Biology contribution
A novel antiangiogenesis therapy using an integrin antagonist or anti–Flk-1 antibody coated 90Y-labeled nanoparticles

https://doi.org/10.1016/j.ijrobp.2003.10.057Get rights and content

Abstract

Purpose

Integrin αvβ3 and vascular endothelial growth factor receptor 2 (Flk-1) have been shown to be involved in tumor-induced angiogenesis. Selective targeting of upregulated αvβ3 and Flk-1 on the neovasculature of tumors is a novel antiangiogenesis strategy for treating a wide variety of solid tumors. In the studies described here, we investigated the potential therapeutic efficacy of two three-component treatment regimens using two murine tumor models.

Methods and materials

The treatment regimens used nanoparticle (NP) based targeting agents radiolabeled with 90Y. The small molecule integrin antagonist (IA) 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethoxy]-benzoyl-2-(5)-aminoethylsulfonylamino-β-alanine, which binds to the integrin αvβ3, and a monoclonal antibody against murine Flk-1 (anti–Flk-1 MAb) were used to target the NPs. Murine tumor models K1735-M2 (melanoma) and CT-26 (colon adenocarcinoma) were used to evaluate the treatment efficacy.

Results

In K1735-M2 and CT-26 tumors, a single treatment with IA-NP-90Y (14.2 μg/g IA, 5 or 6 μCi/g 90Y) caused a significant tumor growth delay compared to untreated control tumors, as well as tumors treated with IA, IA-NP, and NP-90Y, respectively (p < 0.025, Wilcoxon test). In K1735-M2 tumors, a single treatment with anti–Flk-1 MAb-NP-90Y (0.36 μg/g anti–Flk-1 MAb, 5 μCi/g 90Y) also caused a significant tumor growth delay (p < 0.05, Wilcoxon test) compared to untreated tumors, as well as tumors treated with anti–Flk-1 MAb, anti–Flk-1 MAb-NP, and conventional radioimmunotherapy with 90Y-labeled anti-Flk mAb. Anti-CD31 staining showed a marked decrease in vessel density in tumors treated with anti–Flk-1 MAb-NP-90Y, which was associated with a high level of apoptotic death in these tumors, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining.

Conclusions

The present studies provide proof of principle that targeted radiotherapy works using different targeting agents on a nanoparticle, to target both the integrin αvβ3 and the vascular endothelial growth factor receptor. These encouraging results demonstrate the potential therapeutic efficacy of the IA-NP-90Y and anti–Flk-1 MAb-NP-90Y complexes as novel therapeutic agents for the treatment of a variety of tumor types.

Introduction

The development of a supportive vessel network is critical to the survival and growth of solid tumors. Without a sufficient vascular supply, solid tumors can reach a volume of only a few cubic millimeters 1, 2. The dependence of tumors on vessel formation for survival, growth, and spread has created a great deal of enthusiasm for the development of therapeutic approaches that specifically target the neovasculature 3, 4.

One antiangiogenic approach is to target the upregulated cell surface receptors on tumor neovasculature (5). In particular, a number of integrins expressed on the surface of activated endothelial cells regulate distinct biologic events such as cell migration, proliferation, and differentiation during angiogenesis. One of these integrins, αvβ3 integrin, is a receptor for extracellular matrix harboring a tripeptide (RGD) sequence, including vitronectin, fibronectin, and fibrinogen. The αvβ3 integrin is expressed on a variety of tumor cells 6, 7 and is more consistently expressed at higher levels on neovascular endothelial cells 8, 9. However, the αvβ3 integrin is not generally expressed on blood vessels in normal mature tissues, and the expression of αvβ3 integrin on activated endothelial cells suggests that αvβ3 may have an important role in tumor-induced angiogenesis. Recently, antagonists of αvβ3 have been shown to inhibit angiogenesis and induce tumor regression (8), and MRI studies suggest that αvβ3 integrin expression may be useful for diagnostic or prognostic purposes. For example, the angiogenic vasculature in rabbit carcinomas has been imaged with anti-αvβ3 mAb LM609 coupled to a gadolinium-containing liposome using MRI, resulting in improved enhancement and detailed imaging of rabbit carcinomas compared with MRI and images of angiogenic “hot spots” not detectable by conventional MRI imaging (10). Other studies have also reported specific tumor targeting and imaging using radiolabeled cyclic RGD peptides 11, 12, 13, 14.

