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）是一种慢性衰弱性疾病，需要终身治疗干预和患者监测。目前，最广泛施用的免疫抑制剂包括靶向单一炎症因子的抗体，或介导免疫细胞运输至炎症部位的细胞粘附分子。尽管有一系列针对不同免疫组分的现有治疗剂，但很大一部分患者患有治疗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疗法。在我们最近发表在Cell上的工作中，我们使用了最先进的单细胞RNA测序和质谱技术来检测炎症期间的致病性变化。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。我们使用来自健康供体和患有活动性疾病的IBD患者的组织样本来突出IBD病理学的主要驱动因素。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，CCL 19和CCL 21，它们在炎症组织中显示出高水平的异常表达，并且具有强的化学引诱物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稳态而使炎症持续存在。因此，在这个项目中，我们的目标是设计特定的靶向治疗，抑制这些主要趋化因子的作用。考虑到这些靶标之间的功能冗余，我们的目标是通过工程化中和双特异性抗体来破坏这些分子的信号传导活性，所述中和双特异性抗体将同时结合并拮抗CCL 19和CCL 21功能。我们将在抗体生产的第一阶段选择具有高亲和力的靶向CCL 19和CCL 21的抗体。然后，我们将通过证明它们抑制免疫细胞中特征良好的细胞信号传导途径的能力来验证所选抗体的功能。这些抗体的功能性将通过趋化性测定进一步证实，其中细胞通常可以朝向CCL 19/CCL 21趋化因子梯度迁移，但在中和抗体存在下，这种活性将被抑制。有效治疗性双特异性抗体的鉴定将使得能够随后测试其在结肠炎模型中防止免疫细胞迁移至粘膜炎症区域的能力。该项目产生的治疗性抗体将提供一种新型的治疗方法，其作用机制与现有疗法不同，其中大多数疗法侧重于具有明确生物学目的的单一分子靶标。通过使用我们的策略切断免疫细胞募集，我们的目标是同时破坏由不同亚型的免疫细胞介导的多种促炎活性。这包括T细胞，这是慢性肠道炎症的关键驱动因素，以及成熟的树突状细胞，它们携带微生物衍生的蛋白片段来激活T细胞。因此，CCL 19/CCL 21阻断将直接阻碍许多负责维持IBD炎症的免疫刺激途径，从而降低疾病严重程度并可能恢复免疫稳态所需的平衡。这种治疗策略将潜在地有益于对常规治疗无效的患者、对当前治疗产生耐药性的患者，并有助于预防术后患者的疾病复发。靶向这些趋化因子还可以为IBD患者提供更个性化治疗的急需机会，例如表现出高水平异位CCL 19/CCL 21产生的患者，并且还可以用于开发联合治疗以实现更好的临床结果。

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.