YSA Peptide Research Articles

EphA2-specific microvesicles derived from tumor cells facilitate the targeted delivery of chemotherapeutic drugs for osteosarcoma therapy

Despite advances in surgery and chemotherapy, the survival of patients with osteosarcoma (OS) has not been fundamentally improved over the last two decades. Microvesicles (MVs) have a high cargo-loading capacity and are emerging as a promising drug delivery nanoplatform. The aim of this study was to develop MVs as specifically designed vehicles to enable OS-specific targeting and efficient treatment of OS. Herein, we designed and constructed a nanoplatform (YSA-SPION-MV/MTX) consisting of methotrexate (MTX)-loaded MVs coated with surface-carboxyl Fe3O4 superparamagnetic nanoparticles (SPIONs) conjugated with ephrin alpha 2 (EphA2)-targeted peptides (YSAYPDSVPMMS, YSA). YSA-SPION-MV/MTX showed an effective targeting effect on OS cells, which was depended on the binding of the YSA peptide to EphA2. In the orthotopic OS mouse model, YSA-SPION-MV/MTX effectively delivered drugs to tumor sites with specific targeting, resulting in superior anti-tumor activity compared to MTX or MV/MTX. And YSA-SPION-MV/MTX also reduced the side effects of high-dose MTX. Taken together, this strategy opens up a new avenue for OS therapy. And we expect this MV-based therapy to serve as a promising platform for the next generation of precision cancer nanomedicines.Graphical

Cantharidin-loaded functional mesoporous titanium peroxide nanoparticles for non-small cell lung cancer targeted chemotherapy combined with high effective photodynamic therapy.

BackgroundAlthough photodynamic therapy (PDT) has emerged as a potential alternative to conventional chemotherapy, the low reactive oxygen species (ROS) yield of the photosensitizer such as TiO2 nanoparticles has limited its application. In addition, it is difficult to achieve effective tumor treatment with a single tumor therapy.MethodsWe used TiOx nanocomposite (YSA‐PEG‐TiOX) instead of TiO2 as a photosensitizer to solve the problem of insufficient ROS generation in PDT. Benefiting from the desired mesoporous structure of TiOx, Cantharidin (CTD), one of the active components of mylabris, is loaded into TiOx for targeted combination of chemotherapy and PDT. The cellular uptake in human non‐small cell lung carcinoma cell line (A549) and human normal breast cell line (MCF 10A) was evaluated by confocal microscopy. in vitro cytotoxicity was evaluated using Cell Counting Kit‐8 assay. The ROS was detected via a chemical probe DCFH‐DA and the photodynamic treatment effect of YSA‐PEG‐TiOx was further evaluated by a living‐dead staining. The cell apoptosis was detected by the flow cytometry.ResultsOur findings showed that the modification of YSA peptide improved the cytotoxicity of YSA‐PEG‐TiOX/CTD to EphA2 overexpressing A549 non‐small cell lung cancer (NSCLC) than non‐YSA modified counterparts. In addition, TiOx generated adequate ROS under X‐ray irradiation to further kill cancer cells. Flow analysis results also proved the superiority of this combined treatment.ConclusionsYSA‐PEG‐TiOX nanoparticles could significantly increase ROS production under X‐ray exposure and provide a new drug delivery nanocarrier for CTD in combination with PDT to achieve effective NSCLC treatment.

Engineering nanomolar peptide ligands that differentially modulate EphA2 receptor signaling

The EPH receptor A2 (EphA2) tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements. We now report the high-resolution (1.53-2.20 Å) crystal structures of the YSA peptide and several of its improved derivatives in complex with the EphA2 LBD, disclosing that YSA targets the ephrin-binding pocket of EphA2 and mimics binding features of the ephrin-A ligands. The structural information obtained enabled iterative peptide modifications conferring low nanomolar potency. Furthermore, contacts observed in the crystal structures shed light on how C-terminal features can convert YSA derivatives from antagonists to agonists that likely bivalently interact with two EphA2 molecules to promote receptor oligomerization, autophosphorylation, and downstream signaling. Consistent with this model, quantitative FRET measurements in live cells revealed that the peptide agonists promote the formation of EphA2 oligomeric assemblies. Our findings now enable rational strategies to differentially modify EphA2 signaling toward desired outcomes by using appropriately engineered peptides. Such peptides could be used as research tools to interrogate EphA2 function and to develop pharmacological leads.