In our initial studies, αvβ3 was chosen as a target because of its selective expression on angiogenic blood vessels. In the experiments described here, we used the small molecule integrin antagonist (IA) 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethoxy]-benzoyl-2-(5)-aminoethylsulfonylamino-β-alanine for targeting because it specifically binds to the upregulated αvβ3 on tumor neovasculature. Liposomes have been shown to be promising agents for the delivery of radioisotopes (15). We used 90Y as the therapeutic isotope in these studies to increase the efficacy of this therapy. 90Y was chosen because it is a pure beta emitter with an energy of 0.93 MeV, a half-life of 2.7 days, and a mean path length in tissue of 2.5 mm 16, 17. These properties are ideal for the treatment of macroscopic disease because of the significant cross fire that can kill both endothelial cells and tumor cells in close proximity to neovasculature. Studies were then performed to determine the potential therapeutic efficacy of this three-component radiopharmaceutical treatment regimen [which combines a targeting molecule, a nanoparticle (NP), and 90Y] in the murine melanoma K1735-M2 and colon adenocarcinoma CT-26 models.

Another antiangiogenic approach was also assessed to demonstrate the utility of targeting tumor endothelial cells. We investigated the efficacy of another three-component agent, anti–Flk-1 MAb-NP-90Y, which uses the anti–Flk-1 monoclonal antibody as the targeting agent. It is well known that vascular endothelial growth factor (VEGF) plays a unique role in tumor-induced angiogenesis and has emerged as an important target for antiangiogenic therapy 18, 19. In particular, recent studies have shown that Bevacizumab, an antibody against VEGF, can significantly prolong the time to progression of disease in patients with metastatic colorectal and renal cell cancer 20, 21. VEGF induces angiogenesis through the activation of its receptors, fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1), expressed on endothelial cells. Flk-1 (VEGF receptor 2) is expressed at much higher levels on neovascular endothelial cells in tumors than in normal tissues (22), and Flk-1 has been the subject of numerous studies in both animal models and in clinical trials 23, 24, 25, 26. In the studies presented here, anti–Flk-1 MAb was also used to target the tumor neovasculature.

Section snippets

IA-targeted NP preparation

Nanoparticles are polymerized liposomes. They were prepared and chemically linked to the IA targeting moiety (Fig. 1) , as previously described in detail (27). The final IA-NP consists of 10 mole percent of a lipid derivative of an αvβ3 IA (27), 10 mole percent of a lipid derivative of diethylenetriaminepentaacetic acid (DTPA) (28), 79 mole percent 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (Avanti Polar Lipids), and 1 mole percent of the DTPA-based lipid containing europium.

Results

In mice bearing K1735-M2 tumors, treatment with IA-NP-90Y (14.2 μg/g IA, 5 μCi/g) caused a significant tumor growth delay compared to untreated tumors, as well as tumors treated with IA, IA-NP, and NP-, respectively (n = 8, p < 0.025 for all comparisons, Wilcoxon test) (Fig. 2). TVQT was delayed from 5.5 days (median value) in untreated tumors to 16.5 days in tumors treated with the IA-NP-90Y complex (Fig. 2b). In CT-26 tumors, IA-NP-90Y treatment (14.2 μg/g IA, 6 μCi/g 90Y) was also the most

Discussion

Antiangiogenesis therapy that targets the integrin αvβ3 or Flk-1 is a promising strategy for treating a wide variety of solid tumors. Integrin antagonists can cause tumor regression by inducing apoptosis of proliferating angiogenic vascular cells while having no effect on pre-existing quiescent blood vessels (29). Antagonists of αvβ3, such as RGD peptides, as well as monoclonal antibodies directed to αvβ3 (e.g. murine LM 609 and its humanized version, Vitaxin), are under investigation as

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    This research was supported by a grant from Targesome, Inc.

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