Definition & function of Tregs
Regulatory T cells (Tregs) were first identified by Prof. Sakaguchi, a founder & 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. In addition, 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 autoimmune diseases
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 reports of 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 monogenic autoimmune diseases, single nucleotide polymorphisms (SNPs) of the Treg signature gene loci contribute to 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 utilizes ex vivo expanded autologous nTreg cells from patients’ 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. The transfer of such polyclonal Treg cell preparations persisted in recipients for at least one year. 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 that need to be improved:
- Difficulty in expansion in vitro.
- Difficulty in assuming the adequate number of cells sufficient for treatment.
- Antigen specificity cannot be guaranteed.
- Treg function may be abnormal in patients with autoimmune diseases.
- Pharmacokinetics is assumed to be poor.
- Survival duration in vivo remains unclear.
TGF-β-induced conventional iTreg cells do not possess Treg-type DNA hypomethylation, showing unstable expression of FoxP3 and other Treg signature genes, hence they are functionally unstable and occasionally driven to differentiate into effector T cells after in vivo transfer.
One hindrance in using nTreg cells for adoptive cell therapy is that the purification and expansion of nTreg cells are 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 to 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 in autoimmune patients. However, two points generally need to be improved: 1) inability to suppress immunity to unspecified antigens or multiple antigens; and 2) safety concerns due to genetic modification.
S/F-iTregs have solved the issues of nTregs, conventional iTregs, and CAR-Tregs. Applying S/F-iTreg technologies for “autologous” cell therapy enables specific targeting to many autoimmune diseases with high unmet medical needs caused by unknown antigens (eg, neo-antigens) as known antigens, where no treatments 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 has been proven, it will pave the way for the treatment of various autoimmune diseases since the technologies of suppressing immune responses to specific antigens can be applied to various autoimmune diseases. Furthermore, 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. It reduces the burden on patients and contributes to an improved quality of life.
We have 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. The generated iTregs show a high expression of Foxp3 and other Treg-specific genes, with Treg-specific DNA hypomethylation at Treg signature genes, and possess TCR-specificities that recognize specific self-antigens. The technologies have been optimized to maximize the stability and functionality to generate next-generation iTregs designated “S/F-iTreg, ImmuTact®”. The generation of S/F-iTregs has been confirmed at a clinical scale in a GMP-compliant facility. Strong issued process patents and composition of matter patents are pended.
CDK8/19 kinase inhibitor
Chemical inhibition of cyclin-dependent kinase 8 (CDK8) and CDK19 can induce Foxp3 in antigen-stimulated effector/memory and naïve CD4+ and CD8+ T cells. This induction is associated with STAT5 activation, independent of TGF-action, and not affected by inflammatory cytokines. CDK8/19 physiologically represses Foxp3 expression in activated conventional T cells. Its pharmacological inhibition enables the conversion of antigen-specific effector/memory T cells into Foxp3+ Treg cells to treat various immunological diseases.
Original medium “TrSM™”
GMP-grade TrSM is highly optimized to induce iTreg, to expand similarly to nTreg, and to reduce inflammatory cytokine release and activity of conventional T cells.
S/F-iTreg technologies are not undisclosed. To produce former generation iTregs, CD4+ naïve T cells are stimulated with plate-bound anti-CD3 mAb in the presence of human IL-2 and TGF-β1. S/F-iTregs are generated by removing CD28 stimuli for stability, applying the CDK8/19 inhibitor for higher Foxp3 induction, applying novel culture methods & new methods to remove monocytes from PBMC, and using 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, showed more prolonged survival, and effectively suppressed antigen-specific immune responses.
The stability and functionality of S/F-iTregs are comparable to those 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, the induction of infectious tolerance, an indicator of sustained efficacy after treatment, is also being confirmed.
Target of interest
(A) Autoimmune diseases
Lifelong autoimmune diseases are one of the significant causes of mortality. The primary 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 the 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 (e.g. islet cell, liver, kidney) is the gold standard therapy for end-stage organ failure. Although organ transplantation results have been ameliorated in recent decades, chronic rejection and the side effects of immunosuppressants are still ongoing serious issues. None of the present immunosuppressive medications (in contrast to Tregs) have the potential to suppress immune mechanisms specifically. Various strategies are underway to avoid or minimize the use of immunosuppressive drugs. Tregs may represent a promising solution to induce transplantation tolerance and control the immune response in this case.