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1.
Int J Cancer ; 112(2): 279-85, 2004 Nov 01.
Article in English | MEDLINE | ID: mdl-15352041

ABSTRACT

To redirect the lytic activity of cytotoxic T lymphocytes (CTL) toward tumor vascular endothelial cells, we prepared bifunctional proteins with specificity for both alphavbeta3 and CD3. Monocyclic RGD peptides (cRGDfK) were covalently coupled to an anti-CD3 monoclonal antibody at different peptide:protein ratios. The resulting RGDpep-anti-CD3 conjugates bound specifically to alphavbeta3-expressing endothelial cells. Displacement studies with radiolabeled alphavbeta3 ligand demonstrated that the alphavbeta3 binding affinity of RGDpep-anti-CD3 conjugates was elevated as compared to the non-conjugated RGDpep. IC50 values ranged from 150-1.1 nM, depending on the amount of coupled RGDpep molecules per antibody molecule. RGD modification did not affect the ability of anti-CD3 antibodies to bind to CTL. Furthermore, RGDpep-anti-CD3 was fully capable of activating T cells upon CD3 binding as was shown in a Jurkat/NFAT reporter-gene activation assay. All RGDpep-anti-CD3 conjugates were able to induce RGDpep, CD3-dependent lysis of human primary endothelial cells by anti-CD3/IL-2 activated human peripheral blood mononuclear cells (PBMC), with a significant induction of cytotoxicity observed at an E/T ratio as low as 10. Redirecting cytolytic activity reached up to 50% cytotoxicity using the conjugate with the highest RGD peptide load. Combining the good accessibility of tumor blood vessel endothelium for CTL with the efficiency of target cell killing warrants further investigations on anti-tumor effects of this type of conjugates in vivo.


Subject(s)
CD3 Complex/immunology , Endothelial Cells/immunology , Immunotherapy/methods , Integrin alphaVbeta3/immunology , T-Lymphocytes, Cytotoxic/immunology , Antibodies/immunology , Humans , Leukocytes, Mononuclear/immunology , Neoplasms/immunology , Neoplasms/therapy , Oligopeptides
2.
J Control Release ; 91(1-2): 115-22, 2003 Aug 28.
Article in English | MEDLINE | ID: mdl-12932643

ABSTRACT

Angiogenesis is a key process in the growth and metastasis of a tumor. Disrupting this process is considered a promising treatment strategy. Therefore, a drug delivery system specifically aiming at angiogenic tumor endothelial cells was developed. Alpha v beta 3-integrins are overexpressed on actively proliferating endothelium and represent a possible target. For this, RGD-peptides with affinity for this integrin were coupled to the distal end of poly(ethylene glycol)-coated long-circulating liposomes (LCL) to obtain a stable long-circulating drug delivery system functioning as a platform for multivalent interaction with alpha v beta 3-integrins. The results show that cyclic RGD-peptide-modified LCL exhibited increased binding to endothelial cells in vitro. Moreover, intravital microscopy demonstrated a specific interaction of these liposomes with tumor vasculature, a characteristic not observed for LCL. RGD-LCL encapsulating doxorubicin inhibited tumor growth in a doxorubicin-insensitive murine C26 colon carcinoma model, whereas doxorubicin in LCL failed to decelerate tumor growth. In conclusion, coupling of RGD to LCL redirected these liposomes to angiogenic endothelial cells in vitro and in vivo. RGD-LCL containing doxorubicin showed superior efficacy over non-targeted LCL in inhibiting C26 doxorubicin-insensitive tumor outgrowth. Likely, these RGD-LCL-doxorubicin antitumor effects are brought about through direct effects on tumor endothelial cells.


Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/pharmacology , Doxorubicin/administration & dosage , Doxorubicin/pharmacology , Neoplasms/blood supply , Neoplasms/drug therapy , Animals , Antibiotics, Antineoplastic/pharmacokinetics , Doxorubicin/pharmacokinetics , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Excipients , Flow Cytometry , Humans , Liposomes , Male , Mice , Mice, Inbred BALB C , Regional Blood Flow/drug effects , Tissue Distribution
3.
Mol Cancer Ther ; 1(11): 901-11, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12481411

ABSTRACT

The design, synthesis, and initial biological evaluation of a doxorubicin prodrug that contains a dual tumor specific moiety, which allows enhanced tumor recognition potential, is reported. Both a tumor-specific recognition site and a tumor selective enzymatic activation sequence are incorporated in the prodrug. The first tumor-specific sequence is the bicyclic CDCRGDCFC (RGD-4C) peptide that selectively binds alpha v beta 3 and alpha v beta 5 integrins. These integrins are highly overexpressed on invading tumor endothelial cells. The second tumor-specific sequence is a D-Ala-Phe-Lys tripeptide that is selectively recognized by the tumor-associated protease plasmin, which is involved in tumor invasion and metastasis. An aminocaproyl residue was incorporated as a spacer between the two peptide sequences, whereas a self-eliminating 4-aminobenzyl alcohol spacer was inserted between the plasmin substrate and doxorubicin. Although the prodrug showed a decreased binding affinity as compared with the unconjugated reference peptide, it was still a potent ligand for alpha v beta 3 and alpha v beta 5 integrin receptors. The synthesized construct also possessed plasmin substrate properties as demonstrated by doxorubicin release from 1 upon incubation with plasmin. The release of doxorubicin from 1 was not complete, possibly related to low prodrug solubility. In vitro prodrug 1 showed plasmin-dependent cytotoxicity for endothelial cells and HT1080 fibrosarcoma cells. On the basis of these in vitro results, derivatives of 1 with improved water solubility are considered good candidates for additional development and in vivo evaluation of this dual targeting concept.


Subject(s)
Antineoplastic Agents/chemical synthesis , Doxorubicin/pharmacology , Drug Design , Prodrugs/pharmacology , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Benzyl Alcohols/pharmacology , Binding Sites , Cell Adhesion , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Endothelium/cytology , Endothelium, Vascular/cytology , Fibrinolysin/chemistry , Fibrinolysin/metabolism , Humans , Inhibitory Concentration 50 , Integrin alphaVbeta3/metabolism , Integrins/chemistry , Integrins/metabolism , Ligands , Models, Chemical , Molecular Sequence Data , Neoplasm Invasiveness , Neoplasm Metastasis , Oligopeptides , Peptides/chemistry , Prodrugs/chemical synthesis , Protein Binding , Protein Structure, Tertiary , Receptors, Vitronectin/metabolism
4.
Biotechnol Annu Rev ; 8: 133-65, 2002.
Article in English | MEDLINE | ID: mdl-12436918

ABSTRACT

Endothelial cells play a pathological role in cancer and chronic inflammation and are therefore attractive targets for therapeutic intervention. This review focuses on endothelial cell specific drug targeting strategies for the treatment of these diseases. The cellular and molecular processes involved in the activation of endothelial cells in angiogenesis and inflammation will be reviewed. Various target epitopes expressed by activated endothelium suitable for targeting purposes, design and development of drug-carrier complexes, drugs of interest which might interfere with endothelial cell activation, as well as in vitro and in vivo experimental approaches to study (intra) cellular drug delivery will be discussed.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Antineoplastic Agents/administration & dosage , Drug Delivery Systems/methods , Inflammation/drug therapy , Neoplasms/drug therapy , Chronic Disease , Drug Carriers , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiopathology , Humans , Inflammation/immunology , Inflammation/physiopathology , Neoplasms/physiopathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/physiopathology
5.
Int J Cancer ; 102(5): 469-75, 2002 Dec 10.
Article in English | MEDLINE | ID: mdl-12432548

ABSTRACT

Angiogenesis-associated integrin alpha(v)beta(3) represents an attractive target for therapeutic intervention because it becomes highly upregulated on angiogenic endothelium and plays an important role in the survival of endothelial cells. Cyclic RGD peptides were prior shown to have a high affinity for alpha(v)beta(3) and can induce apoptosis of endothelial cells. In our laboratory, monocyclic RGD peptides (cRGDfK) were chemically coupled to a protein backbone. Previous results demonstrated that the resulting RGDpep-HuMab conjugate bound with increased avidity to alpha(v)beta(3)/alpha(v)beta(5) on endothelial cells. In our present study, RGDpep-HuMab was injected intravenously and intraperitoneally in B16.F10 tumor-bearing mice to determine its pharmacokinetics and organ distribution. In the tumor, the RGDpep-HuMab conjugate specifically localized at the endothelium as was demonstrated by immunohistochemistry. The control RADpep-HuMab conjugate was not detected in the tumor. Besides tumor localization RGDpep-HuMab was found in liver and spleen associated with macrophages. This uptake by macrophages is probably responsible for the more rapid clearance of RGDpep-HuMab from the circulation than HuMab and RADpep-HuMab. The half-life of RGDpep-HuMab (90 min) was still considerably longer than that of free RGD peptides (<10 min). This prolonged circulation time may be favorable for drug targeting strategies because the target cells are exposed to the conjugate for a longer time period. Taken together these results indicate that RGD-modified proteins are suitable carriers to deliver therapeutic agents into tumor or inflammation induced angiogenic endothelial cells.


