^99mTc-labeled Mutant Interleukin-2 as a Promising SPECT Probe for in vivo Imaging of Tumor-Infiltrating T Cells

Objectives: T cell infiltration of tumor lesions is a known prognostic factor in several tumor types and is used as treatment mechanism in some of these tumor types. Tumor infiltrating T cells express the high affinity interleukin-2 (IL2) receptor (CD25) on their surface. These T cells could therefore be visualized by molecular imaging with a radiolabeled IL2 for this receptor. For this purpose, [¹⁸F]FB-IL2 was designed as a PET imaging probe for T cell imaging, but strictly limited by its low target-to-background ratios and toxicity, which was resulted from direct binding of IL-2 to αβγ IL-2Rs on lung endothelial cells. Interaction of IL-2 with lung endothelial cells can be abrogated by blocking IL2 and IL-2Rα interaction. Here we developed a novel T cell imaging probe based on mutant Interleukin-2 (mIL2), which reduced binding to IL-2Rα and increased binding affinity for IL-2Rβ and may lead to higher target-to-background ratios and less toxicity in vivo than IL2.

Methods: Mutant Interleukin-2 was expressed in Sf9 cells and purified by Ni-NTA, then concentrated using spin concentrators and purified with an FPLC Superdex 75 sizing column. The bioactivity of purified mIL2 was detected with the CTLL2 cells which were dependent upon IL-2 for growth. IL2/IL-2Rα interaction was detected by ELISA. Then, mIL2 was radiolabeled with ^99mTc-HYNIC-GGGGK conjugated to the LPETG tag through Sortase A. The pharmacokinetic data of ^99mTc-HYNIC-mIL2 was obtained by ex vivo biodistribution experiments. Finally, the tumor infiltrating T cells detecting capability of ^99mTc-HYNIC-mIL2 in MC38 subcutaneous tumor-bearing mice was evaluated by in vivo NanoScan SPECT/CT imaging.

Results: mIL2 showed bioactivity with CTLL2 cells whereas lower CD25 binding compared with IL2. The ^99mTc-labeling procedure was done within 2 h, with a yield of 70%. The radiochemical purity was greater than 98%. As shown in figure 1, the tumor-infiltrating T cells located in the MC38 tumor margin was clearly visualized by ^99mTc-HYNIC-mIL2 NanoScan SPECT/CT imaging with low background except relatively high kidney uptake at all detected time points (0.5 and 2 h p.i.). Specificity of ^99mTc-HYNIC-mIL2 to T cells was demonstrated by competitive blocking of tumor uptake with excess mIL2. Biodistribution results showed that the tumor uptake of ^99mTc-HYNIC-mIL2 at 0.5 and 2 h p.i. was 2.59 ± 0.37 %ID/g and 2.19 ± 0.16 %ID/g, respectively.

Conclusions: ^99mTc-HYNIC-mIL2 is a promising SPECT radiotracer for the noninvasive imaging of tumor infiltrating T cells, which showed high target-to-background ratios. This novel tracer possesses enormous potential for evaluating and guiding immunotherapy in clinical practice, and its further investigation is still in progress.

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