IL-2 immunotherapy for targeting regulatory T cells in autoimmunity

FOXP3+ regulatory T cells (Treg) are indispensable for immune homoeostasis and for the prevention of autoimmune diseases. Interleukin-2 (IL-2) signalling is critical in all aspects of Treg biology. Consequences of defective IL-2 signalling are insufficient numbers or dysfunction of Treg and hence autoimmune disorders in human and mouse. The restoration and maintenance of immune homoeostasis remain central therapeutic aims in the field of autoimmunity. Historically, broadly immunosuppressive drugs with serious side-effects have been used for the treatment of autoimmune diseases or prevention of organ-transplant rejection. More recently, ex vivo expanded or in vivo stimulated Treg have been shown to induce effective tolerance in clinical trials supporting the clinical benefit of targeting natural immunosuppressive mechanisms. Given the central role of exogenous IL-2 in Treg homoeostasis, a new and promising focus in drug development are IL-2-based approaches for in vivo targeted expansion of Treg or for enhancement of their suppressive activity. In this review, we summarise the role of IL-2 in Treg biology and consequences of dysfunctional IL-2 signalling pathways. We then examine evidence of efficacy of IL-2-based biological drugs targeting Treg with specific focus on therapeutic candidates in clinical trials and discuss their limitations.


INTRODUCTION
In 1976, the supernatant of activated T cells was found to contain a potent T cell growth factor, which was cloned in 1983 as interleukin-2 (IL-2) [1][2][3].The identification of IL-2 marked the start of substantial efforts to unravel IL-2-dependent immunological processes, to mechanistically understand IL-2 binding to its receptor and to dissect the signalling pathways downstream of receptor activation.Importantly, with the discovery of IL-2 and an increasing knowledge on IL-2 functions, immense research efforts were launched to develop IL-2-based immunotherapies to exploit its properties in cancer and autoimmune diseases.Here, we provide a brief overview on IL-2 signalling, its relevance in the biology of regulatory T cells (T reg ), and detail recent advances in IL-2-based immunotherapeutics for autoimmune and inflammatory diseases predominantly in clinical stages of development.
Various immune and non-immune cell types express the IL-2R.In humans and mice, the dimeric IL-2R is expressed at low levels by CD4 memory T cells and naïve T cells and at high levels by CD8 memory T cells [15,16] and CD56 low NK cells [17].In mice, the trimeric IL-2R on the other hand is expressed highest on T reg [18,19] and at lower levels on recently activated and effector CD8 T cells, ILC2, and some NKT and CD56 bright NK cells [17,[20][21][22][23]. Similarly, in human peripheral blood mononuclear cells (PBMC), T reg express the highest levels of the trimeric IL-2R whereas other immune cells such as CD45RO pos CD4 T cells, most CD56 high NK cells, few CD4 and CD8 naïve T cells express it at lower levels [24].The trimeric IL-2R is also expressed by endothelial cells, with further CD25 upregulation upon IL-2 treatment [25,26], and signalling can cause the vascular leak syndrome-a known adverse effect of high-dose IL-2 therapy in mice and patients.
IL-2 is a pleiotropic cytokine that can act in an autocrine and paracrine way, with cell type-and context-dependent positive effects on survival, population expansion or lineage stability [17,[27][28][29].The main source of IL-2 are CD4 conventional T cells upon T cell receptor (TCR)/CD28 (co-) stimulation [30,31].Other immune cells such as CD8 T cells, NK(T) cells or dendritic cells can produce IL-2 as well albeit at lower quantities [24,29].T reg are highly dependent on exogenous IL-2 sources as FOXP3 in cooperation with other transcription factors represses Il2 transcription [32][33][34].Yet, a sizable population of T reg in mice is capable of producing IL-2, albeit at a lower per cell level compared to Foxp3 neg CD4 T cells [29].In contrast, IL-2 expression in human peripheral blood FOXP3 pos CD4 T cells is limited to a subset of cells with low expression of FOXP3 likely not representing suppressive T reg (Fig. 1) [35].
