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

Abstract

The clinical translation of theranostic ¹⁷⁷Lu-radiopharmaceuticals based on inhibitors of the prostate-specific 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 ¹⁷⁷Lu-labelled agent with a better pharmacokinetic profile than those previously reported. The labelled conjugated ¹⁷⁷Lu-scFvD2B was obtained in high yield and stability. In vitro, ¹⁷⁷Lu-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 ¹⁷⁷Lu-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 ¹⁷⁷Lu-scFvD2B has great potential in delivering ablative radiation doses to PSMA-expressing tumors, and warrants further studies to evaluate its preclinical therapeutic efficacy.

Figure 1

(A) Overlapping of MALDI-MS spectra of the unconjugated scFvD2B (red line) and DOTA-scFvD2B conjugate obtained, using a 1:5 (scFvD2B:DOTA) molar ratio (black line). (B) Binding affinity studies conducted by flow cytometry on the PSMA-positive PC-3-PIP cells (dots) and PSMA-negative PC-3 cells (triangles).

Figure 2

(A) Radiochemical yield for increasing amounts of DOTA-scFvD2B, incubated at 37 °C for 1 and 2 h. (B) Size-exclusion HPLC radiochromatograms of ¹⁷⁷Lu-scFvD2B obtained with a molar activity of 18 MBq/nmol without dilution (black line) and after 24 and 192 h dilution in 0.1 M phosphate buffer at pH 7.0 (red and blue lines respectively).

Figure 3

(A) Cell uptake and (B) internalization of ¹⁷⁷Lu-labeled scFvD2B, ¹⁷⁷Lu-PSMA-617 and ¹⁷⁷Lu-iPSMA by PSMA-negative PC-3 and PSMA-positive LNCaP cells, after 2 h of incubation with 2 MBq of ¹⁷⁷Lu-radiotracers (16 MBq/nmol) at 37 °C. Bars represent the average value ± SD of one experiment performed in triplicate, **p < 0.005 and ****p < 0.0001, two-way ANOVA.

Figure 4

Biodistribution data (decay-corrected) up to 192 h after injection of 3.7 MBq (6.5 MBq/nmol) of ¹⁷⁷Lu-scFvD2B in healthy nude male mice, and expressed as %ID/g. Bars represent the average value ± SD obtained from each group of mice (n = 3).

Figure 5

(A) ¹⁷⁷Lu-scFvD2B blood clearance. (B) Biokinetic curves of non-target organs with rapid clearance. (C) Biokinetic curves of non-target organs with slower clearance.

Figure 6

(A) SPECT/CT images of mice bearing LNCaP lung microtumors collected at different time points after injection of 37 MBq dose of ¹⁷⁷Lu-iPSMA (upper panels) and ¹⁷⁷Lu-scFvD2B (lower panels). Images are representative of one out three mice selected for each group. (B) ¹⁷⁷Lu-scFvD2B and ¹⁷⁷Lu-iPSMA tumor kinetic curves obtained from the pulmonary microtumors data. CT and SPECT single images, as well as axial and sagittal sections displaying the microtumor locations of each mouse, are shown in Supplementary Information (see Figs. S9–S32).