Antibody-Assisted Delivery of a Peptide-Drug Conjugate for Targeted Cancer Therapy.

A number of cancer-targeting peptide-drug conjugates (PDCs) have been explored as alternatives to antibody-drug conjugates (ADCs) for targeted cancer therapy. However, the much shorter circulation half-life of PDCs compared with ADCs in vivo has limited their therapeutic value and thus their translation into the clinic, highlighting the need to develop new approaches for extending the half-life of PDCs. Here, we report a new strategy for targeted cancer therapy of a PDC based on a molecular hybrid between an antihapten antibody and a hapten-labeled PDC. An anticotinine antibody (Abcot) was used as a model antihapten antibody. The anticancer drug SN38 was linked to a cotinine-labeled aptide specific to extra domain B of fibronectin (cot-APTEDB), yielding the model PDC, cot-APTEDB-SN38. The cotinine-labeled PDC showed specific binding to and cytotoxicity toward an EDB-overexpressing human glioblastoma cell line (U87MG) and also formed a hybrid complex (HC) with Abcot in situ, designated HC[cot-APTEDB-SN38/Abcot]. In glioblastoma-bearing mice, in situ HC[cot-APTEDB-SN38/Abcot] significantly extended the circulation half-life of cot-APTEDB-SN38 in blood, and it enhanced accumulation and penetration within the tumor and, ultimately, inhibition of tumor growth. These findings suggest that the present platform holds promise as a new, targeted delivery strategy for PDCs in anticancer therapy.

Targeted Cancer Therapy Using Fusion Protein of TNFα and Tumor-Associated Fibronectin-Specific Aptide.

Tumor necrosis factor-α has shown potent antitumor effects in preclinical and clinical studies. However, severe side effects at less than therapeutic doses have limited its systemic delivery, prompting the need for a new strategy for targeted delivery of the protein to tumors. Here, we report a fusion protein of mouse tumor necrosis factor (TNF)-α (mTNFα) and a cancer-targeting, high-affinity aptide and investigate its therapeutic efficacy in tumor-bearing mice. A fusion protein consisting of mTNFα, a linker, and an aptide specific to extra domain B (EDB) of fibronectin (APTEDB), designated mTNFα-APTEDB, was successfully produced by expression in Escherichia coli. mTNFα-APTEDB retained specificity and affinity for its target, EDB. In mice bearing EDB-overexpressing fibrosarcomas, mTNFα-APTEDB showed greater efficacy in inhibiting tumor growth than mTNFα alone or mTNFα linked to a nonrelevant aptide, without causing an appreciable loss in body weight. Moreover, in vivo antitumor efficacy was further significantly increased by combination treatment with the chemotherapeutic drug, melphalan, suggesting a synergistic effect attributable to enhanced drug uptake into the tumor as a result of TNFα-mediated enhanced vascular permeability. These results suggest that a fusion protein of mTNFα with a cancer-targeting peptide could be a new anticancer therapeutic option for ensuring potent antitumor efficacy after systemic delivery.

Targeting the tumor microenvironment with amphiphilic near-infrared cyanine nanoparticles for potentiated photothermal immunotherapy.

Despite the potential of photothermal therapy (PTT) for cancer treatments, PTT alone has limitations in treating metastatic tumors and preventing tumor recurrence, highlighting the need to combine PTT with immunotherapy. This study reports tumor microenvironment (TME)-targeting, near-infrared (NIR) dye derivative-based nanomedicine for effective combined PTT-immunotherapy. Amphiphilic NIR dye cyanine derivatives are used not only for constructing the nanoparticle mass, but also for creating a stable complex with CpG adjuvant; a peptide specific to fibronectin extra domain B (APTEDB) is also introduced as a TME-targeting ligand, yielding the TME-targeting nanomedicine, APTEDB-cyNP@CpG. APTEDB-cyNP@CpG shows cancer-targeting ability in EDB-overexpressing CT26 colon tumor-bearing mice. When combined with laser irradiation, it induces immunogenic cell death (ICD) and subsequently leads to significant increase in CD8+ T cell population in the tumor, resulting in greater antitumor therapeutic efficacy than does cyNP@CpG lacking the TME-targeting ligand. Moreover, the combination of APTEDB-cyNP@CpG-based PTT and an immune checkpoint blockade (ICB) antibody leads to remarkable antitumor efficacy against the laser-irradiated primary tumor as well as distant tumor through potentiation of systemic cancer cell-specific T cell immunity. Furthermore, the PTT-immunotherapy combination regimen is highly effective in inhibiting tumor recurrence and metastasis.

