Abstract

Monoclonal IgG antibodies in oncology inhibit cancer cell receptors, preventing tumor proliferation. Trastuzumab targets HER2 in breast cancer, blocking HER2 dimerization and signaling. It also induces ADCC by engaging NK cells via the FcγRIIIA (CD16) receptor, leading to cancer cell lysis. NK cells can directly kill cancer cells and modulate immune responses via cytokines and chemokines. Higher NK cell infiltration in HER2-positive tumors correlates with better responses to HER2-targeted therapies. Studies show increased NK cells in tumors post-trastuzumab treatment. Whole-body imaging tracks NK cell distribution and infiltration, providing spatial and longitudinal data. Techniques include optical imaging, MRI, SPECT/γ-scintigraphy with [111In]In-oxine, and PET with [89Zr]Zr-oxine. A novel PET method using [89Zr]Zr-oxine enables 1–2 week tracking of NK cells. This study uses PET/CT to track [89Zr]Zr-oxine labeled NK cells in HER2-positive HCC1954 tumors, assessing trastuzumab’s effect on NK cell infiltration.

Materials and methods

Human NK cells were isolated from peripheral blood of healthy donors with ethical approval and written consent. The cells were expanded ex vivo and radiolabeled with [89Zr]Zr-oxine, then assessed for retention, viability, growth, chemotaxis, degranulation, and ADCC behaviors. Animal studies used NSG mice inoculated with HCC1954 breast cancer cells. When tumors reached 100–150 mm^3, mice received 89Zr-NK cells, rhIL-15, and either PBS, a control antibody, or trastuzumab. PET/CT imaging was performed on days 1, 3, and 7 post-injection to track NK cell distribution.

Additional rhIL-15 doses supported NK cell survival and expansion in vivo. Ex vivo flow cytometry analyzed human NK cells in mouse liver and spleen, confirming the presence of 89Zr-NK cells. SPECT/CT imaging assessed trastuzumab biodistribution. Data were analyzed using Prism software with appropriate statistical tests. The study demonstrated that trastuzumab enhances NK cell infiltration into HER2-positive tumors.

Results from nanoScan® PET/CT

⁸⁹Zr-NK Cells in HCC1954 Tumor–Bearing Mice Demonstrate Enhanced Tumor Localization with Trastuzumab Treatment

Using PET/CT imaging, the migration and accumulation of human 89Zr-NK cells were studied in female NSG immunodeficient mice with orthotopic HCC1954 breast tumors. HCC1954 cells, despite high HER2 expression, are resistant to trastuzumab’s HER2-binding effects, making this model suitable for studying Fc-mediated effects of the antibody on NK cell accumulation. Mice received 1 × 10^7 89Zr-NK cells intravenously, combined with either PBS (n = 6), trastuzumab (n = 6), or a control antibody (n = 4). NK cells were sourced from three healthy donors, with each group receiving cells from each donor. To support NK cell survival, mice were given rhIL-15 intraperitoneally. PET/CT scans were conducted on days 1, 3, and 7 post-injection. Results showed 89Zr-NK cells initially migrating to the lungs, liver, and spleen within 24 hours, followed by a redistribution primarily to the liver and spleen over seven days.

FIGURE 3. Biodistribution of ⁸⁹Zr-NK cells in female NSG mice bearing orthotopic HER2-expressing HCC1954 tumors. (A) Representative maximum-intensity projection (MIP) and tumor slice PET/CT images of mice administered ⁸⁹Zr-NK cells (10⁷ cells, ∼150–200 kBq) in combination with trastuzumab (5 mg/kg) or PBS only. Tumors are outlined for clarity. (B) PET image quantification of selected organs and tumors (mean ± SD, n = 4–6/group). *P < 0.05. **P < 0.01. Li = liver; Lu = lungs; MIP = maximum-intensity projection; ns = nonsignificant; Sp = spleen; T = tumor.

PET/CT imaging showed that 89Zr-NK cells accumulated in tumors but decreased from day 1 to day 7 post-injection, with a heterogeneous distribution across all groups of mice, predominantly at the tumor periphery. PET quantification revealed that mice treated with trastuzumab had significantly higher 89Zr-NK cell infiltration in tumors at 1 day post-injection (0.66 ± 0.13 %ID·g−1) compared to the sham group (0.38 ± 0.16 %ID·g−1, P = 0.0063) and the isotype group (0.37 ± 0.11 %ID·g−1, P = 0.0499). At 3 days post-injection, the trastuzumab group also showed higher infiltration (0.34 ± 0.12 %ID·g−1) than the sham group (0.21 ± 0.04 %ID·g−1, P = 0.0593) and the isotype group (0.18 ± 0.03 %ID·g−1, P = 0.0268). No significant differences in tumor activity were observed between the isotype-treated and PBS sham-treated groups at these time points. By day 7, only low amounts of 89Zr were detected in tumors across all groups, reflecting the in vitro observed loss of the 89Zr label. Ex vivo biodistribution and γ-counting at days 3 and 7 corroborated PET findings, showing similar radioactivity in major organs and tissues. No significant differences in tumor radioactivity between the trastuzumab, PBS sham, and isotype groups were observed at day 3, likely due to NK cell loss during tissue processing.

Conclusion

In this study, they have demonstrated the effectiveness of [89Zr]Zr-oxine for labeling and tracking human NK cells in a murine model of orthotopic human breast cancer. This method provides a sensitive means to quantitatively analyze changes in the distribution of NK cells in response to antibody treatments. Our results underscore that NK cells effectively migrate to orthotopic HER2-expressing HCC1954 tumors, with trastuzumab enhancing early infiltration, consistent with clinical observations. The integration of 89Zr labeling with PET/CT imaging offers valuable insights into the dynamics of antibody therapies, particularly in relation to the immune microenvironment and Fc-mediated therapeutic mechanisms.

Full article on Journal of Nuclear Medicine