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

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.

Inflammatory bowel disease (IBD) is a highly heterogeneous disease necessitating a range of treatment options, especially for non-responders to existing therapies. The disease complexity also demands new treatments to improve limited response rates associated with the blockade of single cytokine or cell adhesion targets.

In our work recently published in Cell, we used patient tissue biopsies to identify a pathogenic stromal population significantly expanded in active disease, secreting two major T cell chemoattractants, CCL19 and CCL21, which share functional redundancy in their ability to mediate the trafficking of CCR7+ T cells and mature dendritic cells (DCs). Our data indicate aberrantly activated stromal cells create a high lymphocyte-attraction and retention gradient, thus contributing to the chronicity of inflammation. Here we seek to disrupt the T cell recruitment/stimulation circuitry mediated by the CCL19/21-CCR7 axis.

Blockade of CCL19/21 will also have an effect on antigen presentation in secondary lymphoid organs (SLOs), where mature DCs carrying microbiota-derived antigens will migrate towards the chemokine gradients. These gradients also balance the attraction versus egress of CCR7+ naïve and memory T cells to and from SLOs. As >60% of microbiota-reactive T cells express high levels of CCR7, transient disruption of these ligand-receptor interactions will impair T cell priming in SLOs during in both acute and chronic phases of the disease, in addition to cutting off T cell homing to inflamed tissues with high ectopic CCL19/21 production.

We will select CCL19/21 neutralising antibodies with cross-species reactivity and assess their functionality by T cell signalling and chemotaxis assays. Following functional validation, we will use an established chronic inflammatory murine colitis model to assess the effects of CCL19/21 blockade. Pre-clinical pharmacodynamics and efficacy data will then be used to inform subsequent clinical dosing regimen.

2.5 million-3 million people in Europe are affected by Inflammatory bowel disease (IBD), with the highest rates reported in Scandinavia and the UK. The incidence of both ulcerative colitis and Crohn's disease is stable or increasing globally, incurring a direct healthcare cost of 4.6-5.6 billion Euros in Europe, and £1.5 billion to the NHS in the UK per year.

IBD is a highly heterogeneous disease and necessitates a range of treatment options, especially for non-responders to conventional therapies. Anti-TNF therapies have dominated treatment of IBD for close to two decades, but 20-30% of patients discontinue treatment due to primary non-response, and a further 30-40% lose response within the first year of treatment, presenting a large unmet need in cost-effective therapies. Overall, currently available immunotherapies demonstrate limited efficacy - 40-55% of patients demonstrate no response, and 65-80% do not achieve full remission.

This healthcare and economic burden demands significant new insights into reasons behind the limitations of current treatments, as well as strategies for overcoming loss of response to existing therapies.

We have used state of the art single cell RNA sequencing and mass cytometry to detect fundamental changes in healthy and inflamed colon tissues. We mapped disease-associated changes on cellular and population levels, enabling our visualisation of how the inflammatory tissue environment evolves in relation to disease activity and severity. Such insights have allowed us to highlight the most biologically-relevant changes for disease targeting.

We found multiple cell adhesion molecules involved in T cell recruitment upregulated in stromal cells in colitis. This functional redundancy likely accounts for the limited response rates of the new lines of anti-adhesion therapies. Similarly, the inhibition of single inflammatory mediators among a cocktail of cytokines and other factors often proves insufficient for achieving disease remission.

Importantly, we identified a specific pathogenic cellular population involved in recruiting immune cells that propagate chronic inflammation. Two of these key molecules are the chemokines CCL19 and CCL21, both of which are indispensable for T cell migration as well as T cell priming by professional antigen presenting cells. Our goal is therefore to design a specific targeted therapeutic that disrupts these inflammatory drivers. Using this strategy, we aim to dampen the production of a multitude of T cell-derived proinflammatory factors.

We will assess the most optimal route to enable preclinical testing following the generation of bispecific antibodies against our chemokine targets. We will then translate preclinical findings into clinical development for patients. Appropriate clinical indications for CCL19/CCL21 blockade may include:

- 5 ASA or thiopurine refractory ulcerative colitis patients to avoid side effects associated with anti-TNF treatment.
- Patients who have treatment-refractory active disease (such as anti-TNF primary and secondary non-responders).
- Patients requiring maintenance of remission in whom anti-TNF therapy is contra-indicated.
- Patients who progressively lose response to anti-TNF and/or anti-adhesion therapies or other current therapies.
- Prevention of Crohn's disease recurrence in postoperative patients following small bowel resection.
- For prevention of stricturing or fistulizing Crohn's.

Development of new therapies for patients who are non-responsive to currently available treatments is imperative. This project will allow us to explore the therapeutic potential of transiently disrupting T cell recruitment via CCL19/CCL21-CCR7 interactions. This strategy will provide a means to circumvent limitations of current approaches targeting single mucosal-homing cell adhesion molecules and proinflammatory factors, therefore potentially leading to an improved clinical outcome and higher response rates.