Anti-PSMA 124I-scFvD2B as a new immuno-PET tool for prostate cancer: preclinical proof of principle

Abstract

Background: Prostate cancer (PCa) is the second leading cause of cancer-related death in the Western population. The use in oncology of positron emission tomography/computed tomography (PET/CT) with emerging radiopharmaceuticals promises accurate staging of primary disease, restaging of recurrent disease and detection of metastatic lesions. Prostate-specific membrane antigen (PSMA) expression, directly related to androgen-independence, metastasis and progression, renders this tumour associate antigen a good target for the development of new radiopharmaceuticals for PET. Aim of this study was to demonstrate in a preclinical in vivo model (PSMA-positive versus PSMA-negative tumours) the targeting specificity and sensitivity of the anti-PSMA single-chain variable fragment (scFv) labelled with ¹²⁴I.

Methods: The ¹²⁴I-labeling conditions of the antibody fragment scFvD2B were optimized and assessed for purity and immunoreactivity. The specificity of ¹²⁴I-scFvD2B was tested in mice bearing PSMA-positive and PSMA-negative tumours to assess both ex-vivo biodistribution and immune-PET.

Results: The uptake fraction of ¹²⁴I-scFvD2B was very high on PSMA positive cells (range 75-91%) and highly specific and immuno-PET at the optimal time point, defined between 15 h and 24 h, provides a specific localization of lesions bearing the target antigen of interest (PSMA positive vs PSMA negative tumors %ID/g: p = 0.0198 and p = 0.0176 respectively) yielding a median target/background ratio around 30-40.

Conclusions: Preclinical in vivo results of our immuno-PET reagent are highly promising. The target to background ratio is improved notably using PET compared to SPECT previously performed. These data suggest that, upon clinical confirmation of sensitivity and specificity, our anti-PSMA ¹²⁴I-scFvD2B may be superior to other diagnostic modalities for PCa. The possibility to combine in patients our ¹²⁴I-scFvD2B in multi-modal systems, such as PET/CT, PET/MR and PET/SPECT/CT, will provide quantitative 3D tomographic images improving the knowledge of cancer biology and treatment.

Keywords: 124I; Antibody fragment; PCa; PET; scFv.

Fig. 1

Cartoon illustrating the in vivo procedure: 5 × 10⁶ of either PC3-PIP (PSMA positive) or PC3 (PSMA negative) cells were injected subcutaneously (s.c.) in the flank of mice. 20 days later, ¹²⁴I-scFvD2B was administered i.v. and after 15–24 h both ImmunoPET imaging and ex vivo biodistribution were performed

Fig. 2

ImmunoPET imaging and ex vivo biodistribution analysis with ¹²⁴I-scFvD2B of experiment #1: a Immuno-PET images after intravenous administration of 3.7 MBq ¹²⁴I⁻scFvD2B (166.5 MBq/mg) in two mice. PC3 (PSMA negative, right flank) and PC3-PIP (PSMA positive, left flank) tumours evaluated 24 h post injection. Stomach and Thyroid are indicated by an arrow. The imaging is so clean in the irrelevant organ that it is barely visible the shape of the mice. b Ex vivo biodistribution analysis in PC3 and PC3-PIP tumours in the same two mice. Uptake and retention were measured in different organs and illustrated both as decay-adjusted percentage of the injected dose per gram of tissue (% ID/g) and T/B. The background activity observed in the stomach was probably due to the free ¹²⁴I or the possible ingestion of the sawdust contaminated by the radioactive fraction present in the mice urine since was not present in the fourth experiment done in a larger number of animals (Fig. 3b)

Fig. 3

ImmunoPET imaging and ex vivo biodistribution analysis with ¹²⁴I-scFvD2B of experiment #4. a One representative Immuno-PET images after intravenous administration of 7.4 MBq ¹²⁴I⁻scFvD2B (433 MBq/mg). PC3 (PSMA negative, right flank) and PC3-PIP (PSMA positive, left flank) tumours evaluated 15 and 24 h post injection. Bladder is also indicated by an arrow. The imaging is so clean in the irrelevant organ that it is barely visible the shape of the mice. b Ex vivo biodistribution analysis in PC3 and PC3-PIP tumour bearing mice evaluated 15 h (2 mice) and 24 h (3 mice) post injection of ¹²⁴I-scFvD2B. Uptake and retention were measured in different organs and illustrated both as decay-adjusted percentage of the injected dose per gram of tissue (% ID/g) and T/B