Astrid J. Schraa

Anti-tumor efficacy of tumor vasculature-targeted liposomal doxorubicin

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.

Peptide-targeted PEG-liposomes in anti-angiogenic therapy.

Peptides with the RGD amino acid sequence show affinity for the alpha(v)beta(3) integrin, an integrin which is over-expressed on angiogenic endothelium and involved in cell adhesion. A peptide with the sequence ATWLPPR has been demonstrated to show affinity for the vascular endothelial growth factor (VEGF) receptor, a receptor involved in the proliferation of endothelial cells. By coupling these peptides to liposomes, these liposomes can serve as a site-specific drug delivery system to tumor endothelial cells in order to inhibit angiogenesis. In the present study we demonstrate that the coupling of cyclic RGD-peptides or ATWLPPR-peptides to the surface of PEG-liposomes results in binding of these liposomes to endothelial cells in vitro. Subsequent studies with RGD-peptide targeted liposomes in vivo also demonstrate specific binding to the tumor endothelium.

Targeting of RGD-modified proteins to tumor vasculature : A pharmacokinetic and cellular distribution study

Angiogenesis‐associated integrin αvβ3 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 αvβ3 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 αvβ3/αvβ5 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.

Endothelial cells internalize and degrade RGD-modified proteins developed for tumor vasculature targeting

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.

RGD-modified anti-CD3 antibodies redirect cytolytic capacity of cytotoxic T lymphocytes toward αvβ3-EXPRESSING endothelial cells

To redirect the lytic activity of cytotoxic T lymphocytes (CTL) toward tumor vascular endothelial cells, we prepared bifunctional proteins with specificity for both αvβ3 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 αvβ3‐expressing endothelial cells. Displacement studies with radiolabeled αvβ3 ligand demonstrated that the αvβ3 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.

Development of vasculature targeting strategies for the treatment of cancer and chronic inflammatory diseases.

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.

Ligand-targeted liposomes directed against pathological vasculature

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.