograms M2-type tumor-associated macrophages into an M1-like phenotype by upregulating miR-7083-5p.

Reprogramming M2-type, pro-tumoral tumor-associated macrophages (TAMs) into M1-type, anti-tumoral macrophages is a key strategy in cancer therapy. In this study, we exploited epigenetic therapy using the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylation inhibitor trichostatin A (TSA), to reprogram M2-type macrophages into an M1-like phenotype. Treatment of M2-type macrophages with the combination of 5-aza-dC and TSA decreased the levels of M2 macrophage cytokines while increasing those of M1 macrophage cytokines, as compared to the use of either therapy alone. Conditioned medium of M2 macrophages treated with the combination of 5-aza-dC and TSA sensitized the tumor cells to paclitaxel. Moreover, treatment with the combination inhibited tumor growth and improved anti-tumor immunity in the tumor microenvironment. Depletion of macrophages reduced the anti-tumor growth activity of the combination therapy. Profiling of miRNAs revealed that the expression of miR-7083-5p was remarkably upregulated in M2 macrophages, following treatment with 5-aza-dC and TSA. Transfection of miR-7083-5p reprogrammed the M2-type macrophages towards an M1-like phenotype, and adoptive transfer of M2 macrophages pre-treated with miR-7083-5p into mice inhibited tumor growth. miR-7083-5p inhibited the expression of colony-stimulating factor 2 receptor alpha and CD43 as candidate targets. These results show that epigenetic therapy upon treatment with the combination of 5-aza-dC and TSA skews M2-type TAMs towards the M1-like phenotype by upregulating miR-7083-5p, which contributes to the inhibition of tumor growth.

Interleukin-4 Receptor Targeting Peptide Decorated Extracellular Vesicles as a Platform for In Vivo Drug Delivery to Thyroid Cancer.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been demonstrated to deliver therapeutic drugs in preclinical studies. However, their use is limited, as they lack the ability to specifically deliver drugs to tumor tissues in vivo. In the present study, we propose the use of a targeting peptide, IL-4R-binding peptide (IL4RPep-1), to specifically deliver intravenously (i.v.) infused EVs to thyroid tumors. In vivo, a xenograft tumor model was treated with either the control peptide (NSSSVDK) or IL4RPep-1-Flamma; mice were fluorescently imaged (FLI) using an in vivo imaging system at 0-3 h post-treatment. EVs (labeled with DiD dye) were conjugated with IL4RPep-1 through a DOPE-NHS linker and administered to mice intravenously. FLI was performed 0-24 h post-injection, and the animals were sacrificed for further experiments. The morphology and size of EVs, the presence of EV markers such as CD63 and ALIX, and the absence of the markers GM130 and Cyto-C were confirmed. In vivo, FLI indicated an accumulation of i.v. injected IL4RPep-1-Flamma at the tumor site 90 min post-injection. No accumulation of NSSSVDK-Flamma was detected. In vivo, IL4RPep-1-EVs targeted the Cal-62 tumor 2 h post-injection. NSSSVDK-EVs were not even detected in the tumor 24 h post-injection. The quantification of FLI showed a significant accumulation of MSC-EVs in the tumor 2 h, 3 h, and 24 h post-injection. Furthermore, ex vivo imaging and an IF analysis confirmed the in vivo findings. Our results demonstrate the use of the IL4RPep-1 peptide as a targeting moiety of EVs for IL-4R-expressing anaplastic thyroid tumors.

Fibrinolytic nanocages dissolve clots in the tumor microenvironment, improving the distribution and therapeutic efficacy of anticancer drugs.