Targeted delivery of YSA-functionalized and non-functionalized polymeric nanoparticles to injured pulmonary vasculature

Ephrin type-A receptor 2 (EphA2) is a transmembrane receptor which is upregulated in injured lungs, including those treated with bleomycin. YSA peptide (YSAYPDSVPMMS), a mimic of ephrin ligands, binds to EphA2 receptors on cell surface with high affinity. In this study, we assessed the ability of YSA-functionalized and non-functionalized poly (dl-lactide-co-glycolide) (PLGA) nanoparticles to enhance delivery to bleomycin treated cultured vascular endothelial cells and, in a bleomycin induced lung injury mouse model. Nanoparticles were loaded with a lipophilic fluorescent dye. Human umbilical vein endothelial cells (HUVEC) with or without 2-day bleomycin pretreatment (25 µg/ml) and adult mice with or without intratracheal instillation of bleomycin (0.1 U) were dosed with nanoparticles. Mice received nanoparticles via tail vein injection 4 days after bleomycin treatment. Three days after nanoparticle injection, tissues (lung, heart, kidney, spleen, liver, brain, eyes and whole blood) were harvested and quantified for fluorescence using IVIS imaging. Mean particle uptake increased with time and concentration for both types of particles in HUVEC, with the uptake being higher for YSA-functionalized nanoparticles. Bleomycin treatment increased the 3-h uptake of both types of nanoparticles in HUVEC by about two-fold, with the YSA-functionalized nanoparticle uptake being 1.66-fold compared to non-functionalized nanoparticles (p < .05). In mice, bleomycin injury resulted in 2.3- and 4.7-fold increase in the lung levels of non-functionalized and YSA-functionalized nanoparticles (p < .05), respectively, although the differences between the two particle types were not significant. In conclusion, PLGA nanoparticle delivery to cultured vascular endothelial cells and mouse lungs in vivo is higher following bleomycin treatment, with the delivery tending to be higher for YSA functionalized nanoparticles.

Codelivery of doxorubicin and JIP1 siRNA with novel EphA2-targeted PEGylated cationic nanoliposomes to overcome osteosarcoma multidrug resistance.

PurposeOsteosarcoma (OS) mostly affects children and young adults, and has only a 20%–30% 5-year survival rate when metastasized. We aimed to create dual-targeted (extracellular against EphA2 and intracellular against JNK-interacting protein 1 [JIP1]), doxorubicin (DOX)-loaded liposomes to treat OS metastatic disease.Materials and methodsCationic liposomes contained N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and distearoyl-phosphatidylethanolamine–methyl-poly(ethylene glycol) (DSPE–mPEG) conjugate. EphA2 targeting was accomplished by conjugating YSA peptide to DSPE–mPEG. Vesicles were subsequently loaded with DOX and JIP1 siRNA.ResultsCharacteristics assessment showed that 1) size of the bilayered particles was 109 nm; 2) DOX loading efficiency was 87%; 3) siRNA could be successfully loaded at a liposome:siRNA ratio of >24:1; and 4) the zeta potential was 18.47 mV. Tumor-mimicking pH conditions exhibited 80% siRNA and 50.7% DOX sustained release from the particles. Stability studies ensured the protection of siRNA against degradation in serum. OS cell lines showed increased and more pericellular/nuclear localizations when using targeted vesicles. Nontargeted and targeted codelivery caused 70.5% and 78.6% cytotoxicity in OS cells, respectively (free DOX: 50%). Targeted codelivery resulted in 42% reduction in the siRNA target, JIP1 mRNA, and 46% decrease in JIP1 levels.ConclusionOur dual-targeted, DOX-loaded liposomes enhance toxicity toward OS cells and may be effective for the treatment of metastatic OS.

Abstract 3911: Targeting the EphA2-ligand binding domain for the design of innovative cancer therapeutics

