Monitoring CD8a+ T Cell Responses to Radiotherapy and CTLA-4 Blockade Using [64Cu]NOTA-CD8a PET Imaging

Abstract

Purpose: Current response assessment systems for cancer patients receiving immunotherapy are limited. This is due to the associated inflammatory response that may confound the conventional morphological response evaluation criteria in solid tumors and metabolic positron emission tomography (PET) response criteria in solid. Recently, novel PET imaging techniques using radiolabeled antibodies and fragments have emerged as a particularly sensitive and specific modality for quantitative tracking of immune cell dynamics. Therefore, we sought to investigate the utility of Cu-64 labeled F(ab)′2 fragments for in vivo detection of CD8a + T cells as a prognostic imaging biomarker of response to immunotherapy in an immunocompetent mouse model of colorectal cancer. Procedures: [ 64 Cu]NOTA-CD8a was produced by enzymatic digestion of rat-anti-mouse CD8a antibody (clone YTS169.4), purified yielding isolated CD8a-F(ab)′2 fragments and randomly conjugated with the 2-S-(isothiocyanatbenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) chelator. NOTA-CD8a was radiolabeled with Cu-64 and injected into CT26 tumor-bearing mice for longitudinal assessment. To investigate the value of [ 64 Cu]NOTA-CD8a PET imaging for assessment of treatment response, CT26 tumor-bearing mice were subjected to external radiation therapy (XRT) in combination with anti-CTLA-4 therapy. Imaging data was supported by flow cytometry and immunohistochemistry (IHC). Results: Combination treatment with XRT and anti-CTLA-4 effectively inhibited tumor growth until day 22 post-therapy initiation (p = 0.0025) and increased the overall survival of mice compared to control (p = 0.0017). The [ 64 Cu]NOTA-CD8a tumor-to-heart ratio was increased in XRT + anti-CTLA-4-treated mice on day 8 after initiation of therapy (p = 0.0246). Flow cytometry and IHC confirmed the increase in tumor-infiltrating CD8a + cells in XRT + anti-CTLA-4-treated mice. Furthermore, [ 64 Cu]NOTA-CD8a PET imaging distinguished responders and nonresponders prior to treatment-induced changes in tumor volume among mice. Conclusion: In the present study, we demonstrated that [ 64 Cu]NOTA-CD8a was able to detect treatment-induced changes in CD8a + infiltration in murine CT26 colon tumors following a common preclinical combination treatment protocol. Overall, [ 64 Cu]NOTA-CD8a exhibited good prognostic and predictive value. We suggest that [ 64 Cu]NOTA-CD8a PET imaging can be used as an early biomarker of response to therapy in preclinical models.

Positron emission tomography imaging with 89Zr-labeled anti-CD8 cys-diabody reveals CD8+ cell infiltration during oncolytic virus therapy in a glioma murine model

“…The patterns of activity retained in target brain regions of mice in the present studies are consistent with the hypothesis that M002 oHSV treatment mediated the specific retention of [ 89 Zr]-malDFO-169 cDb in the treated glioma tumors. The very low uptake of [ 89 Zr]-malDFO-169 cDb in normal brain regions was consistent with results from alternative radiolabeled polypeptide imaging agents that do not cross the BBB and show below 0.2% ID/g in normal brains of mice 40 , 41 . As all groups of mice received i.c.…”

“…Our work adds to the growing body of studies that evaluate immunotherapy responses in vivo through immune cell-targeted PET imaging agents. Alternative CD8-targeted scaffolds (single-domain or F(ab)′2 antibody fragments) have shown correlations between PET signals and CD8 + cell infiltration as well as response to checkpoint immunotherapy in syngeneic murine non-CNS tumors 40 , 42 , 46 , 47 . A recent study showed that an anti-human CD8α-targeted minibody (IAB22M2C) labeled with 64 Cu specifically localized in orthotopic patient derived xenograft GBM tumors implanted in brain striatum of humanized mice; the PET signal in the tumor directly correlated with the number of human CD8 + cells that had trafficked into the tumors after systemic injection of human peripheral blood monocytes into the humanized mice 41 .…”

The Future of Cancer Diagnosis, Treatment and Surveillance: A Systemic Review on Immunotherapy and Immuno-PET Radiotracers

“…As expected, the minibody has a fast blood clearance due to its small size, which allows for early image acquisition. A [ 64 Cu]labeled antibody was proposed for the in vivo quantification of CD8+ T cells as a prognostic biomarker in an immunocompetent mouse model of colorectal cancer [ 105 ] and breast [ 106 ] cancer. Since [ 64 Cu] has a shorter half-life (12.7 h) compared to [ 89 Zr] (78.4 h), the use of [ 64 Cu] reduces patient radiation exposure, thus improving the “acquisition window” compared to shorter-lived PET isotope such as [ 18 F] and [ 68 Ga].…”

Interfacing Biomaterials with Synthetic T Cell Immunity

“…To selectively target CD8+ T cells that mediate anti‐tumor cytotoxicity, researchers designed CD8‐targeted PET probes based on antibody fragments, such as minibodies, [ 122,148 ] diabodies, [ 50,52 ] single domain antibody (VHH), [ 51 ] and single chain variable fragments (scFv). [ 53 ] For instance, Rashidian et al. developed 89 Zr‐labeled anti‐CD8 VHH probes and showed that homogeneous CD8+ T cell infiltration throughout the tumor is associated with strong responses to immunotherapy across three tumor models.…”