Fibrin, one of the components of the extracellular matrix (ECM), acts as a transport barrier within the core of tumors by constricting the blood vessels and forming clots, leading to poor intratumoral distribution of anticancer drugs. Our group previously developed a microplasmin-based thrombolytic ferritin nanocage that efficiently targets and dissolves clots without causing systemic fibrinolysis or disrupting hemostatic clots. We hypothesized that the thrombolytic nanocage-mediated degradation of fibrin clots in the tumor ECM can lead to enhanced intratumoral drug delivery, especially for nanosized anticancer drugs. Fibrin clot deposition worsens after surgery and chemotherapy, further hindering drug delivery. Moreover, the risk of venous thromboembolism (VTE) also increases. Here, we used thrombolytic nanocages with multivalent clot-targeting peptides and fibrin degradation enzymes, such as microplasmin, to dissolve fibrin in the tumor microenvironment and named them fibrinolytic nanocages (FNCs). These FNCs target tumor clots specifically and effectively. FNCs efficiently dissolve fibrin clots inside of the tumor vessels, suggesting that they can mitigate the risk of VTE in cancer patients. Coadministration of FNC and doxorubicin led to improved chemotherapeutic activity in a syngeneic mouse melanoma model. Furthermore, the FNCs increased the distribution of Doxil/doxorubicin nanoparticles within mouse tumors. These results suggest that fibrinolytic cotherapy might help improve the therapeutic efficacy of anticancer nanomedicines. Thus, microplasmin-based fibrinolytic nanocages are promising candidates for this strategy due to their hemostatic safety and ability to home in on the tumor.

The macrophage odorant receptor Olfr78 mediates the lactate-induced M2 phenotype of tumor-associated macrophages.

Expression and function of odorant receptors (ORs), which account for more than 50% of G protein-coupled receptors, are being increasingly reported in nonolfactory sites. However, ORs that can be targeted by drugs to treat diseases remain poorly identified. Tumor-derived lactate plays a crucial role in multiple signaling pathways leading to generation of tumor-associated macrophages (TAMs). In this study, we hypothesized that the macrophage OR Olfr78 functions as a lactate sensor and shapes the macrophage-tumor axis. Using Olfr78+/+ and Olfr78-/- bone marrow-derived macrophages with or without exogenous Olfr78 expression, we demonstrated that Olfr78 sensed tumor-derived lactate, which was the main factor in tumor-conditioned media responsible for generation of protumoral M2-TAMs. Olfr78 functioned together with Gpr132 to mediate lactate-induced generation of protumoral M2-TAMs. In addition, syngeneic Olfr78-deficient mice exhibited reduced tumor progression and metastasis together with an increased anti- versus protumoral immune cell population. We propose that the Olfr78-lactate interaction is a therapeutic target to reduce and prevent tumor progression and metastasis.

M1 macrophage exosomes engineered to foster M1 polarization and target the IL-4 receptor inhibit tumor growth by reprogramming tumor-associated macrophages into M1-like macrophages.

M2-polarized, pro-tumoral tumor-associated macrophages (TAMs) express the interleukin-4 receptor (IL4R) at higher levels compared with M1-polarized, anti-tumoral macrophages. In this study, we harnessed M1 macrophage-derived exosomes engineered to foster M1 polarization and target IL4R for the inhibition of tumor growth by reprogramming TAMs into M1-like macrophages. M1 exosomes were transfected with NF-κB p50 siRNA and miR-511-3p to enhance M1 polarization and were surface-modified with IL4RPep-1, an IL4R-binding peptide, to target the IL4 receptor of TAMs (named IL4R-Exo(si/mi). IL4R-Exo(si/mi) were internalized and downregulated target gens in M2 macrophages and decreased M2 markers, while increasing M1 markers, more efficiently compared with untargeted and control peptide-labeled exosomes and exosomes from non-immune, normal cells. Whole-body fluorescence imaging showed that IL4R-Exo(si/mi) homed to tumors at higher levels compared with the liver, unlike untargeted and control peptide-labeled exosomes. Systemic administration of IL4R-Exo(si/mi) inhibited tumor growth, downregulated target genes, and decreased the levels of M2 cytokines and immune-suppressive cells, while increasing the levels of M1 cytokines and immune-stimulatory cells, more efficiently than untargeted and control peptide-labeled exosomes. These results suggest that IL4R-Exo(si/mi) inhibits tumor growth by reprogramming TAMs into M1-like macrophages and increasing anti-tumor immunity, thus representing a novel cancer immunotherapy.

Therapeutic Effect of IL-4 Receptor-Targeting Pro-Apoptotic Peptide (AP1-ELP-KLAK) in Glioblastoma Tumor Model.