T reg homoeostasis is essential to preserve the delicate balance of immune activation.Absence of T reg or decreased function will result in autoimmune diseases, while abundance of T reg will lead to overt immune suppression.These events are balanced by the exclusive IL-2 capture sensitivity of T reg , an overall high dependency of T reg on exogenous IL-2 sources and, hence, a reciprocal control between effector T cells and T reg .The preferential and high expression of the trimeric IL-2R renders T reg most sensitive to IL-2 capture thereby outcompeting other cell types.This superior efficiency in IL-2 capture is exploited by lowdose IL-2 therapy to specifically target and expand T reg [50,51].Further, in T reg the cooperation of the trimeric receptor and the serine/threonine phosphatase PP2A confers increased sensitivity to IL-2 [52,53] and PP2A deficiency in T reg results in autoimmunity [54].Consequently, 10-fold lower IL-2 levels are required for STAT5 activation in T reg compared to CD25-expressing non-T reg and optimal IL-2-dependent gene expression in T reg occurs at 100-fold lower IL-2 concentrations compared to other cell types expressing CD25 [55].The high sensitivity to IL-2 signalling allows for sufficient signalling when available CD25 surface levels are reduced [56].If the superior IL-2 capture is strongly compromised such as it is in CD25-deficient mice or in patients with risk alleles for CD25, systemic inflammation and/or autoimmunity are the consequence of the resulting T reg deficiency or disturbed T reg homoeostasis.

IL-2 IN T REG BIOLOGY AND FUNCTION
Initially, and with the assumption that the main function of IL-2 was the activation of effector T cells and NK cells, efforts to exploit IL-2 in immunotherapy were focused on promoting anti-tumour immunity [57].High-dose recombinant IL-2 (aldesleukin; trade name Proleukin) was the first immunotherapy approved by the U.S. Food and Drug Administration (FDA) in 1992 [58,59].The activation of effector T cells as the main function of IL-2 was contested when ablation of Il2, Il2ra and Il2rb expression in mice caused lethal lymphoproliferation and autoimmunity, rather than immunodeficiency [60].Ten years later, these observations were explained with the discovery of T reg as an immunosuppressive CD4 pos T cell subset characterised by high levels of CD25 and a non-redundant function for IL-2 in many aspects of T reg biology [18,61].The absence or dysfunction of T reg results in fatal multiorgan autoimmunity in mice (scurfy [62]) and human (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, IPEX [63]), and their reduced function has been reported in several systemic (auto-)inflammatory diseases [51,[64][65][66][67][68][69].
IL-2 signalling (via JAK-STAT5) has been demonstrated to be important for T reg thymic development, peripheral induction, lineage commitment and stability sustainability, and homoeostasis (Fig. 2).T reg development takes place in the thymus (thymic T reg , tT reg ) but conventional CD4 T cells can convert into T reg upon tolerogenic stimulation in the periphery as well (peripherallyinduced T reg , pT reg ).IL-2 signalling is important in establishing the T reg identity alongside with TCR and TGFβ signalling [70,71].For tT reg development, a two-step model of TCR and cytokine signalling has been proposed in which the main driving cytokine is IL-2 and its induction of the JAK-STAT5 signalling pathway.IL-7 and IL-15 can compensate for the lack of IL-2 but in the presence of IL-2 their receptors are downregulated establishing a dominant role for IL-2 [72][73][74][75].Defective expression of IL-2 or its receptor subunits, caused by single nucleotide polymorphisms in human or via introduced genetic modification in mice, results in a lack of functional T reg and consequently lymphoproliferation, multiorgan infiltration of activated lymphocytes and lethal autoimmunity [15,60,[76][77][78][79][80][81].Similarly, inappropriate regulation of IL-2 signal transduction impairs T reg homoeostasis and functional stability [42,54,[82][83][84][85][86].Although genetic studies using germline deletion cannot ultimately dissect the requirement for IL-2 during (thymic or peripheral) T reg development from the requirement for IL-2 during peripheral survival and expansion, several lines of evidence support both intrathymic and peripheral roles for IL-2 in T reg .

IL-2-or IL-2R-deficient mice
Autoimmunity in IL-2-or IL-2R-deficient mice can be prevented by adoptive transfer of T reg demonstrating that proficiency in IL-2signalling in mature T reg is sufficient and necessary for peripheral tolerance even when thymic T reg development is impaired [61,[87][88][89].Notably, T reg numbers but not their suppressive activity can be rescued in IL-2-or CD25-deficient mice by depletion of the pro-apoptotic protein Bim [90].Also in IL-2sufficient mice, Bim has been shown to mediate T reg apoptosis to regulate T reg numbers.A critical role for IL-2 in T reg peripheral survival is to maintain the pro-survival protein Mcl-1 [91].
Antibody-mediated neutralisation or preferential delivery of IL-2 Further, antibody-mediated neutralisation of IL-2 and studies utilising IL-2:anti-IL-2 immune complexes have illustrated the indispensable role of IL-2 in peripheral T reg maintenance and functional maturation.Neutralisation of IL-2 induces T cellmediated autoimmunity by selectively reducing T reg numbers [92].Conversely, the application of IL-2:anti-IL-2 immune complexes can prevent the binding of IL-2 to effector T cells and preferentially deliver it to T reg to substantially expand these [93].

