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1.
Gene Ther ; 19(9): 899-906, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22011643

ABSTRACT

Human adenoviruses have a great potential as anticancer agents. One strategy to improve their tumor-cell specificity and anti-tumor efficacy is to include tumor-specific targeting ligands in the viral capsid. This can be achieved by fusion of polypeptide-targeting ligands with the minor capsid protein IX. Previous research suggested that protein IX-mediated targeting is limited by inefficient release of protein IX-fused ligands from their cognate receptors in the endosome. This thwarts endosomal escape of the virus particles. Here we describe that the targeted transduction of tumor cells is augmented by a cathepsin-cleavage site between the protein IX anchor and the HER2/neu-binding ZH Affibody molecule as ligand. The cathepsin-cleavage site did not interfere with virus production and incorporation of the Affibody molecules in the virus capsid. Virus particles harboring the cleavable protein IX-ligand fusion in their capsid transduced the HER2/neu-positive SKOV-3 ovarian carcinoma cells with increased efficiency in monolayer cultures, three-dimensional spheroid cultures and in SKOV-3 tumors grown on the chorioallantoic membrane of embryonated chicken eggs. These data show that inclusion of a cathepsin-cleavage sequence between protein IX and a high-affinity targeting ligand enhances targeted transduction. This modification further augments the applicability of protein IX as an anchor for coupling tumor-targeting ligands.


Subject(s)
Adenoviruses, Human/genetics , Capsid Proteins/metabolism , Cathepsins/chemistry , Genetic Vectors , Ligands , Transduction, Genetic , Cell Line, Tumor , Gene Targeting , Humans , Neoplasms/therapy , Recombinant Fusion Proteins/chemistry
2.
Gene Ther ; 15(13): 978-89, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18323790

ABSTRACT

Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen (MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens.


Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Melanoma/therapy , Transduction, Genetic/methods , Antigen Presentation , Antigens, Neoplasm/immunology , Capsid Proteins/genetics , Cell Line, Tumor , Cytotoxicity, Immunologic , Flow Cytometry , Gene Targeting , Genetic Engineering , Genetic Vectors/genetics , HLA-A1 Antigen/immunology , Humans , Male , Melanoma/immunology , Melanoma/metabolism , Melanoma-Specific Antigens , Neoplasm Proteins/immunology , Receptors, Antigen, T-Cell/genetics
3.
Gene Ther ; 14(8): 664-70, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17268536

ABSTRACT

Recombinant adenoviruses are frequently used as gene transfer vehicles for therapeutic gene delivery. Strategies to amend their tropism include the incorporation of polypeptides with high affinity for cellular receptors. Single-chain antibodies have a great potential to achieve such cell type specificity. In this study, we evaluated the efficiency of incorporation of a single-chain antibody fused with the adenovirus minor capsid protein IX in the capsid of adenovirus type 5 vectors. To this end, the codons for the single-chain antibody fragments (scFv) 13R4 were fused with those encoding of pIX via a 75-Angstrom spacer sequence. The 13R4 is a hyper-stable single-chain antibody directed against beta-galactosidase, which was selected for its capacity to fold correctly in a reducing environment such as the cytoplasm. A lentiviral vector was used to stably express the pIX.flag.75.13R4.MYC.HIS fusion gene in 911 helper cells. Upon propagation of pIX-gene deleted human adenovirus-5 vectors on these cells, the pIX-fusion protein was efficiently incorporated in the capsid. Here, the 13R4 scFv was functional as was evident from its capacity to bind its ligand beta-galactosidase. These data demonstrate that the minor capsid protein IX can be used as an anchor for incorporation of single-chain antibodies in the capsids of adenovirus vectors.


Subject(s)
Adenoviridae/genetics , Capsid Proteins/genetics , Genetic Engineering , Genetic Vectors/genetics , Immunoglobulin Fragments/genetics , Adenoviridae/ultrastructure , Blotting, Western/methods , Cell Line, Transformed , Gene Expression , Genetic Therapy , Genetic Vectors/analysis , Humans , Immunohistochemistry , Lentivirus/genetics , Microscopy, Immunoelectron , Recombinant Fusion Proteins/genetics , Transduction, Genetic/methods , beta-Galactosidase/genetics
4.
Acta Astronaut ; 39(1-4): 51-60, 1996.
Article in English | MEDLINE | ID: mdl-11540764

ABSTRACT

The STARDUST Discovery mission will collect samples of cometary coma and interstellar dust and return them to Earth. Five years after launch in February 1999, coma dust in the 1- to 100-micrometers size range will be captured by impact into ultra-low-density silica aerogel during a 6 kms-1 flyby of Comet Wild 2. The returned samples will be investigated at laboratories where the most critical information on these primitive materials is retained. The Jet Propulsion Laboratory will provide project management with Lockheed Martin Astronauts as the spacecraft industrial partner. STARDUST management will aggressively and innovatively achieve cost control through the use of Total Quality Management principles, the chief of which will be organization in a Project Engineering and Integration Team that "flattens" the traditional hierarchical structure by including all project elements from the beginning, in a concurrent engineering framework focusing on evolving Integrated Mission Capability.


Subject(s)
Astronomy/instrumentation , Cosmic Dust/analysis , Extraterrestrial Environment , Meteoroids , Space Flight , Spacecraft/instrumentation , Astronomy/methods , Equipment Design , Research Design , Total Quality Management
5.
J Infect Dis ; 150(4): 535-45, 1984 Oct.
Article in English | MEDLINE | ID: mdl-6386996

ABSTRACT

Staphylococcal toxic shock syndrome toxin (TST) inhibited growth of normal human epithelial (Chang) cells in culture, increasing the generation time 28% and 64% at concentrations of 4 X 10(-7)M and 8 X 10(-7)M, respectively. Fluorescence and electron microscopy of the cells treated with TST revealed the location of TST in the coated pits, specialized areas of the cell membrane known to contain high-affinity receptors for other polypeptide ligands. TST was labeled with 125I without detectable damage to the molecule and was shown to bind specifically to epithelial cells. A 100-fold excess of unlabeled TST inhibited binding of 125I-labeled toxin to the cells. Binding data indicated 10(4) receptor sites per cell for TST and a dissociation constant of 4 X 10(-9)M. Specific high-affinity binding of 125I-labeled TST to epithelial cells and the location of receptor sites in coated pits implies a possibility that the toxin is internalized by receptor-mediated endocytosis.


Subject(s)
Bacterial Toxins/toxicity , Shock, Septic/etiology , Staphylococcal Infections/etiology , Animals , Bacterial Toxins/metabolism , Cells, Cultured , Epithelium/drug effects , Epithelium/ultrastructure , Fluorescent Antibody Technique , Humans , Iodine Radioisotopes , Microscopy, Electron , Rabbits
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