A new generation of regulatory T cell therapy: CAR-Treg

Regulatory T cells (Tregs) are small groups of immune cells that are specifically designed to suppress excessive immune activation and maintain immune homeostasis
.
Over the past two decades, advances in the development of chimeric antigen receptors (CAR) and genome editing have led to the optimization of T cell therapy, and these technologies are now used to enhance the specificity and functionality of Treg cells, and Treg cell-based autoimmunity and transplant therapies are rapidly developing
.

Classification and function of Treg

Treg is a subset of T cells, accounting for 5-10% of the total CD4+ T cells, with the function of maintaining homeostasis and preventing autoimmunity, and is characterized by the co-expression
of CD4, CD25, FOXP3 and low levels of CD127.
High levels of FOXP3 and demethylation of specific demethylation regions (TSDRs) are distinctive features of Treg, a conserved region
in the FOXP3 gene.

Tregs can be divided into two categories: natural regulatory T cells (nTregs) and induced regulatory T cells (iTregs).

Both types of Tregs are commonly expressed as Foxp3
.
nTregs develop naturally in the thymus, their inhibition is achieved through cell-to-cell contact, and their main function is to maintain normal immune tolerance and control the inflammatory response
.

iTregs are derived from peripheral blasts induced by tumor microenvironment signals, including tumor antigens, cytokines (such as IL-10, TGF-β), and other soluble molecules
.
iTregs inhibit the anti-tumor immune effects
of effector T cells (Teff), NK cells, and DCs through multiple mechanisms that promote tumor progression.

Tregs mainly have the following five functional mechanisms: (1) Tregs secrete inhibitory cytokines, including IL-10, TGF-β and IL-35
.
(2) Tregs kill effector cells
by granzyme and perforin.
(3) Tregs affects the function of effector cells: Treg competes with effector T cells to consume IL-2, thereby inhibiting the growth of effector T cells; Tregs promotes the production of adenosine in TME by producing extracellular enzymes CD39 and CD73, inducing inhibitory and antiproliferative effects of effector cell conduction; Tregs transfer large amounts of cAMP to effector T cells through slit-junctions, interfering with their metabolism
.
(4) Tregs induce DC tolerance through stimulatory and inhibitory receptors (CTLA-4 or LAG3), which further inhibits the ability of
T cells through IDO.
(5) The factors produced by MDSCs and Tregs form a positive feedback loop, promote proliferation, and enhance the inhibition environment
.

In addition, one study used a new strategy to define Tregs, Th1-like Tregs (T-234bet+IFNγ+Foxp3+), Th2-like Tregs (Gata3+IRF4+IL4+Foxp3+), and Th17-like Tregs (IL-17+RORγt+Foxp3+), which provides new ideas
for targeted Tregs therapy.

Adoptive Treg: from polyclonal to antigen-specific

Initial clinical trials of adoptive Treg primarily used polyclonal or in vitro amplified Tregs
.
The first report on the treatment of GVHD with in vitro amplified Treg showed symptom relief in patients with chronic GVHD and the possibility of reducing the use of
immunosuppressants.
A few years later, 11 patients with GVHD who underwent stem cell transplantation for hematological malignancies received cord blood-derived Treg, which was amplified and administered
.
Patients treated with Treg had a 5-fold reduction in the incidence of grade II-IV acute GVHD, and the Treg-treated group was free of chronic GVHD after one year, compared with 14%
in the control group.

While polyclonal Tregs have achieved some encouraging results, the number of cells required for infusion is considerable, in addition to the risk of non-specific immunosuppression, in fact, viral reactivation after polyclonal Tregs infusion has been reported
.

These drawbacks can be overcome by using antigen-specific Treg, which requires fewer cells to implement more local and targeted inhibition
than polyclonal Treg.
In addition, many studies have demonstrated that Tregs specific to the desired antigen are functionally superior to polyclonal or unmodified Tregs
in animal models.

Traditional methods of generating antigen-specific Treg rely primarily on amplification with APCs and specific antigens, or engineered Treg
with T cell receptors (TCRs).
However, amplifying Tregs with APCs is inefficient, while Tregs constructed with TCR (TCR-Tregs) are still limited by MHC, limiting modular applications
for different patients.

Design by CAR-Treg

Another way to give Tregs specificity is to transduce these cells
with CAR.
CAR has some unique advantages over TCR-Treg: these CARs-expressing T cells bypass HLA restriction when activated, increased specificity through activation of co-receptor signaling, and the flexibility of targeting CARs (any soluble or surface polyvalent antigen can be targeted).

The most direct application of CAR-Treg cells is GvHD and organ transplant rejection
.
Unlike most autoimmune diseases, there is a very well-defined target in transplantation, the HLA molecule
.
In 2016, HLA-A2 CAR Treg cells were first reported, and it was demonstrated that HLA-A2-CAR-Treg cells inhibited Teff cell proliferation and prevented HLA-A2+ PBMC-mediated GvHD
in an immunodeficient NSG mouse model.