Abstract Eph receptors are the largest family of receptor tyrosine kinases, and are involved in regulating several cellular processes, including cell migration, and angiogenesis. Among the Eph family, EphA2 has attracted great interest as it is overexpressed in several tumor types such as melanoma, breast, prostate, lung, colon, and ovarian cancers, and it is also implicated in various aspects of oncogenesis (proliferation, tumor angiogenesis, cell adhesion, migration and metastasis). Thus, EphA2 is a promising direct chemotherapeutic target and several trials are ongoing targeting its kinase domain. In addition, due to its ability to endocytose upon activation, EphA2 could also be used as a carrier of cytotoxic agents, particularly in antibody-drug conjugates (ADCs) or peptidomimetic-drug conjugates (PDCs), using agonistic agents that target its extracellular ligand binding domain (LBD). Hence, potent and selective EphA2-LBD targeting ADCs or PDCs may lead to a decrease in off-target cytotoxicity while maximizing the efficacy of chemotherapeutic agents. Recently, a 12-mer agonistic peptide (YSAYPDSVPMMS, termed YSA in short) targeting EphA2-LBD with modest affinity has been identified by a phage display. YSA binding leads to EphA2 activation and internalization. Accordingly, we developed an innovative anti-triazole linker for the synthesis of EphA2-targeting peptide-drug conjugates to avoid the compatibility problems of disulfide and hydrazine linkers typical of ADCs. Using this strategy, our laboratory initially coupled the YSA peptide with an azide linker-derived taxane (paclitaxel, PTX), generating the desired YSA-PTX conjugate. Additional and more recent studies with a mildly improved EphA2-targeting agent, named 123B9, were also reported by our laboratory and tested in cellular and in vivo models of prostate cancer, pancreatic cancer and melanoma. While these studies clearly suggest that YSA and 123B9 are capable of targeting cytotoxic agents to EphA2-expressing tumor cells, the pharmacodynamic and pharmacokinetic properties of the targeting agents need to be improved to translate these studies into potential new therapies. Our central hypotheses are that by using structure-based design strategies, and leveraging on our previous preliminary data, it should be possible to derive novel YSA or 123B9 derivatives with nanomolar potency against the receptor. In addition, we will show that such agents could be used to prevent cancer cell migration in several solid tumors including breast, prostate, pancreatic and melanomas when used as single agents. Citation Format: Parima Udompholkul, Luca Gambini, Ahmed F. Salem, Carlo Baggio, Maurizio Pellecchia. Targeting the EphA2-ligand binding domain for the design of innovative cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3911.

Reduction of Circulating Cancer Cells and Metastases in Breast-Cancer Models by a Potent EphA2-Agonistic Peptide-Drug Conjugate.

EphA2 overexpression has been associated with metastasis in multiple cancer types, including melanomas and ovarian, prostate, lung, and breast cancers. We have recently proposed the development of peptide–drug conjugates (PDCs) using agonistic EphA2-targeting agents, such as the YSA peptide or its optimized version, 123B9. Although our studies indicated that YSA– and 123B9–drug conjugates can selectively deliver cytotoxic drugs to cancer cells in vivo, the relatively low cellular agonistic activities (i.e., the high micromolar concentrations required) of the agents toward the EphA2 receptor remained a limiting factor to the further development of these PDCs in the clinic. Here, we report that a dimeric version of 123B9 can induce receptor activation at nanomolar concentrations. Furthermore, we demonstrated that the conjugation of dimeric 123B9 with paclitaxel is very effective at targeting circulating tumor cells and inhibiting lung metastasis in breast-cancer models. These studies represent an important step toward the development of effective EphA2-targeting PDCs.

Secondary nuclear targeting of mesoporous silica nano-particles for cancer-specific drug delivery based on charge inversion.

A novel multifunctional nano-drug delivery system based on reversal of peptide charge was successfully developed for anticancer drug delivery and imaging. Mesoporous silica nano-particles (MSN) ~50 nm in diameter were chosen as the drug reservoirs, and their surfaces were modified with HIV-1 transactivator peptide-fluorescein isothiocyanate (TAT-FITC) and YSA-BHQ1. The short TAT peptide labeled with FITC was used to facilitate intranuclear delivery, while the YSA peptide tagged with the BHQ1 quencher group was used to specifically bind to the tumor EphA2 membrane receptor. Citraconic anhydride (Cit) was used to invert the charge of the TAT peptide in neutral or weak alkaline conditions so that the positively charged YSA peptide could combine with the TAT peptide through electrostatic attraction. The FITC fluorescence was quenched by the spatial approach of BHQ1 after the two peptides bound to each other. However, the Cit-amino bond was unstable in the acidic atmosphere, so the positive charge of the TAT peptide was restored and the positively charged YSA moiety was repelled. The FITC fluorescence was recovered after the YSA-BHQ1 moiety was removed, and the TAT peptide led the nano-particles into the nucleolus. This nano-drug delivery system was stable at physiological pH, rapidly released the drug in acidic buffer, and was easily taken up by MCF-7 cells. Compared with free doxorubicin hydrochloride at an equal concentration, this modified MSN loaded with doxorubicin molecules had an equivalent inhibitory effect on MCF-7 cells. This nano-drug delivery system is thus a promising method for simultaneous cancer diagnosis and therapy.

