Preparation and functional evaluation of RGD-modified proteins as alpha(v)beta(3) integrin directed therapeutics.

Tumor blood vessels can be selectively targeted by RGD-peptides that bind to alpha(v)beta(3) integrin on angiogenic endothelial cells. By inhibiting the binding of these integrins to its natural ligands, RGD-peptides can serve as antiangiogenic therapeutics. We have prepared multivalent derivatives of the cyclic RGD-peptide c(RGDfK) by covalent attachment of the peptide to side chain amino groups of a protein. These RGDpep-protein conjugates inhibited alpha(v)beta(3)-mediated endothelial cell adhesion in vitro, while conjugates prepared with a control RAD-peptide showed no activity. Radiobinding and displacement studies with endothelial cells demonstrated an increased affinity of the RGDpep-protein conjugates compared to the free peptide, with IC(50) values ranging from 23 to 0.6 nM, depending on the amount of coupled RGDpep per protein. Compared to the parental RGD-peptide and the related RGD-peptide ligand c(RGDfV), the RGDpep-protein conjugates showed a considerable increase in affinity (IC(50) parent RGDpep: 818 nM; IC(50) c(RGDfV): 158 nM). We conclude that the conjugation of RGD-peptides to a protein, resulting in products that can bind multivalently, is a powerful approach to increase the affinity of peptide ligands for alpha(v)beta(3)/alpha(v)beta(5) integrins.

[1]  F. Ross,et al.  Superactivation of integrin alphavbeta3 by low antagonist concentrations. , 2001, Journal of cell science.

[2]  E. Sackmann,et al.  Selective adhesion of endothelial cells to artificial membranes with a synthetic RGD-lipopeptide. , 2001, Chemistry.

[3]  E Ruoslahti,et al.  Solution structures and integrin binding activities of an RGD peptide with two isomers. , 2001, Biochemistry.

[4]  Fulvio Magni,et al.  Enhancement of tumor necrosis factor α antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13) , 2000, Nature Biotechnology.

[5]  M. Schwaiger,et al.  Carbohydrate Derivatives for Use in Drug Design: Cyclicαv-Selective RGD Peptides , 2000 .

[6]  D. Schuppan,et al.  Successful Targeting to Rat Hepatic Stellate Cells Using Albumin Modified with Cyclic Peptides That Recognize the Collagen Type VI Receptor* , 2000, The Journal of Biological Chemistry.

[7]  E. Ruoslahti,et al.  An address system in the vasculature of normal tissues and tumors. , 2000, Annual review of immunology.

[8]  G. FitzGerald,et al.  Immunotargeting of glucose oxidase to endothelium in vivo causes oxidative vascular injury in the lungs. , 2000, American journal of physiology. Lung cellular and molecular physiology.

[9]  Horst Kessler,et al.  N-methylated cyclic RGD peptides as highly active and selective αvβ3 integrin antagonists , 1999 .

[10]  Horst Kessler,et al.  Radiolabeled αvβ3 Integrin Antagonists: A New Class of Tracers for Tumor Targeting , 1999 .

[11]  D. Cheresh,et al.  Synergy between an antiangiogenic integrin alphav antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  B. Nies,et al.  Selective RGD-Mediated Adhesion of Osteoblasts at Surfaces of Implants. , 1999, Angewandte Chemie.

[13]  G. Bieler,et al.  Evidence for the involvement of endotheliai cell integrin αVβ3 in the disruption of the tumor vascuiature induced by TNF and IFN-γ , 1998, Nature Medicine.

[14]  E. Ruoslahti,et al.  Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. , 1998, Science.

[15]  Grietje Molema,et al.  Tumor Infarction in Mice by Antibody-Directed Targeting of Tissue Factor to Tumor Vasculature , 1997, Science.

[16]  S. Goodman,et al.  Structural and Functional Aspects of RGD-Containing Cyclic Pentapeptides as Highly Potent and Selective Integrin αVβ3 Antagonists , 1996 .

[17]  M. R. Halie,et al.  Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase. , 1995, Thrombosis research.

[18]  Erkki Ruoslahti,et al.  Phage Libraries Displaying Cyclic Peptides with Different Ring Sizes: Ligand Specificities of the RGD-Directed Integrins , 1995, Bio/Technology.

[19]  A. Habeeb,et al.  Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. , 1966, Analytical biochemistry.

[20]  F. Greenwood,et al.  THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. , 1963, The Biochemical journal.

[21]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.