Competitive advantage of IL-2R WT cells in mixed bone marrow chimeras
In T reg , IL-2-STAT5 signalling is sensed via the conserved noncoding sequence 2 (CNS2) in the FoxP3 locus sustaining FOXP3 expression and controlling stable FOXP3 expression inheritance [94].The requirement for IL-2 in T reg development, homoeostasis and competitive fitness has further been studied in mixed bone marrow chimeric mice co-transplanted with IL-2 signallingdeficient bone marrow and wild-type bone marrow.In these mice, wild-type T reg greatly outnumbered mutant FOXP3 pos CD4 T cells in the thymus and in the periphery illustrating the competitive disadvantage conferred by IL-2 signalling deficiency [19,95].These experimental designs, however, use bone marrow from IL-2R germline knockout mice and hence, despite analysing thymic as well as peripheral T reg , fall short on undoubtedly dissecting the role of IL-2 signalling during development versus its role in homoeostasis of T reg .Indeed, the shared signalling pathway with IL-15 and IL-7 and hence their compensatory potential [74] together with data obtained in TCR-transgenic mice [96], indicate that T reg lineage induction can be IL-2 signalling-independent.
T reg -specific rescue of IL-2 signalling Finally, the intrinsic requirement for IL-2 signalling in T reg maintenance and fitness has been demonstrated in mice with T reg lineage-specific deficiency of CD25 or the IL-2Rβ chain presenting with decreased T reg frequencies and reduced per cell FOXP3 protein levels, and developing fatal autoimmune disease [97].In line with the aforementioned studies, dysfunctional FOXP3 low CD25 neg T reg can be found in mice with germline deficiencies of IL-2 and IL-2R [19,95,96].
Together, these studies demonstrate the relevance for IL-2 in T reg development, maturation, and survival, and suggest that it serves a direct role in T reg suppressive function.
The role of IL-2 in T reg maturation and function can in part be attributed to a positive feedback loop between FOXP3 and CD25 expression.Upon activation of the IL-2-STAT5 signalling pathway in T reg , phosphorylated STAT5 binds the FoxP3 locus to promote its expression.FOXP3 in turn positively regulates expression of CD25 [33,85,98,99].CD25 expression constitutes part of the T reg transcriptional signature and upon loss of T reg lineage fate, CD25 gene expression is lost quickly, further illustrating the interdependency of T reg signature genes such as FoxP3 and CD25 [100].High expression of the high-affinity trimeric IL-2R on T reg , however, is not only necessary for T reg to scavenge the low levels of IL-2 for their homoeostasis; the ability to preferentially capture IL-2 also presents an immunosuppressive mechanism by starving effector T cells and NK cells from IL-2 and hence limiting their activation and proliferation.The requirement for the IL-2-STAT5-FOXP3 axis in T reg suppressive function was elegantly demonstrated in a study using transgenic mice with T reg -specific Fig. 2 Critical roles of IL-2 in T reg .Upon binding of IL-2 to the trimeric IL-2R, JAK1 and JAK3 phosphorylate the IL-2Rβ or IL-2R common γ (c γ ) chain, respectively.STAT5 docks onto the phosphorylated residues and is then phosphorylated by JAK1/3.Phosphorylated STAT5 (pSTAT5) dimerises and translocates to the nucleus to bind its target loci (such as FoxP3/FOXP3).IL-2 signalling is critical in T reg biology.It plays a dominant role in thymic T reg development (bottom, left), during peripheral T reg functional maturation in barrier tissues (bottom, middle), and is indispensable for the survival and functional lineage stability of mature T reg in secondary lymphoid organs (SLO) (bottom, right).
deficiency of the IL-2R with simultaneous expression of constitutively active STAT5 [97].Early fatal autoimmune disease otherwise observed in mice with T reg -specific IL-2R deficiency can be rescued by constitutive STAT5 signalling; however, the mice still succumbed at a later age from uncontrolled CD8 T cell activation and expansion.This demonstrated that IL-2 consumption via the high-affinity trimeric IL-2R expressed by T reg particularly controls the CD8 T cell population size and activity.
Memory T reg (mT reg )-analogous to their non-regulatory counterparts-are long-lived cells which upon secondary exposure however do not respond with proliferation and proinflammatory cytokine production but instead possess increased suppressive function.The induction of mT reg would hence be of therapeutic interest.They are thought to mitigate tissue damage during the rapid and heightened response of effector memory T cells upon secondary antigen exposure or to reinforce foetal tolerance during pregnancy.Accordingly, mT reg present as antigen-experienced, CCR7 low cells, express high levels of antiapoptotic BCL-2 and proliferate less (Ki67 low ) compared to activated T reg [101,102].
