echistatin and Neoplasms

We sought proof of principle that tumor-targeting ligands can be displayed on the surface of vesicular stomatitis virus (VSV) by engineering its glycoprotein. Here, we successfully rescued VSVs displaying tumor vasculature-targeting ligands. By using a rational approach, we investigated various feasible insertion sites on the G protein of VSV (VSV-G) for display of tumor vasculature-targeting ligands, cyclic RGD (cRGD) and echistatin. We found seven sites on VSV-G that tolerated insertion of the 9-residue cRGD peptide, two of which could tolerate insertion of the 49-amino acid echistatin domain. All of the ligand-displaying viruses replicated as well as the parental virus. In vitro studies demonstrated that the VSV-echistatin viruses specifically bound to targeted integrins. Since the low-density lipoprotein receptor (LDLR) was recently identified as a major receptor for VSV, we investigated the entry of ligand-displaying viruses after masking LDLR. The experiment showed that the modified viruses can enter the cell independently of LDLR, whereas entry of unmodified virus is significantly blocked by a specific monoclonal antibody against LDLR. Both parental and ligand-displaying viruses displayed equal oncolytic efficacies in a syngeneic mouse myeloma model. We further demonstrated that single-chain antibody fragments against tumor-specific antigens can be inserted at the N terminus of the G protein and that corresponding replication-competent VSVs can be rescued efficiently. Overall, we demonstrated that functional tumor-targeting ligands can be displayed on replication-competent VSVs without perturbing viral growth and oncolytic efficacy. This study provides a rational foundation for the future development of fully retargeted oncolytic VSVs.

Topics: Animals; Cell Line, Tumor; Female; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Neoplasms; Oncolytic Virotherapy; Oncolytic Viruses; Peptides; Peptides, Cyclic; Protein Engineering; Vesicular stomatitis Indiana virus; Viral Envelope Proteins; Virus Internalization

The dimeric transmembrane integrin, α(v)β(3), is a well-investigated target by different imaging modalities through suitably labeled arginine-glycine-aspartic acid (RGD) containing peptides. In this study, we labeled four cyclic RGD peptides with or without PEG functional groups: c(RGDfK) (denoted as FK), PEG(3)-c(RGDfK) (denoted as FK-PEG(3)), E[c(RGDfK)](2) (denoted as [FK](2)), and PEG(4)-E[PEG(4)-c(RGDfK)](2) (denoted as [FK](2)-3PEG(4)), with (89)Zr (t(1/2) = 78.4 h), using the chelator desferrioxamine-p-SCN (Df) for imaging tumor integrin α(v)β(3).. The Df conjugated RGD peptides were subjected to integrin α(v)β(3) binding assay in vitro using MDA-MB-435 breast cancer cells. The (89)Zr-labeled RGD peptides were then subjected to small animal positron emission tomography (PET) and direct tissue sampling biodistribution studies in an orthotopic MDA-MB-435 breast cancer xenograft model.. All four tracers, (89)Zr-Df-FK, (89)Zr-Df-FK-PEG(3), (89)Zr-Df-[FK](2), and (89)Zr-Df-[FK](2)-3PEG(4), were labeled in high radiochemical yield (89 ± 4%) and high specific activity (4.07-6 MBq/μg). Competitive binding assay with (125)I-echistatin showed that conjugation of the RGD peptides to the Df chelator did not have significant impact on their integrin α(v)β(3) binding affinity and the dimeric peptides were shown to be more potent than the monomers. In agreement with binding results, tumor uptake of (89)Zr-Df-[FK](2) and (89)Zr-Df-[FK](2)-3PEG(4) was significantly higher (4.32 ± 1.73%ID/g and 4.72 ± 0.66%ID/g, respectively, at 2 h post-injection) than the monomers (89)Zr-Df-FK and (89)Zr-Df-FK-PEG(3) (1.97 ± 0.38%ID/g and 1.57 ± 0.49%ID/g, respectively, at 2 h post-injection). Out of the four labeled peptides, (89)Zr-Df-[FK](2)-3PEG(4) gave the highest tumor-to-background ratio (18.21 ± 2.52 at 2 h post-injection and 19.69 ± 3.99 at 4 h post-injection), with the lowest uptake in metabolic organs. Analysis of late time points biodistribution data revealed that the uptake in the tumor was decreased, along with increase in the bone, which implies decomplexation of (89)Zr-Df.. Efficient radiolabeling of peptides with an appropriate chelator such as Df-RGD with (89)Zr was observed. The (89)Zr radiolabeled peptides provided high-quality and high-resolution microPET images in xenograft models. (89)Zr-Df-[FK](2)-3PEG(4) demonstrated the highest tumor-to-background ratio of the compounds tested. Preparation of (89)Zr peptides to take advantage of the longer half-life is unwarranted due to the relatively rapid clearance from the tumor region of peptide tracers prepared for this study and the increased uptake in the bone of transchelated (89)Zr with time (2.0 ± 0.36%ID/g, 24 h post-injection).

Topics: Animals; Binding, Competitive; Cell Line, Tumor; Humans; Integrin alphaVbeta3; Intercellular Signaling Peptides and Proteins; Iodine Radioisotopes; Ligands; Mice; Mice, Nude; Neoplasms; Oligopeptides; Peptides; Positron-Emission Tomography; Tissue Distribution; Zirconium

Radioactive iodine-labeled, cyclic RGD-PEGylated gold nanoparticle (AuNP) probes are designed and synthesized for targeting cancer cells and imaging tumor sites. These iodine-125-labeled cRGD-PEG-AuNP probes are stable in various conditions including a range of pHs and high salt and temperature conditions. These probes can target selectively and be taken up by tumor cells via integrin αvβ3-receptor-mediated endocytosis with no cytotoxicity. The probes show a significant increase in the avidity of αvβ3 integrin compared to the corresponding free cRGD peptides. In-vivo SPECT/CT imaging results show that the iodine-125-labeled cRGD-PEG-AuNP probes can target the tumor site as soon as 10 min after injection, and also that cyclic RGD peptides are needed for efficient and long-term in-vivo monitoring. The results suggest that the probes circulate through the whole body, including renal filtration, and are excretable. These promising results show that radioactive-iodine-labeled gold nanoprobes have potential for highly specific and sensitive tumor imaging or for use as angiogenesis-targeted SPECT/CT imaging probes.

Topics: Animals; Cell Line, Tumor; Diagnostic Imaging; Gold; Humans; Integrin alphaVbeta3; Intercellular Signaling Peptides and Proteins; Iodine Radioisotopes; Metal Nanoparticles; Mice; Mice, Nude; Molecular Probes; Neoplasms; Peptides; Peptides, Cyclic; Polyethylene Glycols; Xenograft Model Antitumor Assays