Subsequent studies began using CAR-Tregs, an alloantigen-specific human Tregs designed to express HLA-A2-specific CAR
.
In vitro and in vivo tests, these A2-CAR Tregs maintained high expression
of FoxP3, CD25, Helios, and CTLA-4.
In addition, they prevented xenograft GvHD
in an immunodeficient mouse model.

Following the success of HLA-A2 targeting CAR-Tregs, recent research has focused on generating CAR-Tregs with different targeting domains, such as CD83, which can prevent GvHD
in mouse models.
There is also CD19, which inhibits the production of B cell antibodies and the pathology
that causes GvHD.

These promising results led to the authorization
of the first CAR-Treg clinical trial (NCT04817774) in the UK and USA for kidney transplant patients.
This trial may support the superiority of CAR-Tregs over polyclonal amplified Tregs in clinical trials, further expanding the possibility
of using CAR-Tregs in other disease conditions.
Other biopharmaceutical companies followed, such as Quell Therapeutics, which focused on CAR-Tregs for liver transplant recipients
.

diabetes

Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by insulin deficiency
due to the destruction of cells β the pancreas.
Studies have shown that Treg has reduced
immunosuppressive function in patients with T1D.
In animal models, infusion of amplified antigen-specific Treg has shown promising results
in blocking and reversing diabetes.
However, due to its rarity in circulation, isolating enough antigen-specific Treg is challenging
.

Therefore, research focuses on polyclonal CD4+ T cells transduced with the FoxP3 gene to convert them into Treg
.
In addition, antigen-specific delivery to polyclonal T cells
can be achieved through engineered TCR or CAR techniques.
Brusko's team found that Treg, transduced by glutamate decarboxylase (GAD)-specific TCR, can inhibit the ability of
antigen-specific T cells to proliferate in vitro.
In another study, Hull et al.
transferred islet-specific TCRs to regulatory T cells and confirmed their ability to
inhibit CD4 and CD8 T cell proliferation in vitro.
Currently, companies such as GentiBio and Abata are developing TCR engineered Treg to treat T1D
.

rheumatoid arthritis

Rheumatoid arthritis (RA) is the most common inflammatory arthritis and is characterized by synovial inflammation, hyperplasia, autoantibody production, and cartilage and bone destruction
.
Multiple immune cell subsets are involved in the development of RA, where the interaction between T cells and macrophages plays a crucial role
.

Many studies have investigated the benefits
of increasing the number of Treg or improving Treg function for RA.
Wright et al.
used ovalbumin (OVA)-specific Treg to inhibit OVA-induced arthritis
by producing TCR-transduced Treg or TCR-FoxP3-transduced CD4+ T cells.
Both engineered Tregs exhibit OVA-dependent inhibition of proliferation of different antigen-specific T cells through bystander inhibition
.
Sonoma Biotherapeutics is currently developing CAR Treg therapy
for RA.

Multiple sclerosis

Multiple sclerosis (MS) is an autoimmune demyelinating and neurodegenerative disease caused
by autoreactive T cells that recognize myelin epitopes.
Preclinical studies using experimental autoimmune encephalomyelitis (EAE) models have confirmed the effectiveness of
Tregs in inhibiting antigen-specific autoreactive immune responses.

Fransson et al.
used CAR to create antigen-specific Treg
by targeting myelin oligodendrocyte glycoprotein (MOG).
MOG-CAR Tregs inhibit the proliferation
of effector T cells in vitro.
In vivo, MOG-CAR Tregs reduced disease symptoms in EAE mice and reduced pro-inflammatory cytokine mRNA levels
in brain tissue.
Later, Kim et al.
constructed Treg using myelin basic protein-specific TCRs derived from MS patients, and in vivo TCR-Tregs significantly reduced disease scores
in mouse models of EAE-MS.
The results of immunomodulation engineered Treg in preclinical studies have led to the development of CAR-Treg by a number of biopharmaceutical companies, such as Abata Therapeutics and TeraImmune
.

Inflammatory bowel disease

Inflammatory bowel disease (IBD) is a disease characterized by chronic inflammation of the gastrointestinal tract, with ulcerative colitis (UC) and Crohn's disease (CD) being the most common forms of
IBD.
Studies have shown that the imbalance between the gut microbiota and immune response plays an important role
in IBD.
As a rule, intestinal inflammation is not associated
with a decrease in the number of Treg.
However, mice with insufficient Treg activity were more likely to develop severe colitis
.
Therefore, multiple studies have attempted to use Treg inhibitory activity to maintain UC tolerance
.

