The presence and activity profile of tumor-infiltrating immune cells may predict responsiveness to (immuno)therapy. More specifically, CD8+ cytotoxic T cells are believed to be the main effector cells during anti-cancer responses. Therefore, non-invasive imaging of these cells is believed to be able to predict and/or follow up immunotherapy efficacy in cancer patients. In this study, we have developed novel human CD8β-targeting nanobody-based immunotracers for non-invasive nuclear imaging of CD8+ T cells during anti-cancer immunotherapy.
Two lead CD8β-targeting nanobodies, and for comparison one irrelevant control nanobody, were selected and characterized in vitro on their affinity towards human and cynomolgus CD8 via surface plasmon resonance and ELISA. Binding to the human CD8+ cell line SUP-T1 and primary CD8+ T cells of donors was analysed via flow cytometry. Thermostability was determined using a thermofluor assay. Site-specific 99mTc labelling efficiency was evaluated with iTLC. SPECT/CT imaging and subsequent ex vivo γ-counting of dissected organs were performed in naïve wild-type and human CD8 transgenic mice, and in s.c. MC38-tumor bearing mice one hour post-injection. The CD8β-targeting nanobodies were converted to PET tracers via NOTA-conjugation and subsequent 68Ga-labeling.
The two lead nanobodies showed high affinity binding, in the low nanomolar range, to the human and cynomolgus but not the mouse CD8β chain. Selective binding to human CD8+ SUP-T1 cells and primary CD8+ T cells was observed for both nanobodies. Thermostability studies showed that both nanobodies had melting temperatures above 55°C, which allowed site-specific 99mTc labelling with labelling efficiencies above 99%. SPECT/CT imaging and subsequent ex vivo γ-counting of the organs showed specific accumulation of the radiolabelled CD8β-targeting nanobodies in T-cell-rich organs of naïve human CD8 transgenic, but not wild-type mice. The radiolabelled anti-CD8β nanobodies targeted intratumoral CD8+ T cells in MC38-tumor bearing human CD8 transgenic mice. Finally, these lead nanobodies were converted to PET tracers, which are currently being tested in vitro and in vivo.
Our results show the generation of novel anti-human CD8β immunotracers. Currently, these nanobodies are being analysed to exclude potential effects on T-cell activation. Their abilities as PET tracers are being tested in vitro and in vivo in mice. Overall, these immunotracers could be interesting tools to allow same-day imaging to predict and/or follow up immunotherapy responses in patients with cancer and other CD8+ T cell driven pathologies.

This work was supported by the EU/EFPIA/Innovative Medicines Initiative 2 Joint Undertaking (Immune Image GA831514) under grant agreement N° 831514.
T.W.M De Groof and Y. Lauwers contributed equally. J.A. Van Ginderachter and N.Devoogdt share senior authorship.
Originele taal-2English
StatusPublished - 2023
EvenementEMIM 2023: 18th European Molecular Imaging Meeting - Salzburg, Austria
Duur: 14 mrt 202317 mrt 2023


ConferenceEMIM 2023: 18th European Molecular Imaging Meeting


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