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

溶瘤病毒(OV)最初是作为细胞毒剂开发的，现在已知其抗肿瘤特性主要是由免疫介导的。然而，除了诱导抗肿瘤免疫反应外，OV还刺激抗病毒反应，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转的，因此目前的看法是，患者中预先存在的抗病毒NAB的存在，或者他们在病毒治疗期间的发展，是全身OV传递的障碍，因为它们将阻止病毒访问肿瘤。这意味着重复的全身性OV给药是无效的。全身给药是首选的临床选择，既适用范围广，又适合于针对内脏或广泛的转移性疾病。因此，如果NAB是一个完全的治疗障碍，OV将被限制为直接肿瘤内注射，这在技术上具有挑战性，只适用于容易接触到的肿瘤，或者被限制为一次治疗方法。然而，临床试验表明，尽管存在NAB，溶瘤呼肠孤病毒仍可进入患者的肿瘤。我们还表明，携带预形成的呼肠孤病毒-NAB复合体(ReoNAb)的人单核细胞可以将功能性复制的呼肠孤病毒运送到肿瘤细胞，导致肿瘤细胞感染和溶解。我们还发现，负载了reoNAb的单核细胞可以刺激自然杀伤细胞介导的细胞毒作用，但OV-NAb在诱导先天和获得性抗肿瘤免疫反应中的作用还缺乏详细的分析。目前有几种OV正在临床开发中，但如果要在患者身上充分利用它们的潜力，就需要详细了解预先存在的抗病毒NAB在诱导抗肿瘤免疫反应方面的效果。本项目将比较OV-NAB和未中和OV对血液中不同免疫细胞亚群的激活表型和功能的影响。OV-NAB复合体将由OV和接受OV治疗的患者的血清生成。OV-NAB对免疫细胞亚群激活表型的影响将与未中和的OV进行FACS表面标志物表达分析。OV-NAB处理的免疫细胞与OV处理的免疫细胞的细胞因子谱将通过ELISA法或Luminex多重试验进行检测。包括51Cr释放细胞毒性试验、混合淋巴细胞反应和我们建立的CTL启动试验在内的功能分析也将用于评估先天和获得性抗肿瘤免疫反应。动物实验可能会被用来证实体外的发现。

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.