Development of 89Zr-anti-CD103 PET imaging for non-invasive assessment of cancer reactive T cell infiltration.

PURPOSE: CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (89Zr) radiolabeling of monoclonal antibody (mAb) clones that specifically recognize human CD103 for non-invasive immune positron-emission tomography (PET) imaging of T cell infiltration as potential biomarker for effective anticancer immune responses. EXPERIMENTAL DESIGN: First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed an 89Zr-anti-murine CD103 PET tracer. Healthy, non-tumor bearing C57BL/6 mice underwent serial PET imaging after intravenous injection, followed by ex vivo biodistribution. Tracer specificity and macroscopic tissue distribution were studied using autoradiography combined with CD103 immunohistochemistry. Next, we generated and screened six unique mAbs that specifically target human CD103 positive cells. Optimal candidates were selected for 89Zr-anti-human CD103 PET development. Nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103 expressing Chinese hamster ovary (CHO) or CHO wild-type xenografts were injected with 89Zr-anti-human CD103 mAbs and underwent serial PET imaging, followed by ex vivo biodistribution. RESULTS: 89Zr-anti-murine CD103 PET imaging identified CD103-positive tissues at clinically relevant target densities. For human anti-human CD103 PET development two clones were selected based on strong binding to the CD103+ CD8+ T cell subpopulation in ovarian cancer tumor digests, non-overlapping binding epitopes and differential CD103 blocking properties. In vivo, both 89Zr-anti-human CD103 tracers showed high target-to-background ratios, high target site selectivity and a high sensitivity in human CD103 positive xenografts. CONCLUSION: CD103 immuno-PET tracers visualize CD103 T cells at relevant densities and are suitable for future non-invasive assessment of cancer reactive T cell infiltration.

89Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment.

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 (89Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on89Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to 89Zr-mAb3481 administration. Subsequently, ex vivo 89Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). 89Zr-mAb3481 BT474 tumor uptake was remarkably high at SUVmean 5.6±0.6 (51.8±7.7%ID/g) using a 10 µg 89Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect 89Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. 89Zr-mAb3481 PET accurately reflected HER3 tumor status. 89Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.