Minerva Imaging, Copenhagen, Denmark.
Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Department of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark.
Mol Imaging Biol. 2020 Aug;22(4):1021-1030. doi: 10.1007/s11307-020-01481-0.
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.
[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 [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).
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 [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, [Cu]NOTA-CD8a PET imaging distinguished responders and non-responders prior to treatment-induced changes in tumor volume among mice.
In the present study, we demonstrated that [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, [Cu]NOTA-CD8a exhibited good prognostic and predictive value. We suggest that [Cu]NOTA-CD8a PET imaging can be used as an early biomarker of response to therapy in preclinical models.
目前用于接受免疫疗法的癌症患者的反应评估系统存在局限性。这是由于相关的炎症反应可能使实体肿瘤的常规形态学反应评估标准和实体瘤代谢正电子发射断层扫描（PET）反应标准复杂化。最近，使用放射性标记的抗体和片段的新型 PET 成像技术已成为定量跟踪免疫细胞动力学的一种特别敏感和特异的方式。因此，我们试图研究 Cu-64 标记的 F(ab')2 片段在体内检测 CD8a T 细胞的效用，作为免疫功能正常的结直肠癌小鼠模型中对免疫疗法反应的预后成像生物标志物。
通过酶促消化大鼠抗小鼠 CD8a 抗体（克隆 YTS169.4）产生 [Cu]NOTA-CD8a，纯化得到分离的 CD8a-F(ab')2 片段，并随机与 2-S-（异硫氰酸苯甲基）-1,4,7-三氮杂环壬烷-1,4,7-三乙酸（p-SCN-Bn-NOTA）螯合剂结合。用 Cu-64 标记 NOTA-CD8a 并注入 CT26 荷瘤小鼠进行纵向评估。为了研究 [Cu]NOTA-CD8a PET 成像在评估治疗反应中的价值，将 CT26 荷瘤小鼠接受外部放射治疗（XRT）联合抗 CTLA-4 治疗。通过流式细胞术和免疫组织化学（IHC）支持成像数据。
XRT 和抗 CTLA-4 的联合治疗有效抑制了肿瘤生长，直到治疗开始后第 22 天（p=0.0025），并与对照组相比增加了小鼠的总生存期（p=0.0017）。在治疗开始后第 8 天，XRT+抗 CTLA-4 治疗的小鼠中 [Cu]NOTA-CD8a 肿瘤与心脏的比值增加（p=0.0246）。流式细胞术和 IHC 证实 XRT+抗 CTLA-4 治疗的小鼠肿瘤内浸润的 CD8a 细胞增加。此外，[Cu]NOTA-CD8a PET 成像在治疗诱导的肿瘤体积变化之前区分了反应者和无反应者。
在本研究中，我们证明了 [Cu]NOTA-CD8a 能够检测到在常见的临床前联合治疗方案后，在小鼠 CT26 结肠肿瘤中 CD8a 浸润的治疗诱导变化。总体而言，[Cu]NOTA-CD8a 表现出良好的预后和预测价值。我们建议 [Cu]NOTA-CD8a PET 成像可作为临床前模型中治疗反应的早期生物标志物。
