CD3ε F(ab’)₂ PET imaging captures systemic and intratumoral T-cell response to immune checkpoint blockade

Immunotherapy has transformed cancer treatment, yet only a subset of patients achieve durable responses. Because the spatial distribution of T cells within tumors and lymphoid tissues shapes therapeutic outcomes, noninvasive methods capable of monitoring these populations could improve the evaluation of antitumor immune responses. Here, we report the validation of a CD3ε-targeted F(ab’) 2 positron emission tomography (PET) probe for noninvasive mapping of T-lymphocytes across tumors and lymphoid tissues and characterize its effects on endogenous T-cell biology. Relative to the full-length IgG construct, the F(ab’) 2 format demonstrated more rapid blood clearance, enabled high-contrast imaging within 24 h, produced limited changes in endogenous T-cell populations, and did not measurably impair T-cell activation at imaging-relevant concentrations. CD3ε F(ab’) 2 PET detected tumor-infiltrating lymphocytes (TILs) and lymphocytes within the tumor-draining lymph nodes (TDLN) across multiple syngeneic tumor models and in a genetically engineered mouse model of lung adenocarcinoma. In a combination immunotherapy study, tumor uptake distinguished responding from non-responding lesions and detected treatment-associated changes across peripheral lymphoid tissues. These results support CD3ε F(ab’) 2 PET as a noninvasive imaging approach that enables assessment of T-cell distribution during tumor growth and therapy, with potential applications in mechanistic studies, biomarker development, and preclinical evaluation of immunomodulatory therapies.