Development of 177Lu-scFvD2B as a Potential Immunotheranostic Agent for Tumors Overexpressing the Prostate Specific Membrane Antigen

Abstract

The clinical translation of theranostic 177 Lu-radiopharmaceuticals based on inhibitors of the prostatespecific membrane antigen (PSMA) has demonstrated positive clinical responses in patients with advanced prostate cancer (PCa). However, challenges still remain, particularly regarding their pharmacokinetic and dosimetric properties. We developed a potential PSMA-immunotheranostic agent by conjugation of a single-chain variable fragment of the IgGD2B antibody (scFvD2B) to DOTA, to obtain a 177 Lu-labelled agent with a better pharmacokinetic profile than those previously reported. The labelled conjugated 177 Lu-scFvD2B was obtained in high yield and stability. In vitro, 177 Lu-scFvD2B disclosed a higher binding and internalization in LNCaP (PSMA-positive) compared to PC3 (negative control) human PCa cells. In vivo studies in healthy nude mice revealed that 177 Lu-scFvD2B present a favorable biokinetic profile, characterized by a rapid clearance from non-target tissues and minimal liver accumulation, but a slow wash-out from kidneys. Micro-SPECT/CT imaging of mice bearing pulmonary microtumors evidenced a slow uptake by LNCaP tumors, which steadily rose up to a maximum value of 3.6 SUV at 192 h. This high and prolonged tumor uptake suggests that 177 Lu-scFvD2B has great potential in delivering ablative radiation doses to PSMA-expressing tumors, and warrants further studies to evaluate its preclinical therapeutic efficacy. Background. Prostate cancer (PCa) is the second leading cause of cancer deaths for adult men in the Western world. Although radical prostatectomy and local radiotherapy are largely successful for patients with localized cancer, available treatments for metastatic PCa have demonstrated weak curative efficacy 1. Consequently, new tools to improve the detection of recurrent PCa, and particularly to identify and treat distant metastases, are imperatively needed. The prostate-specific membrane antigen (PSMA) is one of the most promising targets for the development of PCa theranostic agents. PSMA is overexpressed in 95% of prostate cancers and its expression levels progressively increase in high-grade tumors and in metastatic disease, up to 1,000 times more than in normal cells 2. Among the several PSMA-targeting molecules that have been developed, the radiolabeled Glu-ureido-based PSMA inhibitors are gaining much interest due to their high uptake by PSMA-positive cancer cells, and low background and excellent contrast in cancer imaging, even in small metastases 3,4. Theranostic agents such as 177 Lu-PSMA-617,

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

“…ScFvD2B (MW 26624 g/mol) was produced and purified at the University of Verona, as previously described. 10,11 It was then conjugated to DOTA (S-2-(4-benzyl-isothiocyanate)-1,4,7,10-tetra-azacyclododecane tetraacetic-acid) by incubation in 0.2 M carbonate buffer (pH 9.5) of 1:5 scFv: DOTA molar ratios at 37°C for 1 hr, according to the method described by Carpanese et al 9 The DOTA-scFvD2B conjugate was subsequently purified using a Vivaspin ® centrifugal concentrator (MWCO 5 kDa; Sartorius) and 0.25 M ammonium acetate (pH 7.0) as a washing buffer to remove the unconjugated DOTA excess. Finally, the DOTA-scFvD2B conjugate was radiolabeled as previously reported.…”

“…Finally, the DOTA-scFvD2B conjugate was radiolabeled as previously reported. 9 In brief, 1.5 mg of ScFvD2B were dissolved in 0.5 ml of 0.25 M sodium acetate buffer (pH 7.0) and incubated with 25 µl (900 MBq) of no-carrier-added 177 LuCl 3 (ITG) for 2 hr at 37°C to obtain the 177 Lu-scFvD2B.…”

“…In vitro studies using LNCaP cells showed a significant higher cell uptake and internalization of 177 Lu-scFvD2B compared with 177 Lu-PSMA-617 and 177 Lu-iPSMA. 9 Moreover, biodistribution studies in mice demonstrated the favorable biokinetic profile of 177 Lu-scFvD2B with minimal liver accumulation, rapid clearance from nontarget tissues and high and prolonged tumor uptake. 9 However, 177 Lu-scFvD2B presented a high kidney uptake, a common feature of radiolabeled scFv fragments that are endowed with a molecular weight lower than the renal clearance threshold.…”

“…9 Moreover, biodistribution studies in mice demonstrated the favorable biokinetic profile of 177 Lu-scFvD2B with minimal liver accumulation, rapid clearance from nontarget tissues and high and prolonged tumor uptake. 9 However, 177 Lu-scFvD2B presented a high kidney uptake, a common feature of radiolabeled scFv fragments that are endowed with a molecular weight lower than the renal clearance threshold. This could limit its clinical application as a PCa endoradiotherapy agent.…”

“…This could limit its clinical application as a PCa endoradiotherapy agent. 9 The aim of this study was to estimate main organ and tumor-absorbed doses, using OLINDA software and pharmacokinetic data obtained from biodistribution studies and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of 177 Lu-scFvD2B in healthy and tumor-bearing mice. Dosimetric studies of 177 Lu-PSMA-617 and 177 Lu-iPSMA, two of the agents used in PCa clinical trials, were also carried out under the same conditions in order to compare their absorbed dose values with those of 177 Lu-scFvD2B.…”