ABSTRACT
BACKGROUND: CD4+ T cells are central inflammatory mediators in the pathogenesis of autoimmune rheumatoid arthritis (RA), as they are one of the dominating cell types in synovial inflammation. Molecular imaging of CD4+ T cells has potential role for early detection and monitoring of RA. Here, we developed a new radiotracer for in vivo immunoPET imaging of murine CD4+ T cells and tested it in the collagen-induced arthritis (CIA) mouse model of human RA. RESULTS: The tracer, [64Cu]Cu-NOTA-CD4-F(ab)'2 ([64Cu]Cu-NOTA-CD4), was generated from F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to the 2-S-(isothiocyanatbenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) chelator and radiolabeled with copper-64. Accumulation of the tracer and isotype control was evaluated in the CIA model and mice receiving whole-body irradiation (WBI) (5 Gy). The potential of [64Cu]Cu-NOTA-CD4 for response assessment was evaluated in CIA induced mice treated with dexamethasone (DXM). Imaging data were compared with flow cytometry and immunohistochemistry (IHC) of inflammatory cells including CD4+ T cells. [64Cu]Cu-NOTA-CD4 showed increased accumulation in T cell-rich tissues compared with isotype control (p < 0.0001). In addition, reduced accumulation of [64Cu]Cu-NOTA-CD4 was observed in T cell-depleted tissue (p < 0.0001). Flow cytometry and IHC confirmed the increased infiltration of CD4+ T cells in CIA mice. CONCLUSIONS: We developed and evaluated a new radiotracer, [64Cu]Cu-NOTA-CD4, for immunoPET imaging of murine CD4+ T cells. [64Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that our novel CD4 PET tracer can be used for noninvasive visualization of murine CD4+ T cells.
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: [64Cu]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 [64Cu]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 [64Cu]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, [64Cu]NOTA-CD8a PET imaging distinguished responders and non-responders prior to treatment-induced changes in tumor volume among mice. CONCLUSION: In the present study, we demonstrated that [64Cu]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, [64Cu]NOTA-CD8a exhibited good prognostic and predictive value. We suggest that [64Cu]NOTA-CD8a PET imaging can be used as an early biomarker of response to therapy in preclinical models.
