Evaluation of anti-VEGFR-3 specific scFv antibodies as potential therapeutic and diagnostic tools for tumor lymph-angiogenesis.

Vascular endothelial growth factor receptor-3 (VEGFR-3) plays a major role in lymph-angiogenesis, tumor growth and metastatic tumor cell dissemination. The receptor is over-expressed on lymphatic vessels in the vicinity of tumors and on the tumor vasculature and therefore may be an excellent target for an effective cancer intervention. We generated and characterized single chain antibody fragments (scFv) recognizing VEGFR-3 by phage display technology and expression in P. pastoris and analyzed selected antibodies in vitro and in vivo. The scFvs were functionalized by the introduction of cysteines at the C-terminal end of the proteins. The scFvs are species cross-specific and bind to recombinant human and mouse VEGFR-3. ScFv AFC5 showed specific tumor accumulation in an hVEGFR-3 expressing F9 terato-carcinoma mouse model, which was also used for tumor visualization by combined single proton emission computed tomography (SPECT/CT) and immunohistochemical analysis. This antibody also inhibited binding of hVEGF-C to its receptor and reduced proliferation of human lymphatic endothelial cells. Thus, the generated VEGFR-3 specific scFv antibodies represent a valuable tool for novel cancer therapies and diagnostic applications.

Isolation and characterization of a scFv antibody specific for tumor endothelial marker 1 (TEM1), a new reagent for targeted tumor therapy.

Tumor endothelial marker 1 (TEM1) is a protein predominantly expressed on the cell surface of endothelial cells in newly developing blood vessels and on tumor cells. It is therefore ideally suited as a target for anti-angiogenic tumor therapy. Using phage display technology a single chain antibody fragment (scFv-CM6) was isolated that specifically binds to the extracellular part of TEM1. Antibody specificity was determined in ELISA, by Western analysis, fluorescence microscopy and flow cytometry performed with TEM1-expressing cells. ScFv-CM6 was further functionalized and coupled to liposomes. Such immunoliposomes loaded with the cytotoxic drug N4-octadecyl-1-beta-D-arabinofuranosylcytosine-(5'-5')-3'-C-ethinylcytidine showed increased binding affinity and up to 80% higher cytotoxic activity towards TEM1-expressing IMR-32 tumor cells compared with control liposomes.

Determination and modeling of kinetics of cancer cell killing by doxorubicin and doxorubicin encapsulated in targeted liposomes.

Various mathematical approaches have been devised to relate the cytotoxic effect of drugs in cell culture to the drug concentration added to the cell culture medium. Such approaches can satisfactorily account for drug response when the drugs are free in solution, but the approach becomes problematic when the drug is delivered in a drug delivery system, such as a liposome. To address this problem, we have developed a simple model that assumes that the cytotoxic potency of a drug is a function of the intracellular drug level in a critical compartment. Upon exposure to drug, cell death commences after a lag time, and the cell kill rate is dependent on the amount of drug in the critical intracellular compartment. The computed number of cells in culture, at any time after exposure to the drug, takes into account the cell proliferation rate, the cell kill rate, the average intracellular drug concentration, and a lag time for cell killing. We have applied this model to compare the cytotoxic effect of doxorubicin (DOX), or DOX encapsulated in a liposome that is targeted to CD44 on B16F10 melanoma cells in culture. CD44 is the surface receptor that binds to hyaluronan and is overexpressed on various cancer cells, including B16F10. We have shown previously that the drug encapsulated in hyaluronan-targeted liposomes was more potent than was the free drug. The model required the determination of the cell-associated DOX after the cells were incubated with various concentrations of the free or the encapsulated drug for 3 h, and the quantification of cell number at various times after exposure to the drug. The uptake of encapsulated drug was greater than that of the free drug, and the ratio of cell association of encapsulated:free drug was 1.3 at 0.5 micro g/ml and increased to 3.3 at 20 micro g/ml DOX. The results demonstrate that the enhanced potency of the encapsulated drug could stem from its enhanced uptake. However, in certain cases, where larger amounts of the free drug were added, such that the intracellular amounts of drug exceeded those obtained from the encapsulated drug, the numbers of viable cells were still significantly smaller for the encapsulated drug. This finding demonstrates that for given amounts of intracellular DOX, the encapsulated form was more efficient in killing B16F10 cells than the free drug. The outcome was expressed in the kinetic model as a 5-6-fold larger rate constant of cell killing potency for the encapsulated drug versus the free drug. The model provides a quantitative framework for comparing the cytotoxic effect in cultured cells when applying the drug in the free form or in a delivery system.

Targeting human cancer cells with VEGF receptor-2-directed liposomes.

