Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer.

Lior Rosenfeld,Barak Akabayov,Leslie Lobel,Kalyan Dhara,Amiram Sananes,Anat Shahar,Eyal Arbely,Efrat Ben Zeev,Shira Cohen,Sigal Gelkop,Niv Papo,Yuval Zur

doi:10.1021/acs.jmedchem.0c00418

Abstract

The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug delivery. We developed and characterized four nanobodies that present tight and specific binding and internalization into PSMA+ cells and that accumulate specifically in PSMA+ tumors. We then conjugated one of these nanobodies to the cytotoxic drug doxorubicin, and we show that the conjugate internalizes specifically into PSMA+ cells, where the drug is released and induces cytotoxic activity. In vivo studies show that the extent of tumor growth inhibition is similar when mice are treated with commercial doxorubicin and with a 42-fold lower amount of the nanobody-conjugated doxorubicin, attesting to the efficacy of the conjugated drug. These data highlight nanobodies as promising agents for the imaging of PCa tumors and for the targeted delivery of chemotherapeutic drugs.

Highlights

Prostate cancer (PCa) is commonly detected by antibodybased assays that measure the serum concentration of the prostate-specific antigen (PSA),[1−3] but these assays are prone to high error rates.[4−6] In addition, chemotherapies are often used to treat castration-resistant PCa,[7] some potentially effective chemotherapies against PCa, such as doxorubicin (DOX), do not sufficiently accumulate within tumors and have a large distribution volume, resulting in low treatment efficacy and high nonspecific toxicity.[8]
One promising target that can be employed to address both these issues is the prostate-specific membrane antigen (PSMA);[10] a transmembrane protein that is overexpressed in PCa,[11] possibly due to its folate hydrolase activity, which induces cell proliferation.[11−13] PSMA is mostly expressed on the membranes of PCa cells, it is expressed on the neovasculature of many carcinomas, including PCa.[11]
The chemotherapeutic treatment options for castrationresistant PCa are currently limited to taxanes, as most cytotoxic compounds exhibit high toxicity and severe side effects, even at doses that provide limited efficacy

Summary

■ INTRODUCTION

Prostate cancer (PCa) is commonly detected by antibodybased assays that measure the serum concentration of the prostate-specific antigen (PSA),[1−3] but these assays are prone to high error rates.[4−6] In addition, chemotherapies are often used to treat castration-resistant PCa,[7] some potentially effective chemotherapies against PCa, such as doxorubicin (DOX), do not sufficiently accumulate within tumors and have a large distribution volume, resulting in low treatment efficacy and high nonspecific toxicity.[8]. To test whether the DOX released from the internalized conjugate retains its cytotoxic activity, we incubated PC3-PIP cells for 24 h with 1.5 μg/mL DOX or with an equivalent molar amount of NB7cysDOX or NB7cys, counted the number of cells in each well, and compared it to that of untreated cells (Figure S12A) A TUNEL assay revealed only a few apoptotic cells in tumors obtained from saline-treated mice, as compared with significant apoptosis in tumors obtained from DOX- or NB7cysDOX-treated mice (Figure 6D, bottom) These findings demonstrate that while the number of DOX molecules administered to NB7cysDOXtreated mice is less than 3% of that administered to the DOXtreated mice, the cytotoxic effect of the drug is similar in both groups

■ DISCUSSION AND CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES