Single-domain antibodies targeting the pan-T-cell markers CD2 and CD7 as universal immunotherapy T-cell tracers

Rationale Despite significant advancements in immunotherapeutic approaches, such as immune checkpoint modulation and CAR-T cell therapy, reliable and universally applicable surrogate markers to monitor and evaluate the wide variety of therapeutic responses are still missing. This gap of information can lead to delayed assessment of responders versus non-responders and to misinterpretation, potentially resulting in premature or erroneous treatment decisions. Thus, a universal surrogate marker may be of preeminent value in monitoring immune responses, guiding immunotherapies, and ultimately improving patient outcomes. To address this challenge, we developed two immunoPET tracers based on single-domain antibodies (sdAbs) that target the pan T-cell markers CD2 and CD7 to monitor and visualize T cells distribution in vivo. Methods This study aimed to establish a comprehensive approach for clinical translation by assessing the feasibility and key characteristics of CD2- and CD7-sdAb for safe, effective clinical application. Binding specificity was confirmed through antigen transduction and knockout in tumor and T-cell leukemia cell lines. In vitro, cytokine secretion and T-cell cytotoxicity were evaluated, while a xenogeneic myeloid sarcoma mouse model was used to analyze T-cell cytotoxicity and visualize TCR-transduced CD8 + T cells at the tumor site using PET/MR imaging. Results This study demonstrates the high specificity, affinity, and thermal stability of CD2- and CD7-sdAb, enabling efficient radiolabeling without impairing T-cell functionality. Specifically, binding to human CD8⁺ T cells did not alter cytokine secretion or cytotoxicity in vitro. Furthermore, in vivo, the cytotoxic ability of transferred TCR-transduced human CD8 + T cells was not compromised upon i.v. application of CD2- and CD7-sdAb. Ultimately, intravenously injected 68 Ga-NOTA-CD2-sdAb and 68 Ga-NOTA-CD7-sdAb were respectively able to clearly depict and visualize previously administered TCR-transduced CD8 + T cells at the tumor site by PET/MRI. Conclusions In this study we have developed two pan T-cell tracers that are able to visualize human T cells and clearly differentiate between responding- and non-responding tumors while not impacting T-cell functionality in vitro and in vivo. The results provide a base for clinical use of these tracers as pan T-cell tracers applicable for any form of immunotherapy, making them a crucial tool in guiding immunotherapeutic decision-making.