While memory T reg may be less dependent on IL-2 for long-term maintenance, they express high levels of CD25 and expand in response to low-dose IL-2 therapy [55,101,[103][104][105].However, long-lived (local) tolerance induced by IL-2-based therapy relies on the pre-existence of antigen-specific mT reg or (local) induction of mT reg .Antigen therapy to induce hyposensitivity to allergens might be explained by the induction of mT reg .However, e.g.isletspecific antigen therapy alone has been disappointing in trials with type 1 diabetes patients and it has been suggested that antigen therapy must be combined with a T reg -inducing agent such as low-dose IL-2.While this presents a promising strategy for type 1 diabetes and other autoimmune diseases, clinical trials are needed to establish antigen dosing and boosting regimens, longterm efficacy and its correlation with mT reg induction and persistence.
The roles of IL-2 in T reg biology and suppressive function make IL-2 a highly attractive immunotherapeutic molecule in the context of autoimmunity and transplantation.However, its activities on different (immune) cell types also demand caution in clinical trial design and close monitoring of adverse effects.In the below chapters, we will discuss recent pre-clinical and clinical efforts to develop IL-2-based immunotherapeutic strategies that target T reg to treat autoimmune conditions characterised by low numbers or reduced suppressive activity of T reg as well as to prevent transplant rejection.

IL-2-BASED IMMUNOTHERAPIES
IL-2 was the first cytokine therapy approved by the U.S. FDA [58].The initial indications were in metastatic cancers where IL-2 had to be administered at very high doses (HD IL-2) to achieve clinical benefit.The high doses were necessary because of a very short half-life of IL-2 in vivo and for stimulation the cytotoxic effector T cells and NK cells, presumably, once the high-affinity receptor on T reg has been saturated.The approval was based on the overall objective response rates in up to 20% of patients and durable complete responses for up to 91 months [59].However, the treatment also induced severe treatment-associated toxicities including vascular leak syndrome and clinical manifestations of a cytokine storm.Efforts to reduce toxicities by lowering the dose led to a considerable loss of therapeutic efficacy due to the expansion of immunosuppressive T reg that contain a high-affinity IL-2 receptor and thereby outcompete other cells for IL-2 [106].HD IL-2 remains an important treatment in selected patients, either as a first-line option or in combination with new targeted and immunological therapies [107].
The preferential capture of natural IL-2 via the high-affinity IL-2R expressed by T reg is exploited in low-dose (LD) IL-2 therapy.The short half-life of IL-2 (<10 min [108]) requires daily injections of 0.5-3 million international units (MIU) in repetitive treatment courses with effects on T reg lasting days to weeks, but at the same time its quick clearance allows for fast and flexible dose adjustment to ameliorate possible adverse effects.Overall, LD IL-2 treatment is well-tolerated as documented in animal studies and clinical trials (reviewed in [50,51]).Long-term administration in mice showed no impairment of immune responses or vaccination, nor did it increase cancer occurrence [109].Similarly, a long-term study in children with early onset type 1 diabetes mellitus (T1D) concluded that the treatment was safe and well-tolerated [110].However, inherent to the pleiotropic nature of IL-2, dosedependent mild-to-moderate adverse effects are associated with LD IL-2 treatment.While high-dose IL-2 treatment can induce vascular leak syndrome, LD IL-2 may result in transient influenzalike symptoms, or in eosinophilia driven by ILC2-produced IL-5 [24,51].Overall, data obtained in murine disease models and clinical studies are promising with partial or complete response to treatment.Completed and ongoing clinical trials with LD IL-2 in autoimmune and rheumatic diseases are summarised elsewhere [111].
Fuelled by the therapeutic benefit of LD IL-2 and to overcome its limitations, further efforts have focused on the development of second-generation versions of IL-2 with superior pharmacokinetics and T reg selectivity.Aims of these efforts beyond target cell selectivity and reduced off-target effects are to increase the halflife of the novel molecules (at the expense of fast adjustment of dosing in case of adverse effects), less frequent administration, and increased therapeutic dose range.Several groups and pharmaceutical companies have developed PEGylated IL-2 variants [112,113], IL-2 muteins [114][115][116], fusion proteins of IL-2 linked to CD25 [117,118], and IL-2:anti-IL-2 antibody complexes [93,[119][120][121]] that promote T reg cell expansion in vivo.
Here, we will present promising IL-2-based molecules and clinical translation thereof with focus on selected therapeutic IL-2 molecules with post-translational modifications, IL-2 muteins, fusion proteins of IL-2 with other molecules, alternative delivery methods of IL-2, and IL-2:anti-IL-2 antibody complexes (Table 1).

