Bifunctional phage-based pretargeted imaging of human prostate carcinoma

https://doi.org/10.1016/j.nucmedbio.2009.04.010Get rights and content

Abstract

Introduction

Two-step and three-step pretargeting systems utilizing biotinylated prostate tumor-homing bacteriophage (phage) and 111In-radiolabeled streptavidin or biotin were developed for use in cancer radioimaging. The in vivo selected prostate carcinoma-specific phage (G1) displaying up to five copies of the peptide IAGLATPGWSHWLAL was the focus of the present study.

Methods

The ability of G1 phage to extravasate and target prostate tumor cells was investigated using immunohistochemistry. G1 phages were biotinylated, streptavidin was conjugated to diethylenetriaminepentaacetic acid (DTPA) and biotin was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Biodistribution studies and single-photon emission computed tomography (SPECT)/CT imaging of xenografted PC-3 tumors via two-step pretargeted 111In-labeled streptavidin and three-step pretargeted 111In-labeled biotin were performed in SCID mice to determine the optimal pretargeting method.

Results

The ability of G1 phage to extravasate the vasculature and bind directly to human PC-3 prostate carcinoma tumor cells in vivo was demonstrated via immunocytochemical analysis. Comparative biodistribution studies of the two-step and three-step pretargeting strategies indicated increased PC-3 human prostate carcinoma tumor uptake in SCID mice of 4.34±0.26 %ID g−1 at 0.5 h postinjection of 111In-radiolabeled biotin (utilized in a three-step protocol) compared to 0.67±0.06 %ID g−1 at 24 h postinjection of 111In radiolabeled streptavidin (employed in a two-step protocol). In vivo SPECT/CT imaging of xenografted PC-3 tumors in SCID mice with the three-step pretargeting method was superior to that of the two-step pretargeting method, and, importantly, blocking studies demonstrated specificity of tumor uptake of 111In-labeled biotin in the three-step pretargeting scheme.

Conclusion

This study demonstrates the use of multivalent bifunctional phage in a three-step pretargeting system for prostate cancer radioimaging.

Introduction

Investigations into the use of radioimmunoimaging (RII) and radioimmunotherapy (RIT) have been ongoing for more than two decades. Radiolabeled monoclonal antibodies against CD20 have been successfully utilized in patients with non-Hodgkin's B-cell lymphoma [1], [2]. Another antibody showing clinical promise is trastuzumab (Herceptin), a humanized monoclonal antibody that has been utilized in both RII and RIT studies for many types of solid human tumors expressing the HER2/neu tumor antigen [3], [4], [5], [6], [7], [8], [9]. However, the unfavorable pharmacokinetic properties of whole antibodies and antibody fragments, and the resulting toxicity towards normal tissue, are major drawbacks to the use of directly labeled antibodies for the delivery of radionuclides to solid tumors [10], [11]. An alternative strategy to the use of radiolabeled antibodies is to partition the delivery of the antibody and the radionuclide using pretargeting techniques [12], [13]. One form of pretargeting has focused on the use of the streptavidin (SA)/avidin–biotin system because of the extraordinarily high affinity (KD=1015 M−1) of SA for biotin, as well as the potential for signal amplification due to the tetrameric architecture of SA. Su et al. [15] employed a monoclonal antibody (NR-LU-10)/SA conjugate to pretarget epithelial cellular adhesion molecule, expressed on human colon carcinoma xenografts for the efficient delivery of β-emitting 90Y [14] and α-emitting 212Bi [15]. Other pretargeting methods for the diagnosis and therapy of cancer have been developed [16], [17], [18], [19], [20], [21], [22], [23]. One such approach focuses on the use of phage to pretarget tumor antigens [23]. Phages are multivalent and thus should have an increased avidity for the intended target compared to that of bivalent antibodies or antibody fragments. Phages are also easily derivatized via their coat proteins, which allows multiple reporter tags to be coupled to each virion. Thus, the development of pretargeted, multivalent and bifunctional phage may have benefits over implementation of antibodies for the RII or RIT of cancer.

However, the successful implementation of phage as imaging agents would require a better understanding of phage pharmacokinetics and biodistribution in vivo. Our laboratory previously analyzed the biodistribution properties of commonly employed phage display libraries in mice and demonstrated that phages were primarily cleared through the organs of the reticuloendothelial system [24]. Based on these studies, we devised a new in vivo selection scheme to obtain tumor-homing phage able to extravasate the vasculature and bind directly to the tumor tissue in comparison to the commonly utilized selection procedures that result in vasculature binding phage clones [25], [26]. One phage clone that was obtained in our laboratory, G1, displaying the foreign peptide sequence IAGLATPGWSHWLAL on coat protein III, was developed into a multivalent, bifunctional, biological nanoparticle for the in vivo targeting and optical imaging of prostate cancer [27]. Phage have been employed by other groups to image a variety of disease pathologies. Kelly et al. [28] in vivo imaged Lewis lung carcinoma and inflammation through the use of fluorescently labeled phage targeting osteonectin and vascular cell adhesion molecule-1, respectively. Segers et al. [29] exploited phage displaying a peptide with affinity for phosphatidylserine, by labeling with ultrasmall iron oxide particles for use as a contrast agent for the magnetic resonance imaging of apoptosis. Phage have also been directly radiolabeled with 99mTc for the in vivo imaging of infection [30], [31]. However, because of the previously described unfavorable pharmacokinetics of phage particles, the use of phage directly labeled with radioisotopes would lead to extended exposure of nontarget tissues (such as the liver and spleen) and potential harmful radioactive-induced damage. Nevertheless, we reasoned that phage can be effective radioimaging agents; however, the delivery of the targeting phage and the radiolabel should be segregated as part of a pretargeting strategy.

Formerly, a pretargeting strategy was employed by our laboratory for the development of phage-based biological nanoparticles displaying multiple copies of tumor-homing peptides to be used in cancer imaging [23]. The first pretargeting technique we attempted centered on phage displaying an engineered analog of the natural peptide hormone, α-melanocyte stimulating hormone (α-MSH), that binds the melanocortin-1 receptor overexpressed on malignant melanoma [23]. The engineered phages (MSH2.0) were examined for their ability to target malignant melanoma in a C57 mouse in vivo using a two-step pretargeting scheme. It was theorized that the two-step pretargeting system would allow the clearance of the majority of unbound phage before the injection of the imaging label. However, even at 24 h postinjection of the 111In-radiolabeled SA, there were significant levels of activity within nontargeted tissues. Therefore, we decided to examine the potential use of biotinylated tumor-homing phage for use in a three-step pretargeting protocol.

It was decided to examine the PC-3 human prostate tumor-targeting phage, G1, to determine whether they could function in a pretargeting approach as in vivo SPECT radioimaging agents for the detection of prostate carcinoma. G1 phages were previously selected in vivo in SCID mice bearing human PC-3 prostate carcinomas and were subsequently examined both in vitro and in vivo for use as a biological nanoparticle for the targeting of prostate carcinoma [27]. The less than optimal biodistribution and retention of 111In-radiolabeled SA in C57 mice bearing B16-F1 mouse melanoma tumors observed by our laboratory as well as by others [32], [33], [34] prompted us to explore whether a three-step pretargeting scheme may make the best use of phage for the radioimaging of tumors. The three-step pretargeting system involved, first, administration of biotinylated G1 phage, followed by injection of avidin and, finally, 111In-labeled DOTA-biotin injection. It was hypothesized that the small molecular weight of biotin would favor fast clearance of excess radiolabel as well as reduce nontarget tissue uptake. Biotinylated G1 phages were able to localize to PC-3 carcinomas in SCID within 4 h and were used in both two-step and three-step pretargeting protocols. Analysis of in vivo biodistribution data revealed increased tumor uptake and retention of 111In-labeled biotin used in the three-step method over that of 111In-labeled SA used in the two-step method within xenografted PC-3 tumors in SCID mice. The liver uptake of 111In-labeled biotin was distinctly improved over the two-step pretargeting method even after 24 h postinjection of 111In-labeled SA. Kidney uptake and retention of 111In-labeled SA and 111In-labeled biotin was found to be similar for all time points investigated. SPECT/CT imaging studies revealed the three-step pretargeting method to be a more efficient technique for the visualization of the pretargeted tumor than that of the two-step pretargeting method, and blocking studies verified the specificity of 111In-biotin tumor uptake. These data provide proof of the utility of bifunctional, multivalent phage as a biological nanoparticle to facilitate the three-step pretargeted radioimaging of prostate cancer.

Section snippets

Materials

Cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA), unless otherwise stated.

Cell lines

The human prostate carcinoma cell line PC-3 [35] was grown in Ham's F12K media, 7% fetal bovine serum, 2 mM l-glutamine and 48 μg ml−1 gentamicin at 37°C in 5% CO2. The cell line was tested for pathogens before injection into mice by the Cell and Immunobiology Core Facility at the University of Missouri.

Generation of clonal phage populations

The G1 phage clone was

Immunohistochemical investigation of extravasation of phage from the vasculature

In this study, the previously in vivo selected G1 phages were utilized for the development of a novel phage-based pretargeting strategy for the SPECT/CT imaging of prostate cancer. The G1 phage clone was previously selected under conditions that were predicted to ensure recovery of phage that extravasated the vasculature and bound directly to human PC-3 prostate carcinoma tumor cells. The procedure utilized for the in vivo selection of G1 phage included homogenization, washing and gentle lysing

Conclusion

The ability of G1 phage to bind directly to PC-3 tumor cells in vivo was examined via immunohistochemistry. Resected tumor tissue from PC-3 tumor-bearing SCID mice injected with G1 phage was probed for the presence and localization of the phage. Biodistribution of G1 phage particles was similar to the reported biodistribution data of other types of M13 phage investigated, in that phage particles were found in the liver and tumor but not in the muscle at 4 h postinjection [24], [45], [46], [47],

Acknowledgments

The authors would like to thank and acknowledge the contributions of Samantha Sublett, Lisa Watkinson, Terry Carmack, Marie T. Dickerson and George P. Smith.

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    This work was supported by a Merit Review award from the Veterans Administration and by grant NIH P50 CA103130-01.

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