Nanoparticles targeting extra domain B of fibronectin-specific to the atherosclerotic lesion types III, IV, and V-enhance plaque detection and cargo delivery.

Extra domain B of fibronectin (FN-EDB) is upregulated in the extracellular matrix during tissue remodeling and has been postulated as a potential biomarker for atherosclerosis, yet no systematic test for FN-EDB in plaques has been reported. We hypothesized that FN-EDB expression would intensify in advanced plaques. Furthermore, engineering of FN-EDB-targeted nanoparticles (NPs) could enable imaging/diagnosis and local delivery of payloads to plaques. Methods: The amount of FN-EDB in human atherosclerotic and normal arteries (ages: 40 to 85 years) was assessed by histological staining and quantification using an FN-EDB-specific aptide (APTFN-EDB). FN-EDB-specific NPs that could serve as MRI beacons were constructed by immobilizing APTFN-EDB on the NP surface containing DTPA[Gd]. MRI visualized APTFN-EDB-[Gd]NPs administered to atherosclerotic apolipoprotein E-deficient mice in the brachiocephalic arteries. Analysis of the ascending-to-descending thoracic aortas and the aortic roots of the mice permitted quantitation of Gd, FN-EDB, and APTFN-EDB-[Gd]NPs. Cyanine, a model small molecule drug, was used to study the biodistribution and pharmacokinetics of APTFN-EDB-NPs to evaluate their utility for drug delivery. Results: Atherosclerotic tissues had significantly greater FN-EDB-positive areas than normal arteries (P < 0.001). This signal pertained particularly to Type III (P < 0.01), IV (P < 0.01), and V lesions (P < 0.001) rather than Type I and II lesions (AHA classification). FN-EDB expression was positively correlated with macrophage accumulation and neoangiogenesis. Quantitative analysis of T1-weighted images of atherosclerotic mice revealed substantial APTFN-EDB-[Gd]NPs accumulation in plaques compared to control NPs, conventional MRI contrast agent (Gd-DTPA) or accumulation in wild-type C57BL/6J mice. Additionally, the APTFN-EDB-NPs significantly prolonged the blood-circulation time (t1/2: ~ 6 h) of a model drug and increased its accumulation in plaques (6.9-fold higher accumulation vs. free drug). Conclusions: Our findings demonstrate augmented FN-EDB expression in Type III, IV, and V atheromata and that APTFN-EDB-NPs could serve as a platform for identifying and/or delivering agents locally to a subset of atherosclerotic plaques.

Magnetic Resonance Imaging-Guided Drug Delivery to Breast Cancer Stem-Like Cells.

The feasibility of detecting breast cancer stem-like cells (BCSCs) with magnetic resonance imaging using extradomain-B of fibronectin (EDB-FN)-specific peptide (APTEDB )-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (APTEDB -TCL-SPIONs) is previously demonstrated. Here, doxorubicin (Dox)-loaded APTEDB -TCL-SPIONs (Dox@APTEDB -TCL-SPIONs) are generated and their theranostic ability in a BCSC xenograft mouse model is assessed. The Dox@APTEDB -TCL-SPIONs enable more efficient delivery of Dox to tumors than nontargeted Dox@TCL-SPIONs. Much greater inhibition of BCSC tumor growth is observed after treatment with the Dox@APTEDB -TCL-SPIONs than with either Dox@TCL-SPIONs or free Dox. Hypointense signals are observed in the majority of the mice in postcontrast but not precontrast T2*-weighted MR images of tumors 7 days after treatment with Dox@APTEDB -TCL-SPIONs. An inverse correlation is observed between signal intensity and both EDB-FN expression and response to chemotherapy. The data indicate Dox@APTEDB -TCL-SPIONs can detect BCSCs within tumors by targeting EDB-FN-expressing cells. These nanoparticles thus have theranostic potential in breast cancer.

Effect of PEG pairing on the efficiency of cancer-targeting liposomes.