PEGylated IL-2 variants
PEGylation is a covalent conjugation of proteins to inert polyethylene glycol (PEG) moieties.PEGylation extends the halflife of protein therapeutics by increasing the effective molecular weight of the molecule, while the PEG moieties can also shield the proteins from digestion by proteolytic enzymes via increased steric hindrance.For example, a PEG-modified murine IL-2 increased IL-2 retention in vivo by protection from enzymatic digestion and renal clearance [122].Although PEG is known as a safe, inert and non-immunogenic synthetic polymer, some FDAapproved drugs are associated with the development of antibodies against PEG moieties that accelerate drug clearance and loss of clinical efficacy [123,124].
NKTR-358/LY3471851/rezpegaldesleukin (Nektar/Lilly) is recombinant human IL-2 (aldesleukin sequence) chemically conjugated with stable PEG moieties, which has an attenuated affinity for IL-2Rβ compared with recombinant human IL-2.NKTR-358 promoted selective T reg activation and increased T reg suppressive function in mice.The durability and specificity of the response was greater following a single subcutaneous administration of NKTR-358 compared to five daily administrations of IL-2, and led to disease suppression in a mouse delayed-type hypersensitivity (DTH) model [125].Further, biweekly dosing induced preferential and sustained T reg expansion in mice and non-human primates (NHP) resulting in ameliorated disease progression in a mouse model of systemic lupus erythematosus (SLE), and in a non-human primate cutaneous hypersensitivity model [112].The single ascending dose study in healthy volunteers (NCT04133116) and the multiple ascending dose study with three biweekly subcutaneous doses of  rezpegaldesleukin versus placebo in patients with SLE (NCT03556007) yielded promising results [126].Dose-dependent, selective, and sustained increases in percentages and absolute numbers of total CD4 pos T reg and CD25 bright T reg were observed, with no significant changes in conventional CD4 and CD8 T cells, and low-level increases in NK cells.At the highest dose tested, a 12-17-fold increase in CD25 bright T reg over baseline was sustained for 20-30 days.Most adverse events were grade 1-2 injection-site reactions.Immunogenicity was not observed.SLE disease score was not evaluated due to study limitations, however, data for the follow-up phase 1b randomised studies in psoriasis (NCT04119557) and atopic dermatitis (NCT04081350) have been recently presented [127].Treatment of patients with psoriasis with rezpegaldesleukin resulted in increased T reg numbers, and improved disease score (PASI, psoriasis area and severity index) versus placebo, which was maintained up to week 19 posttreatment [128].In atopic dermatitis, biweekly subcutaneous injections of rezpegaldesleukin increased total T reg and CD25 bright T reg during treatment period (12 weeks), while a dose-dependent improvement was observed in disease-relevant scores (EASI, eczema area and severity index) versus placebo up to 36 weeks following end of treatment [129].Together with a favourable safety profile these data further support clinical development of rezpegaldesleukin in patients with atopic dermatitis [130].Less encouraging data were reported for phase 2 ISLAND study (NCT04433585) that enroled adults with moderate-to-severe SLE.
Although respegaldesleukin led to dose-dependent proliferation of T reg , the primary endpoint of the study-a four-point reduction in the SLE disease activity index (SLEDAI-2K)-was not met [131].THOR-809/SAR444336 (Synthorx/Sanofi) is a site-specific PEGylated IL-2 variant with a PEG moiety attached to an unnatural amino acid at the IL-2Rβ interface designed to increase half-life and enhance selectivity for the trimeric IL-2R.The modified IL-2 has a reduced affinity to the IL-2Rβ chain so that the potency of trimeric IL-2R engagement relies on the IL-2Rα chain binding [132].In mice and NHP, THOR-809 preferentially stimulated proliferation of peripheral T reg relative to effector T cells and NK cells.Expanded T reg had sustained pSTAT5 signalling and upregulated suppression markers FOXP3, CD25, ICOS and HELIOS.Furthermore, THOR-809 administration in mice led to dosedependent expansion of highly suppressive T reg and control of skin inflammation in the DTH mouse model [133].A phase 1 trial in healthy subjects (NCT05876767) is currently ongoing [134].
Another promising IL-2 variant with site-specific PEGylation, designated dual 31/51-20 K, similarly displayed substantially increased clearance half-life, preferentially stimulated T reg over effector T cells compared with unmodified IL-2 by selectively reducing the binding affinity for the β subunit of IL-2R, and significantly reduced disease activity and severity in mouse models of xenogeneic graft-versus-host disease (GvHD), SLE and collagen-induced arthritis.Moreover, a single subcutaneous injection of this PEGylated IL-2 did not induce anti-drug antibody formation, nor did it compromise the host defence against viral infection [113].

