Definition & function of Tregs
Regulatory T cells (Tregs) are first identified by Prof. Sakaguchi, a founder & a technical advisor of RegCell. Tregs are a naturally immunosuppressive population of CD4 T cells. Treg cells can be defined by the expression of the master control gene Foxp3 (forkhead box protein P3) in the nucleus and the alpha chain of the high-affinity interleukin-2 receptor (IL-2Rα or CD25) on the cell surface. Tregs constitute approximately 10% of circulating CD4 T cells in humans. Natural Treg cells are engaged in the maintenance of immune self-tolerance and homeostasis, preventing autoimmune disease, allergy, and inflammatory bowel disease. They are effective in preventing graft rejection in organ transplantation and graft-versus-host disease after hematopoietic stem cell transplantation. Treg cells exert their immunosuppressive effects on diverse immune cell populations, including CD4+ and CD8+ T cells, B cells, dendritic cells, macrophages, mast cells, and natural killer T cells, in a cell-contact manner and by secreting immunosuppressive cytokines such as interleukin-10.
Impairment in immunosuppression by alterations of Tregs in AIDs
A typical example of Treg anomaly as a cause of autoimmune and other immunological diseases is Foxp3 gene mutations, called Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. It accompanies autoimmune diseases such as type 1 diabetes, severe allergy including food allergy, and inflammatory bowel disease. There have been reported many genes (e.g., CD25 and CTLA4) whose mutations affect Treg development and function, thereby causing immunological diseases similar to those seen in Foxp3 mutations. They are now called “Tregopathies”. In addition to such monogenic autoimmune diseases, single nucleotide polymorphisms (SNPs) of Treg signature gene loci contribute to the occurrence of polygenic autoimmune diseases such as rheumatoid arthritis by affecting Treg development and function. Treg-targeting therapy is therefore envisaged to be effective for treating various autoimmune diseases.
Potential & variety of Tregs as a cell therapy
Treg-based cell therapy utilizes natural Tregs (nTregs), induced Tregs (iTregs), or Chimeric Antigen Receptor (CAR)-Tregs.
Current nTreg cell therapy is utilizing ex vivo expanded autologous nTreg cells from patient blood. The expansion is achieved by polyclonal anti-CD3 and anti-CD28 stimulation of naïve nTreg cells with the phenotype of Foxp3-low, CD25-intermediate, and CD45RA-high. Transfer of such polyclonal Treg cell preparations proved to persist in the recipients for at least one year, and phase 1 trials of autologous Treg transfer have already demonstrated that this approach is feasible and safe in patients with T1D or GVHD. However, there are generally six points to be improved: 1) difficulty in expansion in vitro, 2) difficulty in assuming the adequate number of cells sufficient for treatment; 3) antigen specificity cannot be guaranteed; 4) Treg function may be abnormal in patients with autoimmune diseases; 5) pharmacokinetics is assumed to be poor; 6) survival duration in vivo still remains unclear.
TGF-b-induced conventional iTreg cells do not possess Treg-type DNA hypomethylation, showing unstable expression of FoxP3 and other Treg signature genes, hence functionally unstable and occasionally driven to differentiate into effector T cells after in vivo transfer.
A hindrance of the use of nTreg cells for adoptive cell therapy is that purification and expansion of nTreg cells is limited by their relative scarcity in human blood and their slow rate of in vitro expansion. One strategy for circumventing these problems is to generate CAR-Treg cells from nTreg cells by conferring antigen specificity by expressing the Fab region of an antibody specific for a particular target self-antigen. Such mono-specific CAR-Treg cells, whose activation is independent of TCR recognition of peptide/class II MHC, may strongly suppress autoimmune responses with bystander suppression. They can also be expanded to store as off-the-shelf CAR-Treg cells for further treatment of autoimmune patients. However, there are generally two points to be improved: 1) inability to suppress immunity to unspecified antigens or multiple antigens; 2) safety concerns due to genetic modification.