Antibodies are among the most versatile tools used today to characterize and target molecules in cells and in biological tissues. The development of phage display libraries encoding a large repertoire of single chain antibodies, scFv, allows the rapid and efficient isolation of antibodies specific for almost any type of molecule. A great advantage of such recombinant antibodies is the possibility to functionalize them by introducing new amino acid sequences. This leads to new features that would be difficult to introduce into naturally occurring antibody molecules. This approach has been successfully applied to create molecules with new biological activities, e.g. by generating chimeric scFv antibodies carrying sequences derived from other biomolecules such as blood clotting factors or enzymes. Here, we describe a new antibody isolated from an M13 phage library that recognizes vascular endothelial growth factor receptor 2, VEGFR-2. This antibody, scFvVR-2H9 was coupled to liposomes and used to specifically target VEGFR-2-expressing human cancer cells in culture.

Cytotoxic tumor targeting with scFv antibody-modified liposomes.

Specific targeting of liposome-formulated cytotoxic drugs or antigens to receptors expressed selectively on target cells represents an effective strategy for increasing the pharmacological efficacy of the delivered molecules. We have developed a feasible technique to selectively attach antibodies and fragments thereof, but also small-mol-wt ligands such as peptides, carbohydrates, or any molecules that recognize and bind target antigens or receptors to the surface of small unilamellar liposomes. Our concept is based on the site-specific functionalization of the ligands to be attached to the liposomes by thiol groups. These thiol groups can easily be introduced to antibodies or peptides by addition of cysteines, preferably at sites that do not interfere with the receptor binding domains. Optimally, the site-specific modification is introduced at the C-terminal end of the ligand, separated by an inert spacer sequence located between the thiols and the specific part of the ligand. The thiol-reactive molecules on the liposome surface are maleimides that are linked to phospholipids composing the liposome bilayer membrane. We illustrate the coupling method of a functionalized single-chain antibody fragment with binding specificity to ED-B fibronectin, an isoform of fibronectin exclusively expressed in tumor tissues, to long circulating small unilamellar poly(ethylene glycol) liposomes.

Cell uptake and radiotoxicity studies of an nuclear localization signal peptide-intercalator conjugate labeled with [99mTc(CO)3]+.

A trifunctional bioconjugate consisting of the SV40 nuclear localization signal (NLS) peptide, an aliphatic triamine ligand, and the DNA intercalating pyrene has been synthesized and quantitatively labeled with [(99m)Tc(OH(2))(3)(CO)(3)](+). The radiotoxicity of the resulting nucleus-targeting radiopharmaceutical on B16F1 mouse melanoma cells has been investigated to evaluate the activity of Auger and Coster-Kronig electrons on the viability of cells. We found a dose-dependent significant radiotoxicity of the nucleus-targeting radiopharmaceutical clearly related to the low energy decay of (99m)Tc. These principal results imply a possible therapeutic strategy based on the use of the low-energy Auger electron-emitting (99m)Tc radionuclide attached to nucleus-targeting molecules and comprising an intercalator. Highly efficient DNA targeting vectors could complement the usual role of (99m)Tc in diagnostic applications. The Auger electrons emitted by the (99m)Tc nuclide induce DNA damage leading ultimately, through a mitotic catastrophe pathway, to necrotic cell death. Non-DNA-targeting (99m)Tc complexes display much lower radiotoxicity.

Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans.

Protein transduction domains (PTDs) are short basic peptide sequences present in many cellular and viral proteins that mediate translocation across cellular membranes. PTDs have become widely used as tools for the delivery of high Mr polypeptides, polynucleotides, or nanoparticles to cells in culture; and even the transfer of cargo molecules to the tissue of live animals has been reported. These cell-permeable peptides are functional when fused in-frame to recombinant polypeptides or when chemically coupled to their cargo. The mechanism responsible for PTD-mediated membrane translocation is controversially discussed and may vary among the various PTDs reported in the literature. Thus direct physical interaction with membrane lipids resulting in vectorial delivery to cells has been proposed for the Antennapedia (Antp) PTD, whereas uptake by the retroviral TAT (transactivator of transcription) protein PTD seems to require cell surface-expressed glycosaminoglycans. The view that PTD-mediated cellular uptake is energy-independent has been dismissed recently as an artifact resulting from fixation of cells. The data reported here agree with and further extend this work. They support the idea that certain PTDs promote cellular uptake via endocytosis and require the expression of negatively charged glycosaminoglycans on the surface of the target cells. Uptake of Antp PTD conjugates or peptide-derivatized liposomes was blocked by heparan sulfate proteoglycans, whereas TAT-mediated uptake was inhibited by both heparin and dextran sulfate. Mutant cells defective for glycosaminoglycan synthesis showed dramatically reduced Antp- or TAT-mediated transmembrane transport confirming the role of these complex polysaccharides in PTD-mediated cellular uptake. The fact that PTDs selectively interact with distinct glycosaminoglycan species has implications for therapeutic applications and may allow targeting of selective tissues that differ in their surface-expressed glycosaminoglycan patterns.