Crohn’s disease (CD) is a chronic immune-mediated inflammatory bowel disease (IBD), characterised by transmural inflammation of the gastrointestinal (GI) tract. It is typically diagnosed in early adulthood and can cause significant morbidity, with symptoms of abdominal pain, change in bowel habit and bleeding from the GI tract, and may be complicated by intestinal strictures or fistulae. There remains an unmet need to develop novel therapies for CD, as current drug treatments frequently fail to maintain long-term remission and may be complicated by significant side effects. Currently available CD therapies, including corticosteroids, Janus kinase (JAK) inhibitors, anti-Tumor necrosis factor (anti-TNF), anti-integrin and anti-Interleukin-12/23R (anti-IL-12/23R) therapy, seek to reduce immune activation in the gut by targeting effector immune mechanisms.

The maintenance or loss of intestinal homeostasis hinges on the balance between inflammatory effector T cells (Teff), which have been implicated in auto-immunity and transplant rejection, and a population of immunoregulatory T cells (Treg).1–3

Tregs are a unique subset of CD4+ T cells with powerful immunosuppressive action. They are defined by expression of the master transcriptional regulator FOXP3 and high constitutive expression of IL-2RA (CD25), the high-affinity receptor for IL-2.4–6 Tregs are capable of exerting tolerising immune responses through several mechanisms, including direct cell-cell contact and secreted products. The expression of inhibitory cytokines by Tregs, including IL-10, Transforming growth factor beta (TGFβ) and IL-35, has been implicated in regulatory function.7 IL-10 can suppress the ability of antigen-presenting cells (APC) to stimulate T cells by inhibiting APC maturation and expression of costimulatory molecules, and by suppression of cytokine production.8 IL-2 is required for Teff activity. Tregs can decrease interstitial IL-2 and inhibit IL-2 synthesis by Teffs.9 10 Tregs also express the inhibitory molecule CTLA-4, by which they can exert control over effector cell function.11

Lamina propria (LP) Tregs are increased in the mucosa and decreased in the blood of patients with active CD, compared with healthy controls.12–14 LP Tregs taken from the inflamed CD mucosa suppress proliferation of conventional CD4+CD25lo/int Teffs obtained from blood but not from the LP, suggesting that mucosal Teff in active CD may be resistant to Treg-mediated suppression.15 However, our group and others have shown that in vitro-expanded Tregs generated from blood in the presence of rapamycin are more potently suppressive than freshly isolated Tregs.16 17 Thus, in vitro-expanded Tregs from blood may overcome mucosal Teff resistance to suppression. Consequently, parenteral therapy with autologous ex vivo-expanded Tregs generated from Crohn’s blood may be an attractive approach to address defects in, or resistance to, mucosal Treg function in CD. We, and others, have shown that Tregs can be infused into animals with IBD, resulting in prevention or reversal of colitis18–22 Further, expanded human Tregs promote skin transplant tolerance, prevent transplant arteriosclerosis, and graft versus host disease (GvHD), in immunodeficient mice reconstituted with human peripheral blood mononuclear cells.23–25 It has also been shown that Tregs expanded in vitro from umbilical cord blood (UCB) are safe and efficacious at preventing GvHD following UCB transplantation.26 Other recent phase I studies demonstrate that adoptively transferred Tregs are safe in the context of haematopoietic stem cell transplantation, GvHD, and type 1 diabetes, while providing an early signal of efficacy.1 27 28

We and others have optimised the conditions for in vitro expansion of Tregs from peripheral blood (PB).6 29 30 Human Tregs isolated from PB or inflamed CD mucosa contain a subpopulation of IL-17+ effector T cells.31 In addition, human Tregs can be induced to express IL-17 or IFNγ in vitro.32 IL-17 is a proinflammatory cytokine implicated in the development of inflammatory disorders including CD.33 Adoptive transfer of expanded cells with the potential to express effector cytokines and contribute to CD pathogenesis could be deleterious. Our previous work has identified that the starting population for Treg expansion from the PB has a critical effect on the phenotype of the expanded cell population.30 34 Tregs from a highly pure FACS-sorted ‘naïve’ CD4+CD25hiCD127loCD45RA+ precursor population demonstrated enhanced suppressive ability and reduced Th17 plasticity in vitro compared with a FACS-sorted CD4+CD25hiCD127loCD45RA- or MACS-enriched CD8-CD25+ population.

The inclusion of rapamycin in culture during the Treg expansion process also prevents the expansion of Th17 cells, even when expanded Tregs are subsequently exposed to a Th17-favourable environment.29–31 35 Rapamycin-expanded Tregs maintain their in vitro regulatory phenotype after transfer into Nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice despite being exposed to an irradiation-induced proinflammatory environment. No additional rapamycin treatment either in vitro or in vivo is required to maintain their regulatory phenotype.31 Rapamycin added to cell culture appears to imprint a fixed CD4+CD25hi phenotype to cells expanded from a ‘naïve’ CD45RA+ population, as evidenced by retention of demethylation at the Forkhead box P3 (Foxp3) locus.

The expression of theheterodimeric integrin α4β7 confers Treg gut-homing ability, through interaction with its ligand mucosal vascular addressin cell adhesion molecule 1 .36 Inclusion of all-trans-retinoic acid (ATRA) in Treg expansion media induces expression of α4β7 integrin on the Treg surface. Tregs expanded in ATRA alone retain the ability to secrete IL-17, but Tregs cultured in media including both rapamycin and ATRA demonstrated no IL-17 production.29 The combination of rapamycin with ATRA provides an approach for large-scale expansion of functionally potent and phenotypically stable Tregs. We have shown that adoptive transfer of expanded Tregs treated with a highly-specific retinoic receptor α (RARα) receptor agonist leads to improved Treg gut trafficking into human intestinal xenografts in mice.30 34 The RARα agonist-treated cell product forms the basis of TR004.

In order to map the fate of infused cells, Tregs are ‘tagged’ through inclusion of deuterated [6,6-2 H2] glucose in the culture media during the expansion process.27 This also enables confirmation that infused Treg does not convert to Teff in vivo post infusion. Incorporation of deuterium into the deoxyribose moiety of newly synthesised DNA enables infused Treg and their progeny to be distinguished from pre-existing or endogenously synthesised Treg; their different genomic DNA isotopic profiles can be readily discriminated by mass spectrometry.37 Previous clinical studies in human participants with diabetes using a similar approach have demonstrated detectable transferred Tregs in peripheral blood up to 1 year post infusion and shown no detectable Treg to Teff conversion post infusion.27 We will use deuterium enrichment added ex vivo during the expansion phase to permit tracking of transferred Tregs to the gut and to determine longevity of the infused Tregs that comprise the cell product (TR004).

This clinical study will thus use autologous, ex vivo-expanded Tregs as a parenteral cell-based therapy to augment regulatory immune responses in the gut of patients with CD.

To explore the preliminary safety and tolerability of a single dose of TR004 in patients with moderate to severe CD who are refractory or intolerant to standard treatment. Further, this trial will inform the design of a subsequent larger trial.

Feasibility objectives: To pilot the operation of the clinical protocol and inform the design of a subsequent larger trial.

To assess the feasibility of manufacturing the investigational medicinal product (IMP) at the dose selected for this study.

To assess the feasibility of recruiting to time and target.

To assess the feasibility of retaining participants for the duration of the study and completion of study visits and assessments.

To understand the experience of the participants, trial team and data safety monitoring board (DSMB) members, exploring any barriers and challenges to trial performance, recruitment and retention.

To measure the in vivo lifespan of infused cells, evaluate localisation to gut and ensure no Treg to Teff conversion.