The impact of virus-specific neutralizing antibodies on oncolytic virus-mediated anti-tumour immune responses

最初作为细胞毒性剂开发，溶瘤病毒（OV）的抗肿瘤特性现在已知主要是免疫介导的。然而，除了诱导抗肿瘤免疫应答外，OV还刺激抗病毒应答，包括产生OV特异性中和抗体（NAb）。病毒的抗体中和被认为是不可逆的，因此目前认为患者中预先存在的抗病毒NAb的存在或其在病毒治疗期间的发展是全身OV递送的障碍，因为它们将阻止病毒进入肿瘤。这意味着重复全身OV给药将是无效的。全身递送是优选的临床选择，其既广泛适用又适合靶向内脏或广泛转移性疾病。因此，如果NAb是完全的治疗屏障，则OV递送将限于直接肿瘤内注射，这在技术上具有挑战性并且仅适用于容易接近的肿瘤，或者可替代地限于受限的“一次性治愈”方法。然而，临床试验表明，溶瘤呼肠孤病毒可以进入患者的肿瘤，尽管存在NAb。我们还表明，载有预形成的呼肠孤病毒-NAb复合物（reoNAb）的人单核细胞（其中呼肠孤病毒被完全中和）可以将功能性复制型呼肠孤病毒递送至肿瘤细胞，导致肿瘤细胞感染和裂解。我们还发现，单核细胞负载reoNAb刺激自然杀伤细胞介导的细胞毒性，但OV-NAb对诱导先天性和适应性抗肿瘤免疫应答的影响的详细分析是缺乏的。目前有几种OV正在临床开发中，但如果要在患者中充分发挥其潜力，则需要详细了解预先存在的抗病毒NAb对诱导抗肿瘤免疫应答的影响。本项目将比较OV-NAb与非中和OV对血液中不同免疫细胞亚群的活化表型和功能的影响。将使用OV和从接受OV治疗的患者中采集的血清生成OV-NAb复合物。OV-NAb对免疫细胞亚群的活化表型的作用将使用表面标志物表达的FACS分析与未中和的OV的作用进行比较。将通过ELISA或Luminex多重测定来检查OV-NAb处理的免疫细胞与OV处理的免疫细胞的细胞因子谱。功能测定包括51 Cr释放细胞毒性测定、混合淋巴细胞反应和我们建立的CTL引发测定也将用于评估先天性和适应性抗肿瘤免疫应答。动物实验可用于确认体外结果。

Initially developed as cytotoxic agents, the anti-tumour properties of oncolytic viruses (OV) are now known to be largely immune-mediated. However, as well as inducing anti-tumour immune responses, OV also stimulate anti-viral responses, including the production of OV-specific neutralizing antibodies (NAb). Antibody-neutralization of viruses is thought to be irreversible, hence the current perception that the presence of either pre-existing anti-viral NAb in patients, or their development during viral therapy, are a barrier to systemic OV delivery, because they will prevent the virus accessing the tumour. This means that repeat systemic OV dosing would be ineffective. Systemic delivery is the preferred clinical option, being both broadly applicable and suitable for targeting visceral or widespread metastatic disease. Thus, if NAb are a complete therapeutic barrier, OV delivery will be limited to either direct intra-tumoural injection, which is technically challenging and only suitable for readily accessible tumours, or alternatively to a restricted 'one shot cure' approach. However, clinical trials have shown that oncolytic reovirus can access tumours in patients, despite the presence of NAb. We have also shown that human monocytes loaded with pre-formed reovirus-NAb complexes (reoNAb), in which the reovirus is fully neutralized, can deliver functional replicative reovirus to tumour cells resulting in tumour cell infection and lysis. We have also found that monocytes loaded with reoNAb stimulate natural killer cell-mediated cytotoxicity, but a detailed analysis of the effects of OV-NAb on the induction of innate and adaptive anti-tumour immune responses is lacking. There are several OV currently under clinical development but if they are to be used to their full potential in patients, a detailed understanding of the effect that pre-existing anti-viral NAb have on the induction of anti-tumour immune responses is required. This project will compare the effect of OV-NAb vs non-neutralized OV on the activation phenotype and function of different immune cell subsets in the blood. OV-NAb complexes will be generated using OV and serum collected from patients treated with OV. The effect of OV-NAb on the activation phenotype of immune cell subsets will be compared with that of non-neutralized OV using FACS analysis of surface marker expression. The cytokine profiles of OV-NAb-treated vs OV-treated immune cells will be examined by ELISA or Luminex multiplex assay. Functional assays including 51Cr-release cytotoxicity assay, mixed lymphocyte reactions and our established CTL priming assay will also be used to assess innate and adaptive anti-tumour immune responses. Animal experiments may be used to confirm the in vitro findings.