Standardized poly(ethylene glycol)-modified (PEGylated) liposomes, which have been widely used in research as well as in pre-clinical and clinical studies, are typically constructed using PEG with a molecular weight of 2000 Da (PEG(2000)). Targeting ligands are also generally conjugated using various functionalized PEG(2000)). However, although standardized protocols have routinely used PEG(2000), it is not because this molecular weight PEG has been optimized to enhance tumor uptake of nanoparticles. Herein, we investigated the effect of various PEG lipid pairings--that is, PEG lipids for targeting-ligand conjugation and PEG lipids for achieving 'stealth' function--on in vitro cancer cell- and in vivo tumor-targeting efficacy. A class of high-affinity peptides (aptides) specific to extra domain B of fibronectin (APT(EDB)) was used as a representative model for a cancer-targeting ligand. We synthesized a set of aptide-conjugated PEGylated phospholipids (APT(EDB)­PEG(2000))­DSPE and APT(EDB)­PEG(2000))­DSPE) and then paired them with methoxy-capped PEGylated phospholipids with diverse molecular weights (PEG(2000)), PEG(2000)), PEG(2000)), and PEG(2000))) to construct various aptide-conjugated PEGylated liposomes. The liposomes with APT(EDB)­PEG(2000))/PEG(2000)) and APT(EDB)­PEG(2000))/PEG(2000)) pairings had the highest uptake in EDB-positive cancer cells. Furthermore, in a U87MG xenograft model, APT(EDB)­PEG(2000))/PEG(2000)) liposomes retarded tumor growth to the greatest extent, followed closely by APT(EDB)­PEG(2000))/PEG(2000)) liposomes. Among the PEGylated liposomes tested, pairs in which the methoxy-capped PEG length was about half that of the targeting ligand-displaying PEG exhibited the best performance, suggesting that PEG pairing is a key consideration in the design of drug-delivery vehicles.

MRI of breast tumor initiating cells using the extra domain-B of fibronectin targeting nanoparticles.

The identification of breast tumor initiating cells (BTICs) is important for the diagnosis and therapy of breast cancers. This study was undertaken to evaluate whether the extra domain-B of fibronectin (EDB-FN) could be used as a new biomarker for BTICs and whether EDB-FN targeting superparamagnetic iron oxide nanoparticles (SPIONs) could be used as a magnetic resonance imaging (MRI) contrast agent for BTIC imaging in vitro and in vivo. BTICs (NDY-1) exhibited high EDB-FN expression, whereas non-BTICs (MCF-7, BT-474, SUM-225, MDA-MB-231) did not exhibit EDB-FN expression. Furthermore, Cy3.3-labeled EDB-FN specific peptides (APTEDB) showed preferential binding to the targeted NDY-1 cells. To construct an EDB-FN targeted imaging probe, APTEDB was covalently attached to a thermally cross-linked SPION (TCL-SPION) to yield APTEDB-TCL-SPION. In the in vitro MRI of cell phantoms, selective binding of APTEDB-TCL-SPION to NDY-1 cells was evident, but little binding was observed in MCF-7 cells. After the intravenous injection of APTEDB-TCL-SPION into the NDY-1 mouse tumor xenograft model, a significant decrease in the signal within the tumor was observed in the T2*-weighted images; however, there was only a marginal change in the signal of non-targeting SPIONs such as APTscramble-TCL-SPION or TCL-SPION. Taken together, we report for the first time that EDB-FN was abundantly expressed in BTICs and may therefore be useful as a new biomarker for identifying BTICs. Our study also suggests that APTEDB-TCL-SPION could be used as an MRI contrast agent for BTIC imaging.

Pharmacotherapy of metastatic melanoma: emerging trends and opportunities for a cure.

Metastatic melanoma is one of the most deadly forms of cancer and is poorly responsive to standard chemotherapeutics, such as Dacarbazine and Paclitaxel. Recently, the advent of Vemurafenib and Ipilimumab has broadened the spectrum of therapeutic options for advanced melanoma patients but the occurrence of resistance and of high-grade toxicities call for better and more effective treatments. This review focuses on approved and experimental therapies for metastatic melanoma. The mechanism of action and the reported efficacy data for small molecule drugs and biologics are discussed, outlining directions for future pharmaceutical research in this field.