Project ID iCASE2023_08


Co Supervisor 1A Faculty of Life Sciences & Medicine, Department of Inflammation BiologyWebsite

Co Supervisor 1B Faculty of Life Sciences & Medicine, Immunology & Microbial SciencesWebsite

Partner Quell Therapeutics Limited

Optimization Of The Potency And Specificity Of Engineered Regulatory T Cells To Treat Inflammatory And Fibrotic Liver Diseases

Partner: Quell Therapeutics Limited 


The prevalence of chronic liver disease, the 5th most common cause of death in the UK is rising sharply mainly due to the increasing load of viral hepatitis, alcohol- related liver disease and non-alcoholic fatty liver. This increasing burden of liver disease in the UK is in stark contrast to the vast improvements made in health and life expectancy for other chronic disorders and most cancers. There is a pressing need therefore to find novel modalities of treatment to prevent or halt liver disease progression, which unabated leads to complications such as portal hypertension, cancer and ultimately death. Although the molecular and cellular events underlying the progression of liver disease remain poorly understood, dysregulated local and systemic inflammatory responses are involved in amplifying hepatic injury and fibrogenesis, which leads to clinical decompensation and death. In this regard, patients with advanced forms of liver disease have been shown to exhibit multiple innate and adaptive immune deficits. As previously reported in autoimmune hepatitis and primary sclerosing cholangitis, this includes reductions in the number and function of CD4+Foxp3+ regulatory T cells (Tregs), which are a lymphocyte subset that is essential for the maintenance of immunological tolerance and that has the capacity to migrate to sites of inflammation and exert powerful anti-inflammatory, tissue repair and regenerative properties. The prospect of ameliorating immunopathology in chronic inflammatory diseases and re-establishing tolerance in autoimmunity and transplantation, has prompted a growing interest in the development of Treg-based cell therapies. This is despite critical gaps in knowledge regarding how the inflammatory microenvironment controls Treg trafficking, activation, longevity, stability, and suppressive function in vivo. King’s College London has pioneered the use of ex vivo expanded Treg adoptive transfer. Our clinical trials using non-engineered autologous polyclonal Tregs in kidney (ONE and TWO Studies, Gamechanger) and liver transplantation (THRIL) have demonstrated the safety of ex vivo expanded non-engineered polyclonal Treg transfer and provided encouraging evidence for their biological efficacy. However, the limited control over Treg trafficking, potency and longevity means that this strategy is unlikely to fulfil the promise of Treg immunotherapy as a truly transformative cell therapy.

Chimeric antigen receptors (CARs) and gain-of-function genetic engineering provide unique opportunities to reprogram Tregs to address the limitations outlined above. We and others have generated HLA-A2-specific CAR human Tregs and shown their trafficking to organs expressing HLA-A2 with superior anti-inflammatory effects as compared to polyclonal Tregs. These results have generated considerable interest in the use of CAR-Tregs in humans, and a clinical trial sponsored by our spin-off company Quell Therapeutics employing anti-HLA-A2 CAR-Tregs in liver transplantation is currently underway (LIBERATE, NCT05234190).
Anti-HLA-A2 CAR-Tregs, however, can only benefit HLA-A2-negative transplant recipients who have received an HLA-A2-positive liver. There is a need therefore to generate organ- rather than allo-antigen specific CAR-Tregs to treat non-transplant liver patients. We propose now to create a human next generation modular CAR-Treg product specifically designed to treat inflammatory and fibrotic liver diseases. Our hypotheses are: i) rendering Tregs specific for antigens expressed on hepatocytes or activated hepatic stellate cells will enhance their recruitment and intra-hepatic persistence during inflammation and fibrosis, respectively; ii) Tregs can be engineered to release antifibrotic peptides (e.g., Relaxin and Amphiregullin), iii) the extent to which CAR-Tregs accumulate in the liver while maintaining a stable phenotype will determine their capacity to halt progressive liver damage.

The following work underpins our proposal: (i) we have developed a pipeline to screen functional CARs using reporter cell lines, we have identified several promising candidates which cross-react with human and mouse antigens and have developed a proprietary inducible NFAT promoter validated in Tregs that links transgene expression to CAR activation, (ii) we have access to mouse models (CCL4 and Balb/c.Mdr2-/-) that results in liver inflammation and fibrosis.

The PhD studentship outlined here has three aims:

Aim 1 (Year 1): To characterise in vitro and in vivo human Tregs expressing CARs specific for ICAM-1, FAP or ASGPR

Rationale: We have designed and screened several liver-specific CARs by fusing a single-domain antibody (sdAb) recognizing the H1 subunit of asialoglycoprotein receptor 1 (ASGPR) with a CD8 hinge and CD28 and CD3? intracellular domains. ASGPR1 is a transmembrane C-type lectin very abundantly expressed in hepatocytes and minimally present elsewhere and previously employed as a target for T cell therapies. In addition, we have designed CARs recognising intercellular adhesion molecule-1 (ICAM-1), and fibroblast activation protein (FAP), to provide Treg activation in response to inflammatory cues: ICAM-1 is highly expressed by hepatocytes and endothelial cells in the inflamed liver while FAP is expressed by activated hepatic stellate cells.

Aim 2 (year 2) : To engineer human Tregs with the ability to produce anti-fibrotic factors in response to CAR activation

Rationale: To confer enhanced antifibrotic effects to CAR-Tregs, we will engineer them to over-express genes encoding for mediators previously shown to have antifibrotic effects (Amphiregulin or Relaxin) and investigate if this interferes with their phenotype and expansion

Aim 3 (year 3): To determine the capacity of engineered anti-fibrotic CAR-Tregs to ameliorate chronic liver fibrosis.

Rationale: The use of humanized immunodeficient mice to assess chronic liver damage and fibrosis is challenging, due to the different kinetics of liver damage in the absence of endogenous immune cells and the development of xeno-GVHD when mice are reconstituted with human effector T cells. Literature reports indicate that repeated administration of either CCL4 (4 weeks) or thioacetamide (TA; 4 months) induce significant liver fibrosis in immunodeficient mice. While we propose to use the chronic CCL4 model in NSG mice infused with human CAR-Tregs, our plans could be modified based on current research at Quell evaluating alternative humanised mouse models.

One representative publication from each co-supervisor:

Putnam A.L*., Safinia N*., Medvec A et al. Clinical grade manufacturing of human alloantigen reactive regulatory T cells for use in transplantation. Am J Transplant 2013; 13(11): 3010-20 (*co-first authors)

Sánchez-Fueyo A, et al. Applicability, safety, and biological activity of regulatory T cell therapy in liver transplantation. Am J Transplant 2020;20(4):1125-1136. doi: 10.1111/ajt.15700.