Disruption of a T cell recruitment and retention gradient for the treatment of chronic intestinal inflammation in inflammatory bowel disease

炎症性肠病(IBD)是一种慢性衰弱疾病，需要终身治疗干预和患者监测。目前，应用最广泛的免疫治疗药物包括针对单一炎症因子的抗体，或介导免疫细胞向炎症部位运输的细胞黏附分子。尽管现有的一系列治疗药物针对不同的免疫成分，但相当大比例的患者患有难治性疾病或在一年内对治疗失去反应，因此需要新的治疗选择或联合治疗。在我们最近发表在《细胞》杂志上的工作中，我们使用了最先进的单细胞RNA测序和质量细胞术技术来检测炎症过程中的致病变化。这些技术使我们能够仔细检查细胞基因和蛋白质表达谱的变化。我们使用来自健康捐赠者和患有活动期疾病的IBD患者的组织样本来强调IBD病理的主要驱动因素。从这些研究中获得的重要见解使我们能够选择两个关键的功能相关分子，CCL19和CCL21，它们在炎症组织中显示出高水平的异常表达，并具有强大的趋化特性来招募免疫细胞。因此，这些趋化因子分子的分泌通过阻止免疫动态平衡来维持炎症。因此，在这个项目中，我们的目标是设计特定的靶向治疗药物，以抑制这些主要趋化因子的作用。鉴于这些靶点之间的功能冗余，我们的目标是通过设计一种中和双特异性抗体来干扰这些分子的信号活性，该抗体将同时结合并拮抗CCL19和CCL21功能。我们将在抗体生产的第一阶段筛选出针对CCL19和CCL21的高亲和力抗体。然后，我们将通过展示所选抗体抑制免疫细胞中特征良好的细胞信号通路的能力来验证它们的功能。这些抗体的功能将通过趋化试验得到进一步证实，在趋化试验中，细胞通常可以向CCL19/CCL21趋化因子梯度迁移，但在存在中和抗体时，这种活性将被抑制。鉴定一种有效的治疗性双特异性抗体将使后续测试其防止免疫细胞迁移到结肠炎模型的粘膜炎症区域的能力成为可能。该项目产生的治疗性抗体将提供一种新型的治疗方法，其作用机制与现有的治疗方法不同，现有的治疗方法大多专注于具有明确生物目的的单分子靶点。通过使用我们的策略切断免疫细胞的招募，我们的目标是同时干扰由不同免疫细胞亚型介导的多种促炎活动。这包括T细胞和成熟的树突状细胞，T细胞是慢性肠道炎症的关键驱动因素，成熟的树突状细胞携带微生物衍生的蛋白片段来激活T细胞。因此，阻断CCL19/CCL21将直接阻碍许多负责维持IBD炎症的免疫刺激途径，从而减轻疾病的严重性，并有可能恢复免疫平衡所需的平衡。这一治疗策略可能会使对传统治疗无效的患者、对当前治疗产生抵抗力的患者受益，并有助于防止术后患者的疾病复发。针对这些趋化因子也可能为IBD患者提供急需的个性化治疗机会，例如那些表现出高水平异位CCL19/CCL21产生的患者，并可能有助于开发联合治疗以获得更好的临床结果。

Inflammatory bowel disease (IBD) is a chronic debilitating disorder requiring lifelong therapeutic intervention and patient monitoring. Currently, the most widely administered immunotherapeutic agents include antibodies targeting single inflammatory factors, or cell adhesion molecules that mediate immune cell trafficking to sites of inflammation. Despite a range of existing therapeutic agents addressing different immune components, a significant proportion of patients suffer from treatment-refractory disease or lose response to treatments within a year, thereby necessitating new treatment options or combination therapies. In our work recently published in Cell, we used state of the art single cell RNA sequencing and mass cytometry technologies to detect pathogenic changes during inflammation. These technologies allowed us to closely examine alterations in cellular gene and protein expression profiles. We used tissue samples from healthy donors and IBD patients with active disease to highlight major drivers of IBD pathology. Important insights derived from these studies have allowed us to select two key functionally-related molecules, CCL19 and CCL21, which display high levels of aberrant expression in inflamed tissues, and have strong chemoattractant properties to recruit immune cells. The secretion of these chemokine molecules therefore perpetuates inflammation by preventing immune homeostasis. In this project we therefore aim to design specific targeted therapeutics that inhibit the actions of these major chemokines. Given the functional redundancy between these targets, we aim to disrupt the signalling activities of these molecules by engineering a neutralising bispecific antibody, which will bind to and antagonise CCL19 and CCL21 functions simultaneously. We will select for antibodies targeting CCL19 and CCL21 with high affinity at the first stage of antibody production. We will then validate the functions of selected antibodies by demonstrating their ability to inhibit a well-characterised cellular signalling pathway in immune cells. The functionality of these antibodies will be further confirmed by chemotaxis assays, in which cells can normally migrate towards a CCL19/CCL21 chemokine gradient, but such an activity will be inhibited in the presence of neutralising antibodies. Identification of an effective therapeutic bispecific antibody will enable subsequent testing of its ability to prevent immune cell migration to areas of mucosal inflammation in colitis models. The therapeutic antibody generated by this project will provide a new type of treatment that differs in its mechanism of action from existing therapies, most of which focus on single molecular targets with a defined biological purpose. By cutting off immune cell recruitment using our strategy, we aim to simultaneously disrupt multiple proinflammatory activities mediated by different subtypes of immune cells. This includes T cells, which are key drivers of chronic intestinal inflammation, and mature dendritic cells, which carry microbial-derived protein fragments to activate T cells. CCL19/CCL21 blockade will therefore directly impede many immune-stimulating pathways responsible for sustaining inflammation in IBD, thereby reducing disease severity and potentially restoring the balance required for immune homeostasis. This therapeutic strategy will potentially benefit patients who fail to respond to conventional therapies, patients who develop resistance to current treatments, and help to prevent disease recurrence in post-operative patients. Targeting these chemokines may also provide much needed opportunities for more personalised therapies for IBD patients, such as those who exhibit high levels of ectopic CCL19/CCL21 production, and may also be useful for the development of combination treatments in order to achieve a better clinical outcome.