Antibody and fragment-based PET imaging of CTLA-4+ T-cells in humanized mouse models

Imaging of immunotherapy targets using positron emission tomography (PET) can allow for noninvasive monitoring of their dynamic expression and may allow for patient stratification in the future. Therefore, two tracers targeting CTLA-4, one a full antibody and the other a F(ab')₂ fragment, were radiolabeled with ⁶⁴Cu and validated in humanized mouse models. Ipilimumab was digested to develop ipilimumab-F(ab')₂, and both the intact antibody and the fragment were conjugated with NOTA to chelate ⁶⁴Cu for PET. The tracers were administered to both control NBSGW mice and humanized mice (PBL mice, engrafted with human peripheral blood lymphocytes), and PET was conducted out to 48 h post-injection. PET region-of-interest analysis, ex vivo biodistribution studies, and tissue staining were used to confirm that the tracers specifically accumulated in CTLA-4+ tissues. Following injection of tracers (n = 3-5 per group), specific uptake was noted in the salivary gland tissues of the humanized mice. This uptake, a result of graft-versus-host disease onset, was proven to be due to human T-cells through staining of the tissues for human CD3 and CTLA-4. ⁶⁴Cu-NOTA-ipilimumab demonstrated the highest absolute uptake in the salivary glands of PBL mice, peaking at 7.00 ± 2.19 %ID/g. In contrast, ⁶⁴Cu-NOTA-ipilimumab-F(ab')₂ uptake was 2.40 ± 0.86 %ID/g at the same time point. However, the F(ab')₂ agent cleared from circulation more quickly than the intact antibody, providing higher salivary gland-to-blood ratios, which reached 1.78 ± 0.72 at 48 h post-injection, compared to ⁶⁴Cu-NOTA-ipilimumab at 1.19 ± 0.49. Uptake of the tracers in the salivary glands of control mice, and the nonspecific tracer in the PBL mice, was lower at all time points as well. PET imaging with both ⁶⁴Cu-NOTA-ipilimumab and ⁶⁴Cu-NOTA-ipilimumab-F(ab')₂ was able to localize CTLA-4+ tissues. These tracers may thus help elucidate the mechanisms of response to CTLA-4-targeted checkpoint immunotherapy treatments.