IL-2 muteins
The elucidation of the crystal structure of IL-2 bound to its trimeric receptor [11,135,136] facilitated the informed introduction of mutations into IL-2 with the aim to increase its affinity or direct its binding.Such targeted mutagenesis allows to uncouple the pleiotropic effects of IL-2 on different immune cells and to target IL-2 activity toward specific cell populations that express either the dimeric IL-2R to boost tumour immunity or the trimeric IL-2R expressed by T reg to increase tolerance in autoimmunity and to transplanted grafts.IL-2 variants with increased binding to CD25 and/or decreased binding to CD122 and/or CD132 preferentially activate and expand T reg .These cytokines are further fused to either the fragment crystalisable (Fc) domain of immunoglobulin  (IgG) or the full IgG, which results in significantly extended half-life due to increased hydrodynamic radius and hence decreased renal clearance but also due to the recycling of the protein via the neonatal Fc receptor.In the following paragraphs, we discuss a selection of promising IL-2 mutein molecules, each designed for T reg selectivity and application in autoimmune or inflammatory disease, that are currently in clinical development.
AMG592 or efavaleukin alpha (Amgen) is an IL-2 mutein with V91K/C125A mutations that confers high CD25-binding affinity, and that is expressed as a fusion to the C-terminus of an Fc homodimer [137].In a first-in-human study, efavaleukin alpha single subcutaneous administration resulted in dose-dependent T reg expansion, which was highly selective relative to conventional T cells (T conv ) and NK cells.T reg -to-T conv ratio peaked at day 8 (4fold vs baseline) and remained elevated up to day 29, while no increases in serum proinflammatory cytokines IL-6, TNFα or IFNγ were detected.The expanded T reg displayed increased CD25 and FOXP3 levels and were enriched for CD31 pos recent thymic emigrants.Treatment was well tolerated [138] and several earlyphase studies were initiated to further evaluate safety and efficacy in subjects with rheumatoid arthritis (RA) (NCT03410056), steroidrefractory chronic GvHD (NCT03422627), and SLE (NCT03451422).Amgen ended the trials in RA due to insufficient therapeutic benefit for the use of efavaleukin alpha plus standard of care therapy in the assessed study population (NCT03410056); and chronic GvHD [139].Data from a multiple ascending dose phase 1b study in patients with SLE demonstrated that efavaleukin alpha was well tolerated and induced a robust and prolonged dosedependent T reg expansion, with minimal changes in CD4 and CD8 T conv , NK cells or serum levels of pro-inflammatory cytokines [140].The biweekly administration resulted in a 50-fold increase in CD25 bright T reg above baseline, and the T reg numbers remained above baseline for an average of 42 days after the last dose.Despite these promising results, a phase 2b study of efavaleukin alpha in patients with SLE (NCT04680637) has been discontinued as it met pre-defined criteria for futility, i.e., it was unlikely to achieve its objectives [141].However, a phase 2 study in ulcerative colitis (NCT04987307) is still ongoing.
RG7835 (Roche) is a bivalent conjugate of human IL-2 mutein (T3A, N88D, C125A) and a human IgG1 with abolished binding to Fcγ receptors.Due to its reduced affinity to IL-2Rβγ, IgG-(IL-2N88D) 2 has a 6-9-fold reduced ability to stimulate T reg in human whole blood pSTAT5 activation assays compared to a wild-type IL-2 dimer but had no effect on other cell types except some activity on CD56 bright NK cells.Treatment of cynomolgus monkeys and humanised NSG mice (engrafted with human foetal liver CD34 pos cells) with a single dose of IgG-(IL-2N88D) 2 induced sustained 10-14-fold expansion of CD4 pos and CD8 pos CD25 pos FOXP3 pos T reg with no effect on other cell types.The in vivo activated and expanded cynomolgus and human T reg had demethylated epigenetic signatures for FOXP3 and CTLA4 characteristic of functionally suppressive cells.However, neither mouse disease models nor multiple-dose studies in NHP could be performed due to the immunogenicity of the molecule in both species [115].Phase 1b study initiated to assess safety, efficacy, pharmacokinetics, and pharmacodynamics of RG7835 in patients with ulcerative colitis (NCT03943550) was terminated after 8 weeks based on the lack of robust clinical improvement in the underlying condition, according to ClinicalTrials.gov.Following the failure in ulcerative colitis, a phase 2 clinical trial designed to evaluate the effect of RG7835 on time to relapse following forced corticosteroid tapering in patients with autoimmune hepatitis (NCT04790916) was also terminated.
