Development and Preclinical Evaluation of [⁶⁴Cu]Cu-NOTA-ABDB6: A CD70 and Albumin Dual-Binding Tracer with Improved Pharmacokinetics.

Abstract

CD70 is an emerging biomarker for both solid tumors and hematologic malignancies, highlighting the urgent need for a molecular imaging tracer capable of visualizing CD70 with favorable pharmacokinetics. <b>Methods:</b> ABDB6 was prepared by fusing the albumin-binding domain ABD035 with the CD70-targeting single-domain antibody RCCB6, which we previously reported. The resulting ABDB6 was then conjugated to the bifunctional chelator <i>p</i>-SCN-NOTA and labeled with ⁶⁴Cu to produce [⁶⁴Cu]Cu-NOTA-ABDB6. Flow cytometry was used to screen 6 lymphoma cell lines with varying CD70 expression levels. Cell uptake and in vivo immuno-PET imaging studies were conducted to fully evaluate the pharmacokinetic properties and tumor-targeting efficacy of [⁶⁴Cu]Cu-NOTA-ABDB6. An ABDB6 blocking study was performed to validate the targeting specificity of [⁶⁴Cu]Cu-NOTA-ABDB6, followed by immunohistochemistry and fluorescent immunostaining studies to correlate tracer uptake with CD70 expression. <b>Results:</b> ⁶⁴Cu labeling of ABDB6 achieved a high radiochemical yield and specific activity. Significant CD70 expression was observed in 5 lymphoma cell lines (TMD8, HBL1, OCI-LY10, LCL-EBV, and type III latency Burkitt lymphoma [BL] cells) but not in type I latency BL cells, which served as the negative control. [⁶⁴Cu]Cu-NOTA-ABDB6 exhibited good affinity for CD70 protein at the nanomolar level (inhibitory concentration of 50%, 91.57 nM) and specificity in binding to human CD70. Immuno-PET imaging of [⁶⁴Cu]Cu-NOTA-ABDB6 demonstrated excellent tumor uptake and retention in various CD70-positive lymphoma models (TMD8, type III latency BL, and LCL-EBV), with the highest tumor uptake values recorded as 24.67 ± 1.36, 18.02 ± 4.29, and 14.68 ± 1.20 percentage injected dose per gram of tissue (%ID/g) at 48 h after injection, respectively. These tumor uptake values were significantly higher than that of the CD70-negative type I latency BL tumor, which had an uptake of 3.59 ± 0.28 %ID/g at the same scanning time point (<i>P</i> < 0.05). In the TMD8 blocking group, tumor uptake was 5.99 ± 1.20 %ID/g at 48 h after injection, significantly lower than in the TMD8 control group (<i>P</i> < 0.01). Both biodistribution and histology results corroborated these imaging findings. <b>Conclusion:</b> [⁶⁴Cu]Cu-NOTA-ABDB6 immuno-PET effectively visualized varying levels of CD70 in different lymphoma models. Its clinical potential may provide insights into CD70 expression in lymphoma patients.