Development and Characterization of 99mTc-scFvD2B as a Potential Radiopharmaceutical for SPECT Imaging of Prostate Cancer.

Previously, we demonstrated that the 177Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to 177Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a 99mTc-/177Lu-scFvD2B theranostic pair, this research aimed to synthesize and biochemically characterize a novel 99mTc-scFvD2B radiotracer. The scFvD2B-Tag and scFvD2B antibody fragments were produced and purified. Then, two HYNIC derivatives, HYNIC-Gly-Gly-Cys-NH2 (HYNIC-GGC) and succinimidyl-HYNIC (S-HYNIC), were used to conjugate the scFvD2B-Tag and scFvD2B isoforms, respectively. Subsequently, chemical characterization, immunoreactivity tests (affinity and specificity), radiochemical purity tests, stability tests in human serum, cellular uptake and internalization in LNCaP(+), PC3-PIP(++) or PC3(-) PCa cells of the resulting unlabeled HYNIC-scFvD2B conjugates (HscFv) and 99mTc-HscFv agents were performed. The results showed that incorporating HYNIC as a chelator did not affect the affinity, specificity or stability of scFvD2B. After purification, the radiochemical purity of 99mTc-HscFv radiotracers was greater than 95%. A two-sample t-test of 99mTc-HscFv1 and 99mTc-HscFv1 uptake in PC3-PIP vs. PC3 showed a p-value < 0.001, indicating that the PSMA receptor interaction of 99mTc-HscFv agents was statistically significantly higher in PSMA-positive cells than in the negative controls. In conclusion, the results of this research warrant further preclinical studies to determine whether the in vivo pharmacokinetics and tumor uptake of 99mTc-HscFv still offer sufficient advantages over HYNIC-conjugated peptides to be considered for SPECT/PSMA imaging.

Preclinical dosimetric studies of 177 Lu-scFvD2B and comparison with 177 Lu-PSMA-617 and 177 Lu-iPSMA endoradiotherapeutic agents.

PURPOSE: Internal dosimetry has become a very important tool to evaluate the risks and benefits of new endoradiotherapeutic agents. Nowadays, some of the most successful targeted radionuclide therapy (TRT) agents are 177 Lu-DOTA conjugates based on low molecular weight (LMW) Glu-ureido PSMA inhibitors. It has, however, been demonstrated that the DOTA chelating moiety reduces the internalization of the LMW-PSMA agent and its radiation dose to the tumor. Previously, we reported that 177 Lu-scFvD2B, an antibody-based construct, demonstrated statistically significant higher cell uptake and internalization in LNCaP prostate cancer (PCa) cells (PSMA-positive) when compared to the LMW-PSMA agents, 177 Lu-PSMA-617 and 177 Lu-iPSMA, two of the endoradiotherapeutic agents which currently are the most used in PCa therapy. The aim of this study is to estimate the preclinical 177 Lu-scFvD2B organ and tumor-absorbed doses, and to compare the values with those of 177 Lu-PSMA-617 and 177 Lu-iPSMA. METHODS: 177 Lu-scFvD2B, 177 Lu-PSMA-617, and 177 Lu-iPSMA were prepared and their radiochemical purity determined. Biodistribution studies of each radiopharmaceutical were then carried out in healthy mice to define the main source organs (SO) and to calculate the number of disintegrations in each source organs per unit of administered activity (NSO ). Absorbed dose in the main organs were then calculated for each 177 Lu-conjugate by means of OLINDA/EXM 2.1.1 software, using the calculated NSO for both the adult male and the mouse phantoms as program inputs. Images of mice bearing micropulmonary tumors injected with 177 Lu-conjugates were also obtained. Tumor standardized uptake values (SUV) for the different conjugates, obtained from the 3D SPECT image reconstruction of these mice, were used as the number of disintegrations in a tumor site per unit of administered activity (NT ). The tumor-absorbed dose was calculated using the published electron dose S-values for sphere models with diameters ranging from 10 µm to 10 mm and considering a uniform activity distribution and tumor density equivalent to water density. RESULTS: All 177 Lu-labeled agents were obtained in high yield (98%). Dosimetric studies carried out using mouse phantoms demonstrated that organ absorbed doses of 177 Lu-scFvD2B were from 1.4 to 2.3 times higher than those for 177 Lu-iPSMA and from 1.5 to 2.6 times higher than those for 177 Lu-PSMA-617. However, the 177 Lu-scFvD2B values of tumor-absorbed doses for all investigated tumor sizes were from 2.8 to 3.0 times greater than those calculated for 177 Lu-iPSMA and 177 Lu-PSMA-617, respectively. Moreover, 177 Lu-scFvD2B showed the highest tumor/kidney ratio when compared to those reported for 177 Lu-albumin conjugates. CONCLUSIONS: In this preclinical study, we demonstrated the potential of 177 Lu-scFvD2B as a therapeutic agent for PSMA-expressing tumors, due to its higher tumor-absorbed dose when compared with 177 Lu-LMW agents.