Using a structure-guided approach, several mutations in IL-2 were introduced that significantly decreased CD122 binding affinity in addition to other mutations that increased CD25 binding affinity (L118I, N88D, V69A, Q74P, C125S) [142].The resulting Fc-fusion molecules, PT101/MK-6194 (Pandion/Merck), selectively activated and expanded T reg in preclinical studies in humanised NSG mice and NHP without significant effects on other immune cell types, and without eliciting proinflammatory cytokine production [143].These expanded T reg had increased expression of FOXP3 and CD25, suggesting enhanced function and stability.In a phase 1a single ascending dose clinical trial in healthy volunteers, PT101 was safe and well-tolerated, and a dosedependent expansion of CD25 bright T reg cells was observed with a mean maximum increase of 72.5-fold for CD25 bright T reg by day 8-10 (and an overall 3.6-fold increase in total T reg ) [144].T conv and NK cells were not increased while increases in eosinophil counts were transient.A phase 1 clinical trial in ulcerative colitis (NCT04924114) was initiated by Merck & Co. in 2021 to further evaluate PT101/MK-6194.
A similar molecule, an IL-2 mutein (T3A, N88R, C125S) fused to a human IgG Fc domain, DEL106/CC-92252 (Delinia/Celgene/BMS) also preferentially binds to IL-2Rα.A single intravenous dose of the compound in cynomolgus monkeys resulted in dose-dependent and selective T reg expansion and activation, which was better compared to IL-2 [145].An increase in total circulating T reg cells was 15-fold on day 5, while no change in the number of circulating T conv or CD8 cells was detected.The compound also stimulated expression of suppression and proliferation markers CD25, FOXP3 and Ki67 on T reg .IL-2 induced selective STAT5 phosphorylation of T reg over a narrow dose range, also activating T conv , CD8 T, NK and B cells; in contrast, DEL106 demonstrated over 1000-fold-greater selectivity for T reg over other immune cells.In addition, subcutaneous administration showed that DEL106 exhibited a lower serum clearance and had a longer circulating half-life than IL-2.A phase 1 first-in-human study with this molecule was conducted in three parts: as a single ascending dose or multiple ascending dose study in healthy volunteers and a multiple ascending dose study in psoriasis patients (NCT03971825).CC-92252 was found safe and well-tolerated across studies with adverse effects of mild to moderate intensity.The treatment resulted in a selective but modest (maximum 2fold) T reg expansion in circulation of healthy participants and in skin lesions of participants with psoriasis.However, as for RG7835 (Roche), no apparent trend of clinical improvement compared to placebo was observed in patients, indicating that the achieved T reg expansion may be insufficient for robust efficacy in active disease.Mechanistic studies revealed that although highly selective, CC-92252 is a weak partial agonist with only a subset of T reg responding to this IL-2 mutein [146].Given limited evidence for clinical efficacy, the CC-92252 programme has been discontinued, although BMS is pursuing alternative approaches to T reg selectivity with IL-2 constructs (see below, with IL-2/CD25 fusion).
The therapeutic molecule CUG252 (Cugene/Abbvie) is an IL-2 mutein (L19H, C125I, Q126E) Fc-fusion protein designed for biased binding activity to IL-2Rα but attenuated binding to the IL-2Rβγ complex [147].In mice and cynomolgus monkeys, administration of CUG252 resulted in dose-dependent increases in T reg expansion by 10-to 30-fold, with largely abolished activities in effector T cells and NK cells [148].T reg had enhanced expression of functional and inhibitory markers (CD25, FOXP3, PD-1, CTLA-4, TIM3 and ICOS) and increased suppressive capacity in DTH.In T cell-dependent antibody response models, CUG252 strongly inhibited antigendriven inflammation, B cell maturation, and antibody production.The molecule is currently in phase 1 study, which aims to evaluate the safety and tolerability of single escalating subcutaneous doses of CUG252 in healthy adult subjects, and multiple escalating subcutaneous doses of CUG252 in patients with mild to moderate SLE (NCT05328557).
MDNA209 (Medicenna) is an IL-2 mutein (L18R, Q22E, Q126T, S130R) with increased affinity to the IL-2Rβ and greatly decreased affinity for IL-2Rγ, resulting in attenuated IL-2Rβγ heterodimerization and reduced signalling.The design of MDNA209 is based on the scaffold of IL-2 'superkine' variants that had an increased affinity for the β chain of the IL-2 receptor [114].Rather than triggering IL-2 signalling, however, MDNA209 acts as an antagonist, blocking the receptor and preventing it from transmitting the signal.When targeted to T cell subsets, this IL-2 variant could be clinically translated in the context of controlling T-cell mediated (auto)immune disorders where it is essential to prevent effector T cell activation and expansion resulting in effector cell-mediated tissue damage, such as during acute GvHD.The mutein and its Fcfusion version have been characterised ex vivo and in vivo.MDNA209 prevented IL-2-and IL-15-induced signalling via STAT5 and blocked proliferation of CD8 pos T cells and NK cells, while inhibiting helper T cell type (T H ) 1, T H 9 and T reg cells but promoting T H 17 cell differentiation.Mice treated with an Fc-fusion version of MDNA209 for 10 days showed prolonged survival in a full MHC-mismatched acute GvHD model compared to control IgG [149].
