SITE-SPECIFIC [99mTc][Tc(CO)3(OH2)3]-LABELED scFvD2B-HysTag FOR SPECT IMAGING OF PSMA IN PROSTATE CANCER

Introduction scFvD2B is a single chain variable fragment (29 kDa) of the monoclonal antibody IgGD2B specific to the extracellular domain of PSMA, especially overexpressed in prostate cancer (PCa) [1,2]. scFvD2B has shown promising properties, especially in terms of high stability, favorable pharmacokinetics and specificity, efficiently accumulating in PSMA-expressing PCa tumors. Moreover, the small size of scFvD2B allows both faster penetration into tumor tissue and rapid clearance from nontarget organs, thus permitting the achievement of good contrast and sensitivity on the day of injection or the day after injection. This allows the use of relatively short-lived radionuclides such as 99mTc or 64Cu with the benefit of an appreciable reduction in the dose absorbed by patients compared to other radionuclides. Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metalaffinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3] + framework. Thus, in order to obtain a radioimmunoconjugate (RIC) suitable for PCa SPECT imaging, a His-Tag sequence was engineered at the C-terminal portion of scFvD2B and labeled using the [99mTc(CO)3] + approach [3]. Methods scFvD2B-HisTag was synthesized by cloning the IgGD2B VH and VL chain genes into an E. Coli vector5 . [99mTc][Tc(CO)3(OH2)3] + was produced through the commercial IsoLink® kit and the radiolabeling reaction was carried out at 37°C for 2 hours using 100-150 μg of scFvD2B-HysTag in a final volume of 250 μL. RIC was characterized by RP-HPLC, purified by gel filtration, and evaluated for stability in PBS, human serum, and transchelation (His, Cys, GSH, EDTA 10 mM). In vitro, uptake and internalization was assessed in PSMA + (LNCaP and PC3-PIP) and PSMA – (PC3) cell lines. Results Under labeling conditions, the [99mTc][Tc(CO)3(scFvD2B-HisTag] returned a RCY in the range 28- 43%; after purification the RCP > 98%. The RIC showed a steady stability profile over time. According to cell studies, the uptake and internalization values were encouraging for future in vivo biodistribution insights: in LNCaP cells 6% and 4% respectively, in PC3-PIP 30% and 15% and in PC3 3% and 0,7%. Furthermore, blocking studies, with an excess of native scFvD2B-HisTag, confirmed the specificity of [ 99mTc][Tc(CO)3(scFvD2B-HisTag] for PSMA receptor. Conclusion [99mTc][Tc(CO)3(OH2)3]-labeled scFvD2B-HisTag, was easily produced with a high RCP. The reaction was site-specific and RIC was stable in vitro and possessed a high cellular uptake in PSMA(+) cells. The process was receptor mediated. Studies are in progress to determine the in vivo performance of RIC. 21 Funding: The authors acknowledge Associazione Italiana per la Ricerca sul Cancro (AIRC) for financial support (AIRC, IG 2020 ID 24528). References [1] Carpanese D, Zorz A, Evangelista L., Salvarese N. Targeting prostate cancer with the anti-PSMA scFvD2B: a theranostic promise for nuclear medicine. Clinical and Translational Imaging 2019 7:4 7, 295–301 (2019). [2]Carpanese D. et al. Development of 177Lu-scFvD2B as a Potential Immunotheranostic Agent for Tumors Overexpressing the Prostate Specific Membrane Antigen. Scientific Reports 10, (2020). [3] Williams, JD et al. Optimal His-Tag Design for Efficient [99mTc(CO)3] + and [188Re(CO)3] + Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays. Bioconjugate Chemistry 32, 1242–1254, 2000.