BACKGROUND: Thermal-responsive self-assembled elastin-like polypeptide (ELP)-based nanoparticles are an emerging platform for controlled delivery of therapeutic peptides, proteins and small molecular drugs. The antitumor effect of bioengineered chimeric polypeptide AP1-ELP-KLAK containing an interleukin-4 receptor (IL-4R) targeting peptide and pro-apoptotic peptide (KLAKLAK) was evaluated in glioblastoma (GBM) in vitro and in vivo. METHODS AND RESULTS: Herein, the therapeutic effect of AP1-ELP-KLAK was tested in advanced, and less curable glioblastoma cells with higher expression of IL-4R. Glioblastoma cell lines stably expressing different reporter systems i.e., caspase-3 sensor (surrogate marker for cellular apoptosis) or effluc/enhanced firefly luciferase (cellular viability) were established to measure cell death non-invasively. Bioluminescence imaging (BLI) of D54/effluc and U97MG/effluc treated with AP1-ELP-KLAK exhibited higher cell death up to 2~3-fold than the control. Treatment with AP1-ELP-KLAK resulted in time-dependent increase of caspase-3 sensor BLI activity in D54/C cells and D54/C tumor-bearing mice. Intravenous injection of AP1-ELP-KLAK dramatically reduced tumor growth by inducing cellular apoptosis in D54/effluc tumor-bearing mice. Further, the immuno-histological examination of the excised tumor tissue confirmed the presence of apoptotic cells as well as caspase-3 activation. CONCLUSION: Collectively, AP1-ELP-KLAK effectively induced cellular apoptosis of glioblastoma cells and non-invasive imaging provides a window for real-time monitoring of anti-tumor effect with the provision of improving therapeutic efficacy in a glioblastoma mice model.

Anticancer nanocage platforms for combined immunotherapy designed to harness immune checkpoints and deliver anticancer drugs.

The interaction of programmed cell death 1 ligand 1 (PD-L1) with its receptor, programmed cell death 1 (PD-1), inhibits T cell responses. Monoclonal antibodies that block this interaction have been shown effective as immunotherapy. However, only a subset of cancers exhibits a durable response to PD-1/PD-L1 blockade. Moreover, antibody-based immune checkpoint blockade is costly and is occasionally accompanied by systemic side effects. To overcome these limitations of antibody-based immune checkpoint blockade, an immune checkpoint-blocking ferritin nanocage displaying 24 PD-L1 binding peptides (PD-L1pep1) on its surface was designed and constructed. These ferritin nanocages displaying PD-L1pep1 (PpNF) specifically bind to PD-L1 expressed on cancer cells or to purified PD-L1 with a ~30 nM binding affinity. The addition of PpNF to co-cultures of T cells and cancer cells inhibited PD-1/PD-L1 interactions and restored T cell activities. In a mouse model of syngeneic colon cancer, PpNF specifically targeted tumors and showed antitumor activity. Moreover, PpNF nanocages encapsulating the chemotherapeutic drug doxorubicin had more potent antitumor activity than a monoclonal antibody against PD-L1. These results demonstrate that ferritin nanocages displaying surface PD-L1pep1 can be efficiently applied for immunotherapy, especially when encapsulating small chemotherapeutic drugs. These nanocages may have promise as an immunotherapeutic nanomedicine against various solid tumors.

Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice.

CD44v6, a splice variant of the cell surface glycoprotein CD44, acts as a co-receptor for c-Met and is upregulated in tumors with high metastatic potential. Methods: We screened a phage-displayed peptide library for peptides that selectively bind to CD44v6-overexpressing cells and exploited them to block CD44v6 and deliver a pro-apoptotic peptide to tumors for cancer therapy. Results: CNLNTIDTC (NLN) and CNEWQLKSC (NEW) peptides bound preferentially to CD44v6-high cells than to CD44v6-low cells. The binding affinities of NLN and NEW to CD44v6 protein were 253 ± 79 and 85 ± 18 nM, respectively. Peptide binding to CD44v6-high cells was inhibited by the knockdown of CD44v6 gene expression and competition with an anti-CD44v6 antibody. A pull-down assay with biotin-labeled peptides enriched CD44v6 from cell lysates. NLN and NEW induced CD44v6 internalization and inhibited hepatocyte growth factor-induced c-Met internalization, c-Met and Erk phosphorylation, and cell migration and invasion. In mice harboring tumors, intravenously administered NLN and NEW homed to the tumors and inhibited metastasis to the lungs. When combined with crizotinib, a c-Met inhibitor, treatment with each peptide inhibited metastatic growth more efficiently than each peptide or crizotinib alone. In addition, KLAKLAKKLAKLAK pro-apoptotic peptide guided by NLN (NLN-KLA) or NEW (NEW-KLA) killed tumor cells and inhibited tumor growth and metastasis. No significant systemic side effects were observed after treatments. Conclusions: These results suggest that NLN and NEW are promising metastasis-inhibiting peptide therapeutics and targeting moieties for CD44v6-expressing metastases.

Development of a Noninvasive KIM-1-Based Live-Imaging Technique in the Context of a Drug-Induced Kidney-Injury Mouse Model.

The development of reliable methods to diagnose acute kidney injury is essential to allow the adoption of early therapeutic interventions and evaluate their effectiveness. Based on the fact that kidney injury molecule-1 (KIM-1) expression levels in kidneys are markedly upregulated early after a damage event, here we developed a noninvasive KIM-1-based molecular imaging technique to detect kidney injury. First, we took advantage of a phage-display platform to select small peptides demonstrating a specific high binding affinity to KIM-1. The promising candidate was conjugated with fluorescent probes, and its imaging potential was validated in vitro and in vivo. This peptide, with the sequence CNRRRA, not only showed a high imaging potential in vitro, allowing a strong detection of KIM-1 expressing cells by microscopy and flow cytometry but also generated a strong kidney-specific signal in live-imaging in vivo experiments in the context of a drug-induced kidney-injury mouse model. Our data overall suggest that the CNRRRA peptide is a promising probe to use in the context of in vivo imaging for the detection of KIM-1 overexpression in damaged kidneys.

Designed ferritin nanocages displaying trimeric TRAIL and tumor-targeting peptides confer superior anti-tumor efficacy.

TRAIL is considered a promising target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors, DR4 or DR5. Although recombinant human TRAIL has shown high potency and specificity for killing cancer cells in preclinical studies, it has failed in multiple clinical trials for several reasons, including a very short half-life mainly caused by instability of the monomeric form of TRAIL and rapid renal clearance of the off-targeted TRAIL. To overcome such obstacles, we developed a TRAIL-active trimer nanocage (TRAIL-ATNC) that presents the TRAIL ligand in its trimer-like conformation by connecting it to a triple helix sequence that links to the threefold axis of the ferritin nanocage. We also ligated the tumor-targeting peptide, IL4rP, to TRAIL-ATNC to enhance tumor targeting. The developed TRAIL-ATNCIL4rP showed enhanced agonistic activity compared with monomeric TRAIL. The in vivo serum half-life of TRAIL-ATNCIL4rP was ~ 16-times longer than that of native TRAIL. As a consequence of these properties, TRAIL-ATNCIL4rP exhibited efficacy as an anti-tumor agent in vivo against xenograft breast cancer as well as orthotopic pancreatic cancer models, highlighting the promise of this system for development as novel therapeutics against cancer.

Inhibition of Tumor Growth against Chemoresistant Cholangiocarcinoma by a Proapoptotic Peptide Targeting Interleukin-4 Receptor.