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

BackgroundThe EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands. MethodsHere, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays. ResultsWe find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration. ConclusionsThe experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer. General significanceThis work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor.

Combination Approach of YSA Peptide Anchored Docetaxel Stealth Liposomes with Oral Antifibrotic Agent for the Treatment of Lung Cancer.

Therapeutic efficacy of nanocarriers can be amplified by active targeting and overcoming the extracellular matrix associated barriers of tumors. The aim of the present study was to investigate the effect of oral antifibrotic agent (telmisartan) on tumor uptake and anticancer efficacy of EphA2 receptor targeted liposomes. Docetaxel loaded PEGylated liposomes (DPL) functionalized with nickel chelated phospholipid were prepared using a modified hydration method. DPL were incubated with various concentrations of histidine tagged EphA2 receptor specific peptide (YSA) to optimize particle size, zeta potential, and percentage YSA binding. Cellular uptake studies using various endocytosis blockers revealed that a caveolae dependent pathway was the major route for internalization of YSA anchored liposomes of docetaxel (YDPL) in A549 lung cancer cell line. Hydrodynamic diameter and zeta potential of optimized YDPL were 157.3 ± 11.8 nm and -3.64 mV, respectively. Orthotopic lung tumor xenograft (A549) bearing athymic nude mice treated with oral telmisartan (5 mg/kg) for 2 days showed significantly (p < 0.05) higher uptake of YDPL in tumor tissues compared to healthy tissue. Average lung tumor weight of the YDPL + telmisartan treated group was 4.8- and 3.8-fold lower than that of the DPL and YDPL treated groups (p < 0.05). Substantially lower expression (p < 0.05) of EphA2 receptor protein, proliferating cell nuclear antigen (PCNA), MMP-9, and collagen 1A level with increased E-cadherin and TIMP-1 levels in immunohistochemistry and Western blot analysis of lung tumor samples of the combination group confirmed antifibrotic effect with enhanced anticancer activity. Active targeting and ECM remodeling synergistically contributed to anticancer efficacy of YDPL in orthotopic lung cancer.

Cell-penetrating peptide-siRNA conjugate loaded YSA-modified nanobubbles for ultrasound triggered siRNA delivery

Due to the absence of effective in vivo delivery systems, the employment of small interference RNA (siRNA) in the clinic has been hindered. In this paper, a new siRNA targeting system for EphA2-positive tumors was developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, a CPP–siRNA conjugate (CPP–siRNA) was entrapped in an ephrin mimetic peptide (YSA peptide)-modified NB (CPP–siRNA/YSA-NB) and the penetration of the CPP–siRNA was temporally masked; local ultrasound stimulation triggered the release of CPP–siRNA from the NBs and activated its penetration. Subsequent research demonstrated that the CPP–siRNA/YSA-NBs had particle sizes of approximately 200nm and a siRNA entrapment efficiency of more than 85%. The in vitro release results showed that over 90% of the encapsulated CPP–siRNA released from the NBs in the presence of ultrasound, while less than 1.5% of that (30min) released without ultrasound. Cell experiments showed a the higher CPP–siRNA cellular uptake of CPP–siRNA/YSA-NB among the various formulations in human breast adenocarcinoma cells (MCF-7, EphA2 positive cells). Additionally, after systemic administration in mice, CPP–siRNA/YSA-NB accumulated in the tumor, augmented c-Myc silencing and delayed tumor progression. In conclusion, the application of CPP–siRNA/YSA-NB with ultrasound may provide a strategy for the selective and efficient delivery of siRNA.

Homing peptide-conjugated gold nanorods: the effect of amino acid sequence display on nanorod uptake and cellular proliferation.