Subject(s)
Endothelium, Vascular/metabolism , Melanoma, Experimental/blood supply , Oligopeptides/pharmacokinetics , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacokinetics , Liver/metabolism , Macromolecular Substances , Macrophages/metabolism , Male , Melanoma, Experimental/metabolism , Mice , Mice, Inbred C57BL , Oligopeptides/administration & dosage , Spleen/metabolism , Tissue Distribution
6.
J Control Release ; 83(2): 241-51, 2002 Oct 04.
Article in English | MEDLINE | ID: mdl-12363450

ABSTRACT

Tumor vasculature can be targeted by peptides containing an RGD (Arg-Gly-Asp) sequence, which bind to alpha(v)beta3 and alpha(v)beta5 integrins on angiogenic endothelial cells. By covalently attaching cyclic RGD-peptides (cRGDfK) to a protein backbone, we prepared a multivalent peptide-protein conjugate with increased affinity for alpha(v)beta3/alpha(v)beta5 integrins. We demonstrated that RGDpep-protein conjugate bound to HUVEC, whereas the conjugate prepared with the control RAD peptide was devoid of any binding. RGDpep-protein conjugate was furthermore functional in inhibiting the adhesion of HUVEC to alpha(v)beta3/alpha(v)beta5 ligand vitronectin, and direct binding of the radiolabeled conjugate to HUVEC was inhibited by alpha(v)beta(3)/alpha(v)beta5-specific RGD peptides. Finally, RGDpep-protein conjugate was shown to be internalized and degraded by HUVEC, a process that could be inhibited by lysosomal degradation inhibitors chloroquine and ammonium chloride. This cellular handling was significantly influenced by the presence of cations, which strongly inhibited internalization. This is the first study that shows direct evidence that primary endothelial cells are capable of internalizing RGD-containing macromolecular proteins. This feature makes them attractive carriers for the intracellular delivery of potent anti-angiogenic drugs into endothelial cells for the treatment of cancer and chronic inflammatory diseases.


Subject(s)
Drug Delivery Systems/methods , Endothelium, Vascular/metabolism , Neoplasms/blood supply , Neoplasms/metabolism , Oligopeptides/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Oligopeptides/administration & dosage
8.
Bioconjug Chem ; 13(1): 128-35, 2002.
Article in English | MEDLINE | ID: mdl-11792188

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.


Subject(s)
Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/pharmacology , Oligopeptides/chemistry , Proteins/chemistry , Proteins/pharmacology , Receptors, Vitronectin/drug effects , Cell Adhesion/drug effects , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Humans , Immunoglobulin G/chemistry , Peptides/chemistry
9.
J Liposome Res ; 12(1-2): 129-35, 2002.
Article in English | MEDLINE | ID: mdl-12604046

ABSTRACT

The development of liposomes targeted to angiogenic endothelial cells offers exciting prospects for intervention in cancer and inflammation. Several proteins are (strongly) over-expressed on angiogenic endothelial cells as compared to the quiescent endothelium, and could potentially serve as targets for site-specific drug delivery. In this contribution particular attention is given to the design of targeted long-circulating liposomes directed against the alpha v beta 3-integrin protein.


Subject(s)
Liposomes/metabolism , Neovascularization, Pathologic/drug therapy , Cells, Cultured , Doxorubicin/pharmacology , Endothelium, Vascular/cytology , Humans , Inflammation , Integrin alphaVbeta3/metabolism , Integrins/metabolism , Ligands , Oligopeptides/pharmacology , Polyethylene Glycols/metabolism , Umbilical Veins/cytology
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