Cholangiocarcinoma (CCA) has a poor prognosis and high chemoresistance. Interleukin-4 receptor (IL-4R) is overexpressed in several cancer cells and plays a crucial role in tumor progression and drug resistance. IL4RPep-1, an IL-4R-binding peptide, has been identified by phage display and used for tumor targeting. In this study, we exploited IL4RPep-1 to guide the tumor-specific delivery of a proapoptotic peptide to chemoresistant CCA, thereby inhibiting tumor growth. Immunohistochemistry of human primary CCA tissues showed that IL-4R levels were upregulated in moderately to poorly differentiated types, and higher levels of IL-4R are correlated with lower survival rates in patients with CCA. IL4RPep-1 was observed to preferentially bind with high IL-4R-expressing KKU-213 human CCA cells, whereas it barely bound with low IL-4R-expressing KKU-055 cells. A hybrid of IL4RPep-1 and a proapoptotic peptide (KLAKLAK)2 (named as IL4RPep-1-KLA) induced cytotoxicity and apoptosis in KKU-213 cells and increased those levels induced by 5-fluorouracil (5-FU). IL4RPep-1-KLA was internalized in the cells and colocalized with mitochondria. Whole-body fluorescence imaging and immunohistochemical analysis of tumor tissues showed the homing of IL4RPep-1-KLA as well as IL4RPep-1 to KKU-213 tumor in mice. Systemic administration of IL4RPep-1-KLA efficiently inhibited KKU-213 tumor growth, whereas treatment with 5-FU alone did not significantly inhibit tumor growth in mice. No significant systemic side effects including liver toxicity and immunotoxicity were observed in mice during peptide treatments. These findings suggest that IL4RPep-1-KLA holds potential as a targeted therapeutic agent against chemoresistant CCA.

Hispidulin alleviates imiquimod-induced psoriasis-like skin inflammation by inhibiting splenic Th1/Th17 cell population and keratinocyte activation.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes accompanied by increased infiltration of immune cells. Previous studies have demonstrated that hispidulin (4',5,7-trihydroxy-6-methoxyflavone, HPD) has various pharmacological benefits such as anti-fungal, anti-inflammation, and anti-allergic effects. This study investigated the effectiveness of HPD to treat psoriasis using an imiquimod (IMQ)-induced mouse model and activated keratinocytes. IMQ was topically applied to the back skin of mice for six consecutive days, and the mice were orally administered HPD. Based on the histological observation and immunological analysis, oral administration of HPD suppressed psoriatic characteristics including skin thickness, psoriasis area severity index, transepidermal water loss, and neutrophil infiltration. HPD alleviated pathologically increased levels of immunoglobulin G2a, myeloperoxidase, and tumor necrosis factor-α. Splenic Th1 and Th17 cell populations were also reduced by HPD in the murine model. In addition, in activated keratinocytes, HPD inhibited gene expression of Th1- and Th17-associated cytokines and chemokines, and phosphorylation of mitogen-activated protein kinases and nuclear factor-κB. In summary, HPD alleviates psoriasis skin inflammation in vivo and in vitro. Therefore, we suggest that HPD would be a potent therapeutic candidate for the treatment of psoriasis.

Phage display-identified PD-L1-binding peptides reinvigorate T-cell activity and inhibit tumor progression.

Blockade of programmed cell death ligand-1 (PD-L1) restores T-cell activity and enhances anti-tumor immunity. Screening a phage-displayed peptide library for peptides that selectively bind to PD-L1-overexpressing cells identified two peptides, CLQKTPKQC and CVRARTR (PD-L1Pep-1 and PD-L1Pep-2, respectively) that appeared to block PD-L1. PD-L1Pep-1 and PD-L1Pep-2 preferentially bound to high PD-L1-expressing cells over low PD-L1-expressing cells; binding was further enhanced by interferon-γ, an inducer of PD-L1 expression. Binding affinities of PD-L1Pep-1 and PD-L1Pep-2 were approximately 373 and 281 nM, respectively. Cellular binding of the PD-L1-binding peptides was reduced by silencing PD-L1 gene expression or competition with anti-PD-L1 antibody. PD-L1Pep-1 and PD-L1Pep-2 induced the internalization and downregulated cell surface levels of PD-L1. The PD-L1-binding peptides restored cytokine secretion and T-cell proliferation to cells inhibited by co-culture with tumor cells or culture on PD-L1-coated plates. Intravenously injected PD-L1Pep-1 and PD-L1Pep-2 efficiently homed to tumor tissues, inhibited tumor growth, and increased CD8+/FoxP3+ ratio in mice. The PD-L1-binding peptides in combination with doxorubicin or PD-L1-targeted liposomal doxorubicin inhibited tumor growth and increased CD8+/FoxP3+ ratio more efficiently than doxorubicin alone and untargeted liposomal doxorubicin, respectively. These results suggest that PD-L1Pep-1 and PD-L1Pep-2 block PD-L1 and reinvigorate T-cell activity, inhibiting tumor growth by enhancing anti-tumor immunity.