Introduction Diffuse Large B-Cell Lymphoma (DLBCL), a prevalent form of non-Hodgkin’s lymphoma (NHL), poses challenges for accurate diagnosis using molecular imaging. The utility of PET imaging, particularly employing [¹⁸F]FDG, in DLBCL is constrained, impeding its reliability as a diagnostic modality due to difficulties in discerning between indolent and aggressive subtypes, as well as the occurrence of false-positive outcomes.¹ B-cell lymphoma 6 (BCL6) manifests dysregulation across multiple subtypes of NHL, featuring prominently in DLBCL. Previous investigations have underscored the pivotal role of BCL6, with its loss of function linked to DLBCL cell death.² Consequently, the functional activity of BCL6 is dependent on its interaction with transcriptional corepressors. The concept of inhibiting BCL6 has emerged as a promising therapeutic avenue, with BCL6 inhibitors demonstrating the capacity to disrupt this co-repressor binding, thereby presenting as potential diagnostic and therapeutic agents for DLBCL treatment.

The current research aims to develop a PET probe that shows enhanced specificity for BCL6 and demonstrates its relevant radiochemical properties, binding affinity, and biodistribution.

Method The precursor (OICR-01) for radiofluorination to develop a BCL6 PET tracer was supplied by the Ontario Institute for Cancer Research (OICR) and subjected to radiofluorination through nucleophilic substitution, as depicted in the provided figure. Quality control assessment included examinations of formulation and serum stability, purity, lipophilicity, and stickiness. The biological activity of the radiolabeled compounds ([¹⁸F]F-OICR) was evaluated in vitro, including the binding assay with Karpas422 cells to determine binding affinity. Subsequently, preliminary imaging and biodistribution studies, performed at one-hour post injection, were undertaken in non-tumor bearing NCr nude mice for preliminary in vivo analysis.

Result The radiolabeling of OICR-01 was reproducible across multiple attempts, resulting in 11.5 ± 4.0% radiochemical conversion (RCC) (n = 4). The final [18F]F-OICR, exhibited radiochemical purity (RCP) exceeding 99%. The molar activity (MA) was calculated at 0.89 ± 0.04 mCi/μmol.

[¹⁸F]F-OICR exhibited stability upon 99% in the formulation buffer for up to 4 hours and 97% in mouse serum for up to 2 hours. The hydrophobic character of [18F]F-OICR, as indicated by a Log P test, elucidated the substantial accumulation observed in the gallbladder during PET imaging studies 60 minutes post-injection (P.I.).

The Micro-PET studies in non-tumor bearing NCr mice (n=3) revealed renal and hepatobiliary excretion of [¹⁸F]F-OICR, with gallbladder accumulation consistent with its apparent lipophilicity. Notably, the blood uptake remained minimal, and blood clearance was rapid, suggesting that high tumor-to-background ratios are achievable using OICR-01. Additionally, limited uptake in the bone emphasized the in vivo stability of [¹⁸F]F -OICR-01. Subsequent in vitro saturation assays revealed concerns regarding the potential limitations of the MA value (0.89 mCi/μmol) in terms of binding efficiency to Karpas422 cells. The observed reduction in binding percentage was caused by the low MA leading to the competitive binding between radioactive and non-radioactive OICR and the self-blocking. Consequently, the declined binding percentage indirectly signifies the specificity of OICR-01 for Karpas422.

Conclusion The DLBCL probe named OICR-01 was successfully radiofluorinated with a RCC of 11.5 ± 4.0 (n=4) and RCP of >99%. The micro-PET study revealed renal and hepatobiliary excretion, along with swift blood clearance and minimal blood uptake, indicating the prospect of tumor uptake in xenograft mouse models. Nonetheless, subsequent binding assays have highlighted potential limitations in the binding efficiency with Karpas422 cells due to inherent self-blocking at the calculated MA of 0.89 mCi/μmol. This prompts further consideration for improving the MA value to enhance the efficiency of tumor cell uptake.

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Image/Figure Caption:

The OICR-01 radiolabeling and biological assessment including in vivo and in vitro.

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