In one study, CAR-Tregs from transgenic mice against a known colitis antigen (TNP) inhibited the proliferation
of effector T cells in vitro.
In vivo, after induction of colitis, an increase
in survival of CAR-Treg transgenic mice was observed compared to wild-type animals.
In addition, transferring TNP-CAR Tregs to a mouse model of colitis can reduce symptoms and improve survival
.
CAR-Treg cell-based therapy may be a promising and effective way to
relieve UC and CD.

asthma

Asthma is a chronic respiratory disease in which Treg is impaired and reduced
in people with asthma.
Therefore, the new approach focuses on preventing airway inflammation
by transferring regulatory T cells in preclinical models of asthma.
In addition, one study used modulated T cell epitopes (Tregitopes) to induce highly inhibitory allergen-specific Treg
.

Treatment with Tregitopes inhibits allergen-induced airway hyperresponsiveness and lung inflammation
.
To direct Treg to asthma-associated antigens, Skuljec et al.
applied CAR technology to isolate second-generation Treg against CEA, a glycoprotein
found on the epithelial surface of the lung and gastrointestinal glands, from transgenic mice.
Their results showed activation and homing
of CEA-CAR Tregs in the inflammatory lungs of asthmatic mice.
In addition, CEA-CAR Tregs improved inflammation
to a greater extent compared to unmodified Tregs.

The next generation of engineered Treg

Synthetic biology

The development of Treg cells as a drug for the treatment of autoimmune diseases is not limited to the application of TCRs and CARs, synthetic immunology has spawned many artificial receptors and systems that are being tested
in Treg cells.

These systems include T cell antigen conjugators that recruit endogenous TCR complexes to non-MHC targets by linked single-chain antibodies, CARs that can be bound and activated by soluble ligands, and separated, universal and programmable CAR (SUPRA).

Cytokines

There is no doubt that cytokines play a key role
in the immune response.
Treg cells constitutively express the high affinity strand CD25 of the IL-2 receptor, effectively depriving Teff cells of IL-2
.
In addition, by engineering the modification of CAR-Treg cells, pro-inflammatory cytokine signals can be converted into IL-2 or IL-10 signals to increase inhibition
of inflammation.

Gene editing

Some preclinical studies have been published using CRISPR–Cas9 to edit human T cell genes, including knocking out the CCR5 gene in CD4+ T cells to produce HIV-resistant T cells; knock out the CD7 gene in CD7-CAR T cells, as the T cells themselves express CD7, thus preventing cannibalism; and knocking out the PD1 gene in CD19-CAR T cells to improve tumor clearance in humanized mouse models
.

Improve delivery systems

Currently, CAR T cells are manufactured using retroviruses and lentiviral transduction to deliver and integrate genetic material into T
cells.
However, these methods are time-consuming, expensive, and problematic
.

Some non-viral delivery methods are being developed, such as CRISPR-RNPs co-electroporation, which can knock more than 1 kb of DNA into specific genomic sites in human T cells, which is simple, safe, and the double-stranded DNA template is not toxic.

This method was used to correct pathogenic CD25 mutations in cells of patients with single-gene autoimmune diseases, demonstrating its potential application, but this method is also limited by the size of
the insert.

Design Treg cells

One obstacle to Treg cell therapy is to ensure cell survival after infusion, using gene editing to knock out the JNK1 gene in Treg cells to make them resistant to apoptosis, and JNK1-deficient Treg cells secrete higher levels of IL-10 and TGFβ, which can protect transplanted islets from rejection for 100 days
longer than controls.

In addition, Treg cells may also become unstable in an inflammatory environment and transform into pathogenic TH17 cells
.
By knocking out the PRKCQ gene, the tendency of Treg cells to differentiate to TH17 cells can be reduced, while maintaining the inhibitory function
of Treg cells.

Finally, access to the latest generation of low-immunogenicity human pluripotent stem cells through gene editing, coupled with ongoing efforts to differentiate stem cells into Treg cells, could revolutionize the way engineered Treg cell therapy is done
.

brief summary

At present, the use of Treg cells to design the treatment of autoimmune diseases is in the ascendant, and engineered CAR-Treg has shown unique advantages in this regard and has great potential
.
However, there are still many questions
in the field of Treg cell biology and Treg cell therapy.

Over the next decade, as these unanswered questions are resolved, expect to see continued improvements in Treg cell manufacturing, as well as widespread applications in synthetic biology and gene editing, that will enable Treg cell therapies to play an increasingly important role
.

References:

1.
 The Future of Regulatory T Cell Therapy: Promises and Challenges of Implementing CARTechnology.
Front Immunol.
 2020; 11: 1608.

2.
Chimeric AntigenReceptor (CAR) Treg: A Promising Approach to Inducing Immunological Tolerance.
Front Immunol.
2018 Oct 12; 9:2359.

3.
 Next-generation regulatory T cell therapy.
Nat Rev Drug Discov.
2019 October ; 18(10): 749–769.

4.
 Chimeric Antigen Receptor (CAR) Regulatory T-Cells in Solid Organ Transplantation.
Front Immunol.
2022; 13: 874157.

5.
CAR-T Regulatory (CAR-Treg) Cells: Engineering and Applications.
Biomedicines.
 2022 Feb; 10(2): 287.