MicroPET imaging of breast cancer α_v-integrin expression with ⁶⁴Cu-labeled dimeric RGD peptides

Purpose

Alpha_vβ₃ and α_vβ₅ integrins are cell adhesion molecules that play a vital role in tumor angiogenesis and metastasis. The ability to visualize and quantify integrin expression in vivo will foster our understanding of the role of integrins α_vβ₃ and α_vβ₅ in tumor angiogenesis and allow for direct assessment of anti-angiogenic treatment efficacy based on integrin antagonists. This study compared the tumor targeting characteristics of two dimeric ⁶⁴Cu-labeled RGD peptide agonists of α_vβ₃ integrin.

Procedures

Dimeric RGD peptides E[c(RGDyK)]₂ and E[c(RGDfK)]₂ were conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N″′-tetraacetic acid (DOTA) and labeled with positron emitter ⁶⁴Cu(t_1/2 = 12.8 h, β⁺ = 19%). Both ⁶⁴Cu-DOTA-E[c(RGDyK)]₂ and ⁶⁴Cu-DOTA-E[c(RGDfK)]₂ were used in biodistribution, microPET imaging and whole-body autoradiography studies in athymic female nude mice with orthotopically growing MDA-MB-435 breast carcinoma xenografts.

Results

At all time points, activity accumulation of ⁶⁴Cu-DOTA-E[c(RGDyK)]₂ in tumors was significantly higher compared to the D-Phe analog. Liver uptake of the D-Tyr derivative was lower than the D-Phe derivative at early time points but the difference became marginal with time. Overall, ⁶⁴Cu-DOTA-E[c(RGDyK)]₂ yielded better position emission tomography (PET) images in orthotopic MDA-MB-435 bearing mice than did ⁶⁴Cu-DOTA-E[c(RGDfK)]₂. Both radiotracers had α_v-integrin specific tumor activity accumulation, as demonstrated by significant reduction of uptake with a coinjected blocking dose of c(RGDyK).

Conclusions

The radiolabeled dimeric RGD peptides ⁶⁴Cu-DOTA-E[c(RGDyK)]₂ and ⁶⁴Cu-DOTA-E[c(RGDfK)]₂ have high and specific tumor uptake in a human breast cancer tumor xenograft, with the D-Tyr derivative showing better in vivo kinetics than the D-Phe derivative, most likely due to the increased hydrophilicity of the D-Tyr. Both dimeric peptides showed better tumor retention than the previously tested monomeric RGD counterparts, presumably because of bivalency and increase in apparent molecular size.

Introduction

Most solid tumors are angiogenesis dependent. Without the formation of neovasculature to provide oxygen and nutrients as well as to remove waste products, tumors cannot growth beyond 1mm to 2 mm in size.1., 2. Anti-angiogenic therapy that aims at halting proliferation of vascular endothelium in tumors has been proven effective in both preclinical tumor models and Phase I/II clinical trials.3., 4., 5. Angiogenesis is a complex process involving extensive cross-talk among tumor cells, soluble factors, and extracellular membrane (ECM) components.⁶ The construction of a vascular network requires multiple sequential steps including basement membrane degradation, endothelial cell migration and proliferation, and finally formation of new blood vessels.⁷ Each of these processes presents possible targets for diagnostic and therapeutic interventions. Members of the integrin class of cell adhesion receptors play a key role in cell-cell and cell-ECM interactions during the growth of new blood vessels.8., 9. This is especially true of the α_v-integrins, α_vβ₃ and α_vβ₅, which are highly expressed on sprouting endothelial cells and tumor cells but not on quiescent endothelial cells and normal cells. These integrins have been demonstrated to be necessary for tumor growth and spread.¹⁰ Expression of α_vβ₃ is required for fibroblast growth factor 2 (FGF-2) and tumor necrosis factor α (TNF-α) induced angiogenesis.¹⁰ Expression of α_vβ₅ is responsible for vascular endothelial growth factor (VEGF) and transforming growth factor α (TGF-α) induced angiogenesis.¹⁰ Both integrins recognize a variety of ECM proteins (e.g. vitronectin, fibrinogen, thrombospontin, proteolyzed collagen, von Willebrand factor and osteopontin) with exposed Arg-Gly-Asp (RGD) sequences.¹¹ Consequently, monoclonal antibody (mAb), RGD peptide and peptidomimetic antagonists of α_v-integrins have been used to inhibit in vitro endothelial tube formation or microvessel growth¹² and to impair angiogenesis, growth and metastasis of solid tumors in vivo.13., 14., 15.

Since this type of anti-integrin treatment is cytostatic, traditional toxicity-based selection of dose may not be optimal for in vivo activity. The ability to visualize and quantify α_v-integrin expression in vivo would allow for individualized optimization of dosage and dose interval for suitable integrin antagonists. For this purpose, cyclic RGD peptide antagonists of α_v-integrins have been labeled with different radionuclides for tumor imaging and internal radiotherapy. Both c(RGDyV)¹⁶ and c(RGDyK)¹⁷ have been labeled with ¹²⁵I and shown to target α_v-integrin positive tumors. Introducing a sugar18., 19. or poly (ethylene glycol)¹⁷ moiety increases water solubility of the RGD peptides and thus improves in vivo kinetics of the resulting radiotracers without compromising their tumor targeting ability. Cyclic RGD peptides have also been labeled with ¹⁸F through both nucleophilic (via prosthetic labeling group 2-[¹⁸F]fluoropropionate¹⁹ or 4-[¹⁸F]fluorobenzoyl20., 21.) and electrophilic substitution.²² While these types of PET tracers are able to image tumors in the brain and breast, they have limited potential for visualizing tumors in the lower abdomen due to the unfavorable hepatobiliary excretion of the ¹⁸F-labeled RGD peptides. On the other hand, RGD peptides have also been conjugated with macrocyclic chelating agents for labeling with metallic radionuclides. For example, various RGD peptide antagonists of α_v-integrins have been coupled with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for ^99mTc,23., 24., 25., 26. with diethylenetriamepentaacetic acid (DTPA) for ¹¹¹In,²⁷ and with 1,4,7,10-tetraazadodecane-N,N′,N″,N″′-tetraacetic acid (DOTA) for ¹¹¹In,^{25 90}Y,25., 28. ¹⁷⁷Lu,²⁸ and ⁶⁴Cu.²⁹

Although suitably labeled monomeric RGD peptides have the ability to target and image α_v-integrin expressing tumors, they have limited clinical potential due to their modest tumor uptake and unfavorable in vivo pharmacokinetics. It has been recently reported that dimeric and polymeric RGD peptides enhance the receptor binding affinity based upon polyvalency, wherein the RGD sequence is locally enriched and might bind simultaneously to several integrins, thus increasing the affinity of RGD ligands. Targeting ratios were enhanced and tumor washout was slowed with this approach³⁰. ⁶⁴Cu (t_1/2 = 12.8 h, 40% β⁻; 19% β⁺; 38% EC), which has diverse applications in radiopharmaceutical chemistry for PET imaging as well as therapy,31., 32. was used to label monomeric RGD peptide c(RGDyK) for breast cancer imaging studies, which showed modest tumor uptake.²⁹ In this study, we coupled dimeric RGD peptides E[c(RGDyK)]₂ and E[c(RGDfK)]₂ with DOTA and labelled the conjugates with ⁶⁴Cu for breast cancer targeting. The tumor targeting efficacy and in vivo kinetics of the two dimers will be presented.