Preparation and Functional Evaluation of RGD-Modified Proteins as α_vβ₃ Integrin Directed Therapeutics

Abstract

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.

Targeting of angiogenic endothelial cells at sites of inflammation by dexamethasone phosphate–containing RGD peptide liposomes inhibits experimental arthritis

“…In comparison with the monovalent RGD-apoptotic peptide construct, RGD-PEG-L may well benefit from enhanced affinity for ␣v␤3 integrins on angiogenic VECs, since it is a multivalent RGD-exposing system. Multivalent RGD exposure on a protein backbone has been shown to result in enhanced receptor affinity compared with the monovalent free peptide (30).…”

“…The cyclic 5-mer RGD c(RGDf[⑀-S-acetylthioacetyl])K and control RAD peptide c(RADf[⑀-S-acetylthioacetyl])K (both described in ref. 30) were synthesized at a purity of 95% by Ansynth Service BV (Roosendaal, The Netherlands). The thioacetyl group was used for thioether linkage to the liposomes (32).…”

“…To direct the antiinflammatory activity to VECs at the site of inflammation, we encapsulated water-soluble dexamethasone phosphate (DEXP) in long-circulating, polyethylene glycol-coated liposomes (28) to which a cyclic RGD peptide was covalently attached (RGD-PEG-L). We selected an RGD peptide with high chemical stability and high affinity for ␣v␤3 integrin, to target angiogenic VECs at sites of arthritis involvement (29,30). The peptide is equipped with a functional group that allows for covalent thioether linkage at the terminal end of the PEG chains on the surface of the long-circulating liposomes, thereby creating a multivalent RGD-exposing drug delivery system.…”

Preparation of integrin α(v)β(3)-targeting Ab 38C2 constructs

“…Their effect can be similar; however, unlike the diketone or vinylketone strategy, a conventional conjugate is not homogenous 10 . In another example, an Ab can be retargeted using a targeting agent-hapten conjugate, against which the Ab has been elicited without forming a formal conjugate 11 .…”

Differences in binding of 99mTc-disintegrins to integrin αvβ3 on tumor and vascular cells

“…VLO4 is also a homodimer, and thus has two RGDcontaining binding domains, which could improve its binding avidity over monomers. In other studies, molecules containing multiple RGD motifs showed higher potency than molecules with single RGD motifs [35].…”