Gold nanorods (GNRs) have attracted significant interest in the field of medicine as theranostic agents for both imaging and photothermal ablation of cancerous cells/tissues. Targeting theranostic GNRs specifically to cancer cells is necessary to enhance treatment efficacy and minimize undesired side effects. In this study, targeting functionalized GNR to EphA2 receptors that are overexpressed on prostate cancer cells was investigated as a strategy to achieve enhanced GNR uptake by cancer cells. In addition, the influence of targeting peptide orientation on functionalized GNR uptake by PC-3 cells was explored. GNRs of aspect ratio 4 were functionalized with an EphA2 homing peptide, YSA, using a layer-by-layer polypeptide wrapping approach. In parallel, an analogous population of YSA-modified GNRs, which display a reversed YSA peptide, with the N- and C- termini reversed, was also prepared. GC-MS analysis of the YSA-GNRs indicated that functionalized GNRs displayed approximately 3000 peptides/GNR. The functionalized GNRs remained well-dispersed in biological media for short times (<24 h). An increase in GNRs uptake of the YSA-GNRs by PC-3 cells, compared to the reversed YSA-GNRs, was observed under identical incubation conditions. Lastly, the effect of the YSA-GNRs binding to EphA2 receptors on prostate cancer cell proliferation was also studied. The YSA-functionalized GNRs inhibit PC-3 proliferation at a significantly lower effective dose than free YSA. Overall, the polypeptide LBL deposition technique provides a facile route to target nanoparticles to overexpressed cellular receptors, with the caveat that the specific orientation and display of the targeting moiety plays a critical role in the interaction between the nanoparticle and the cell.

Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.

Efficient siRNA Delivery into Tumor Cells by p19-YSA Fusion Protein

For the efficient cytoplasmic delivery of siRNA in a receptor-specific fashion, we designed a p19-YSA fusion protein composed of p19 RNA binding protein and ephrin mimetic peptide (YSA peptide). The resulting recombinant protein had the high affinity for EphA2 receptor overexpressed on cancer cells as well as the complexing ability with siRNA, thus leading to tumor-targeted delivery of siRNA. The buried structure of siRNA within p19-YSA/siRNA complexes allowed the bound siRNAs to be protected from the external RNases, resulting in the enhanced stability of siRNA in serum conditions. The p19-YSA carriers could complex with siRNA in a size-dependent and sequence-independent manner and showed the pH-dependent complexing/dissocation behaviors with siRNA. In contrast to electrostatic interaction-mediated siRNA delivery systems such as cationic polymers/siRNA or cationic polypeptides/siRNA complexes, the bound siRNA within p19-YSA/siRNA complexes showed enhanced stability against large polyanions found outside cells, due to the nanomolar levels of affinity. Here, we demonstrated the superior efficiency of p19-YSA/siRNA complexes in RFP gene silencing, compared to untreated cells. These results provide an alternative approach to enhance the stability of siRNA as well as to achieve the targeted siRNA delivery.

A fiber modified adenovirus vector that targets to the EphrinA2 receptor reveals enhanced gene transfer to ex vivo pancreatic cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis. To improve treatment options new treatments, such as adenoviral (Ad) gene therapy are necessary. However, low expression of the coxsackie and adenovirus receptor (CAR) in pancreatic cancer cells (PC) limits the therapeutic efficacy of these vectors. The aim of this study was to improve transduction of PC by recombinant adenoviruses by inserting peptides into the HI loop that binds to receptors highly expressed on pancreatic cancer and were shown to target these carcinomas in vivo. We report the successful incorporation into the HI loop of peptide Tyr-Ser-Ala (YSA), a peptide ligand targeting the EphrinA2 (EphA2) receptor, and K237, a peptide targeting to the vascular endothelial growth factor receptor-II (VEGFRII). Subsequently, we showed that both peptides enhanced the transduction of a number of human PC lines that abundantly express the targeted receptor. Additional competition studies confirmed that the YSA peptide redirects Ad-YSA from CAR and specifically targets the EphA2 receptor. Due to this transduction efficiency of Ad-YSA is increased not only in human pancreatic cancer cell lines but more importantly also in pancreatic cancer resection specimens. Since the YSA peptide has been shown to specifically target pancreatic cancer in patients, it may be expected that Ad-YSA will also display increased tropism for this tumour.

Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo. YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To further increase the specificity of this vector, binding sites for native adenoviral receptors, the coxsackie and adenovirus receptor (CAR) and integrin, were ablated from the viral capsid. The ablated retargeted adenoviral vector was produced on 293T cells. Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines. Upon demonstrating specific in vitro targeting, in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector. Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold. Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor. YSA-mediated transduction was inhibited by addition of synthetic YSA peptide. The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro. In a subsequent in vivo study in a nude (nu/nu) mouse model however, no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein, nor upon injection into the peritoneum. Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.