⁶⁴Cu-labeled Nanobodies Monitor CD4+ T-Cell Populations in Response to Immunotherapy Using PET Imaging

Take-Away Points

• ■ Major Focus: To develop a radiolabeled nanobody to noninvasively track CD4⁺ T-cell populations and monitor response or resistance to immunotherapy by using PET.

• ■ Key Results: In vivo PET using the CD4 receptor targeting copper 64–radiolabeled nanobody, or ⁶⁴Cu-CD4-Nb1, revealed cell density changes within the tumor microenvironment in response to immune checkpoint inhibitor (ICI) therapy.

• ■ Impact: ⁶⁴Cu-CD4-Nb1 PET enables tracking of CD4⁺ cell densities to monitor patient response to immunotherapy and facilitate earlier intervention in patients with cancer.

Response of the immune system to tumor cells, both before and after traditional chemoradiation or immunotherapy, plays a crucial role in treatment planning and patient outcomes. Among the various antitumor immune cells, CD4⁺ T cells are particularly important as they stimulate the response of other immune cells and can serve as biomarkers of immune activation or suppression. Although immune checkpoint inhibitors are a primary treatment option for certain cancers, tracking therapeutic efficacy and resistance remains a challenge. Identifying a biomarker to track the efficacy of immunotherapy would facilitate earlier intervention and adjustments to treatment.

Pezzana et al developed alpaca-derived single-domain antibodies specific to human CD4 receptors (CD4-Nb1) to visualize CD4⁺ T-cell populations in response to immunotherapy using PET. Single-domain antibodies, or nanobodies, enable rapid tissue penetration and blood clearance. CD4-Nb1 was radiolabeled with ⁶⁴Cu to produce ⁶⁴Cu-CD4-Nb1. Human CD4 receptor knock-in mice (CD4-KI) and wild-type C57BL/6J mice were orthotopically inoculated with PyMT breast cancer cells and treated with αPD-1/α4-1BB immune checkpoint inhibitor prior to imaging. ⁶⁴Cu-CD4-Nb1 PET visualized CD4⁺ T-cell populations and densities at the tumor core and periphery, which were confirmed with quantitative ex vivo biodistribution and immunohistochemistry.

⁶⁴Cu-CD4-Nb1 uptake in CD4⁺ T cells was higher in ICI-treated PyMT CD4-KI mice compared to ICI-treated PyMT wild-type mice (0.44 %ID/mL ± 0.04 vs 0.23 %ID/mL ± 0.02). Additionally, untreated tumors exhibited ⁶⁴Cu-CD4-Nb1 accumulation at the tumor periphery, whereas immune checkpoint inhibitor treated tumors showed greater accumulation within the core, indicating enhanced CD4⁺ T-cell infiltration into the tumor. These findings demonstrate that ⁶⁴Cu-CD4-Nb1 effectively targets CD4 T-cell populations and that its spatial distribution provides insights into T-cell dynamics and responses to immunotherapy.

⁶⁴Cu-CD4-Nb1 PET imaging enables early, noninvasive assessment of CD4⁺ T cell–mediated responses to immunotherapy with potential to guide treatment adaptation and improve outcomes in patients with cancer.

Highlighted Article

• Pezzana S, Blaess S, Traenkle B, et al. PET-based immunomapping of intratumoral CD4+ cells to monitor acquired resistance to checkpoint inhibitors. Sci Adv 2025;11(26):eadw1924. doi: https://doi.org/10.1126/sciadv.adw1924