CD4+ and CD8a+ PET imaging predicts response to novel PD-1 checkpoint inhibitor: studies of Sym021 in syngeneic mouse cancer models
CD4+ and CD8a+ PET imaging predicts response to novel PD-1 checkpoint inhibitor: studies of Sym021 in syngeneic mouse cancer models
复制标题
DOI：
10.7150/thno.37513
复制
发表时间：
2019-01-01
期刊：
影响因子：
12.4
通讯作者：
Kjaer, Andreas
中科院分区：
文献类型：
--
作者：
Kristensen, Lotte K.;Frohlich, Camilla;Kjaer, Andreas
预测免疫治疗的结果对于有效治疗至关重要。最近免疫疗法的临床成功正在日益改变癌症治疗的范式。因此，基于免疫的药物的开发正在加速，全球免疫肿瘤学管道中的药物数量在过去一年中增长了60-70%。然而，尽管在一些患者中具有显著的临床疗效，但只有少数患者达到持久的临床应答。治疗失败可归因于不吸引肿瘤浸润淋巴细胞（TIL）的免疫原性差的肿瘤。因此，我们开发了正电子发射断层扫描（PET）放射性示踪剂，用于临床前研究的同基因小鼠肿瘤模型中CD 4（+）和CD 8a（+）TIL的非侵入性检测。7种同系小鼠肿瘤模型使用流式细胞术和免疫组织化学（IHC）定量（B16 F10、P815、CT 26、MC 38、Renca、4 T1、Sa 1 N）的CD 4（+）和CD 8a（+）TIL，以及对Sym 021（与小鼠PD-1交叉反应的人源化PD-1抗体）的肿瘤生长应答。放射性示踪剂由与p-SCN-Bn-去铁胺（SCN-Bn-DFO）螯合剂偶联并用锆-89放射性标记的大鼠抗小鼠CD 4和CD 8a抗体的F（ab）2片段产生（Zr-89-DFO-CD 4/Zr-89-DFO-CD 8a）。对示踪剂进行优化，用于CT 26荷瘤小鼠体内PET/CT成像，并通过耗竭研究和同种型对照成像评价特异性。在用Sym 021进行免疫治疗前，对一组同基因小鼠模型进行Zr-89-DFO-CD 4和Zr-89-DFO-CD 8a PET/CT成像。结果：根据流式细胞术和免疫组化检测的TIL数量，将同基因小鼠模型分为“热”和“冷”两类。成功地产生了Zr-89-DFO-⑶ 4和Zr-89-DFO-⑶ 8a，放射化学纯度>99%，免疫反应性> 85%。最佳成像时间点是注射类似于1 MBq示踪剂与30 μ g非标记共同剂量后24小时。在CD 8a（+）耗竭小鼠中观察到Zr-89-DFO-CD 8a的肿瘤和脾脏摄取减少，且摄取与同种型对照（Zr-89-DFO-IgG 2b）相当，证实了特异性。同系肿瘤模型中的PET成像揭示了不同肿瘤类型和受试者之间Zr-89-DFO-CD 4和Zr-89-DFO-CD 8a的不同最大肿瘤-心脏比，其与相对于治疗开始第10天对Sym 021的个体响应相关（分别为p=0.0002和p=0.0354）。最大（8）9 Zr-DFO-CD 4肿瘤与心脏比率可用于根据Sym 021治疗反应对小鼠进行分层，并且在Zr-89-DFO-CD 4比率>9的小鼠中总存活率得到改善（p=0.0018）。这些放射性示踪剂可用于临床前同基因小鼠肿瘤模型的表型，并预测对免疫检查点抑制剂的反应。我们预见了这种非侵入性体内生物标志物的发展，用于预测和评估免疫抑制剂如Sym 021的临床疗效。
Predicting the outcome of immunotherapy is essential for efficient treatment. The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment. Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year. However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response. Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs). Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4(+) and CD8a(+) TILs in syngeneic mouse tumor models for preclinical studies.Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4(+) and CD8a(+) TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 (Zr-89-DFO-CD4/Zr-89-DFO-CD8a). Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. Zr-89-DFO-CD4 and Zr-89-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021.Results: Syngeneic tumor models were characterized as "hot" or "cold" according to number of TILs determined by flow cytometry and IHC. Zr-89-DFO-CD4 and Zr-89-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%. The optimal imaging time-point was 24 hours post-injection of similar to 1 MBq tracer with 30 mu g non-labeled co-dose. Reduced tumor and spleen uptake of Zr-89-DFO-CD8a was observed in CD8a(+) depleted mice and the uptake was comparable with that of isotype control (Zr-89-DFO-IgG2b) confirming specificity. PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of Zr-89-DFO-CD4 and Zr-89-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively). The maximum (8)9Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a Zr-89-DFO-CD4 ratio >9 (p=0.0018).Conclusion: We developed Zr-89-DFO-CD4 and Zr-89-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4(+) and CD8a(+) status. These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor. We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.