HER2-targeted dual radiotracer approach with clinical potential for noninvasive imaging of trastuzumab-resistance caused by epitope masking

Rationale: The decreased HER2-accessibility by epitope masking is a primary trastuzumab-resistance mechanism. In this study, we developed a HER2-targeted dual radiotracer approach to predict the HER2-trastuzumab engagement noninvasively. Methods: Two novel HER2-specific VHHs, MIRC208 and MIRC213, were acquired by immunizing alpaca with human HER2 protein, and were site-specifically labeled with (99m)Tc. Biodistribution and SPECT/CT imaging studies were performed in mice bearing HER2-positive and HER2-negative tumors. The HER2 binding sites of (99m)Tc-MIRC208 and (99m)Tc-MIRC213 were investigated by cell binding and SPECT/CT imaging studies. We evaluated the therapeutic predictive ability of our dual-radiotracer imaging approach for trastuzumab treatment in mice bearing MUC4-positive tumors (trastuzumab-resistant JIMT-1 and 87MUC4) and MUC4-negative tumors (trastuzumab-sensitive 7HER2 and NCI-N87). The preliminary clinical studies of (99m)Tc-MIRC208 were performed in two patients with HER2-positive breast tumors. Results: (99m)Tc-MIRC208 and (99m)Tc-MIRC213 clearly visualized HER2-positive tumors, but not HER2-negative tumors. (99m)Tc-MIRC208 competes with trastuzumab for HER2-binding while (99m)Tc-MIRC213 recognizes HER2 on an epitope that is not masked by MUC4. The SPECT/CT studies with (99m)Tc-MIRC208 and (99m)Tc-MIRC213 clearly showed that the MUC4-negative and trastuzumab-sensitive 7HER2 and NCI-N87 tumors had very similar tumor uptake with the SUV(208)/SUV(213) (2 h) ratios of 1.11 ± 0.17 in 7HER2 and 1.25 ± 0.22 in NCI-N87. However, the MUC4-positive JIMT-1 tumors showed the decreased SUV(208)/SUV(213) (2 h) ratio (0.63 ± 0.07), which correlated well with the low response rate to trastuzumab therapy. The SUV(208)/SUV(213) (2 h) ratio was reduced to 0.72 ± 0.02 in MUC4-expressing NCI-N87 cells, and resulting in the decreased trastuzumab sensitivity, further supporting the correlation between the SUV(208)/SUV(213) (2 h) ratio and trastuzumab-sensitivity. The primary and metastatic HER2-positive lesions of patients were clearly visualized by (99m)Tc-MIRC208 SPECT at 2 h post injection. Conclusion: Overall, we demonstrated that the dual radiotracer imaging strategy is a valid noninvasive approach for the cancer patient selection before trastuzumab therapy. (99m)Tc-MIRC213 SPECT is utilized to quantify the tumor HER2 expression and screen HER2-positive cancer patients, while (99m)Tc-MIRC208 SPECT is used to determine the HER2-accessibility of trastuzumab. The SUV(208)/SUV(213) (2 h) ratio is an important biomarker to determine the responsiveness of trastuzumab therapy.