S/F-iTregs have solved the issues of nTregs, conventional iTregs, and CAR-Tregs. Application of S/F-iTreg technologies for “autologous” cell therapy enables specifically to target a lot of autoimmune diseases with high unmet medical needs caused by unknown antigens (eg neo-antigens) as well as known antigens, where no treatment are applicable, without suppressing the overall immune system. Our therapy is based on a completely different approach than the conventional approach of identifying the antigens recognized by T cells and then developing a therapy. Once our therapeutic concept is proven, it will pave the way for the treatment of various AIDs, since the technologies of suppressing immune responses to specific antigens can be applied to a variety of AIDs. Because it is derived from autologous cells and does not require gene transfer or modification, it is expected to be a safe treatment with no side effects and to reduce the burden on patients, contributing to an improved quality of life.
(Ref) Modified from a figure in “Preparation of natural or induced Treg cells for treating autoimmune diseases Mikami, Kawakami and Sakaguchi 37, Curr Opin Immunol, 2020 67:36-41”
We established a series of technologies to robustly generate iTregs with a high conversion rate from PBMCs including memory/effector T cells as well as naïve conventional T cells which highly induce expression of foxp3 and other Treg-specific genes, with Treg-specific DNA hypomethylation at Treg signature genes, and with stable TCR-specificity that recognize specific self-antigen. The technologies have been optimized to maximize the stability and functionality to generate next-gen iTregs “S/F-iTreg, ImmuTact®”. GMP-compliant facility has confirmed robustly to generate S/F-iTregs at a clinical scale. Strong issued process patents and composition of matter patents pended.
CDK8/19 kinase inhibitor
Chemical inhibition of the cyclin-dependent kinase 8 (CDK8) and CDK19 can induce Foxp3 in antigen-stimulated effector/memory as well as naïve CD4+ and CD8+ T cells. The induction was associated with STAT5 activation, independent of TGF-action, and not affected by inflammatory cytokines. CDK8/19 is physiologically repressing Foxp3 expression in activated conventional T cells. Its pharmacological inhibition enables conversion of antigen-specific effector/memory T cells into Foxp3+ Treg cells for the treatment of various immunological diseases.
Original medium “TrSM™”
GMP-grade TrSM was optimized highly to induce iTreg, to expand in a similar way to nTreg, as well as to reduce inflammatory cytokine release and activity of conventional T cell.
S/F-iTreg technologies are not undisclosed. For producing former generation iTregs, CD4+ naïve T cells are stimulated with plate-bound anti-CD3 mAb in the presence of human IL-2 and human TGF-β1. S/F-iTregs are generated by a combination of removing CD28 stimuli for stability, applying CDK8/19 inhibitor for higher Foxp3 induction, applying novel culture methods & new methods for removing monocyte from PBMC, and by a variety of know-how for higher function. See details in Mikami et al., PNAS 117(22), 12258-68, 2020.
S/F-iTreg cells acquired stable Treg-specific DNA hypomethylation, stably expressed Foxp3 following in vivo transfer, longer survival, and effectively suppressed antigen-specific immune responses.
The stability and functionality of S/F-iTregs are comparable to that of nTregs, while overcoming the difficulties of Treg expansion in vitro. Antigen-specific immunosuppressive activity in vitro and therapeutic effect in vivo have been suggested. In addition, induction of infectious tolerance, which is an indicator of sustained efficacy after treatment, is also being confirmed.
Target of interest
(A) Autoimmune diseases
Autoimmune diseases as lifelong disorders are one of the major causes of mortality. The main etiology of autoimmune diseases is not fully understood; however, failure of immunological tolerance is believed to be a common cause of each autoimmune condition. Due to the discovery of involvement of Tregs in these patients, Treg-based cellular therapies are opening the door to new therapeutic options for autoimmunity.
(B) Inflammatory diseases
(C) transplant rejection
Organ transplantation (eg islet cell, liver, kidney) is the gold standard therapy for end-stage organ failure. Although, the results of organ transplantation have been ameliorated in recent decades, chronic rejection and the side-effects of immunosuppressants are still an ongoing serious issue. None of the present immunosuppressive medications (in contrast to Tregs) have the potential to specifically suppress immune mechanisms. Various strategies are currently underway to avoid or minimize the use of immunosuppressive drugs. In this case, it may be possible that Tregs represent a promising solution to induce transplantation tolerance and control the immune response