Other IL-2 fusion proteins An alternative approach to increase the selectivity of IL-2 for T reg is through fusion with CD25.The mouse IL-2/CD25 fusion protein forms a tight inactive dimer that slowly releases the active monomer to stimulate the high-affinity IL-2R [117].The longacting biologic expands T reg in vivo more potently than IL-2, but also increases their activation and migration into lymphoid tissues as well as non-lymphoid tissues as shown for the pancreas and its inhibition of anti-insulin autoantibodies.Moreover, the IL-2/CD25 fusion protein was effective in treating diabetes and inhibiting lupus nephritis in mouse models [118,150].The human version of the compound is a full agonist, which maintains high selectivity on T reg over other cell types in whole blood pSTAT5 assays [151].The human IL-2/CD25 had a prolonged half-life and induced a dosedependent selective increase in T reg in cynomolgus monkeys compared to IL-2 or IL-2 mutein Fc-fusion molecules [146].The first-in-human study is still ongoing, but preliminary single-dose pharmacodynamics data confirm robust and prolonged T reg induction in humans with no expansion of inflammatory CD8 or T conv cells [152,153].
CUE-401 (Cue Biopharma) is a tolerogenic IL-2/TGFβ Fc-fusion protein designed to activate and induce FOXP3 expression in CD4 T cells (iT reg ).In mouse CD4 T cells, it induces FOXP3 expression in vitro (iT reg ).Also, in human CD4 T cells from healthy donors, inflammatory bowel disease and RA patients, it results in increased number of FOXP3-expressing cells, however, induction of FOXP3 expression versus preferential expansion of containing T reg has not been dissected.The in vitro induced/expanded iT reg suppress polyclonal T cell proliferation and express comparable phenotypic markers as iT reg induced with combination of TGFβ and IL-2 (CD25, CTLA-4, PD-1, GITR, CD38, CD73, GARP).A single dose of CUE-401 administered to TxA23 mice with ongoing autoimmune gastritis increased FOXP3 pos CD4 T cells in blood and lymph nodes and inhibited autoreactive T cell proliferation in gastric lymph nodes [154,155].
TNF signalling via TNFR2 enhances expansion, function and stability of T reg [156].A dual-acting fusion protein, with IL-2 fused to a TNFR2-selective TNF mutein (IL2-EHD2-sc-mTNFR2) promoted strong activation and expansion of CD4 and CD8 T reg cells in vitro compared to either IL-2 or TNFR2 stimulation alone, with both components necessary for superior biological activity [157].The combination of IL-2 and a TNFR2 agonist is therefore a promising approach for selective T reg expansion in vivo [158].

Alternative IL-2 delivery methods
The therapeutic IL-2 molecules described above are expressed in living cells and are administered as formulations of recombinant protein.Novel technologies are instead based on the in situ expression of encoded proteins and include lipid nanoparticle (LNP)-mediated mRNA delivery, DNA vaccines and gene transfer using viral vectors.Nucleic acid therapeutics are considered safe, well-tolerated and efficacious with major advantages over proteinbased therapeutics including simple and cost-effective production processes and opportunities to improve the drug characteristics [159,160].However, several challenges remain.The greatest challenge for mRNA nanomedicine is immunogenicity both against the LNP itself as well as against the mRNA-encoded proteins.With gene therapies, which are designed for permanent integration of the viral vector into genome, the uncertainty about delayed adverse events remains the greatest risk factor [160].
mRNA-6231 (Moderna) is a lipid nanoparticle (LNP)-encapsulated mRNA encoding a T reg -specific IL-2 mutein fused to human serum albumin (HSA).Two triple-mutant molecules (V69A/Q74P/N88D or V69A/Q74P/V91K) showed the highest difference in pSTAT5 signal between T reg and other cell subsets in human PBMC in vitro and selectively activated and expanded T reg in mice.LNP-formulated mRNA encoding HSA fused to wild-type IL-2 elevated the percentage of T reg in cynomolgus monkeys and was also effective in preclinical models of murine acute GvHD and collagen-induced rat arthritis [161,162].The first-in-human trial of mRNA-6231 in healthy adult participants (NCT04916431) was stopped after early clinical data became available [163].
A tolerogenic immunotherapy NNC0361-0041 (Novo Nordisk) involves a DNA plasmid which encodes for pre-proinsulin (PPI), TGFβ1, IL-10, and IL-2 [164].The combination of antigen (PPI) with the three immune response modifiers (TGFβ1, IL-10, and IL-2) is intended to induce antigen-specific T reg accumulating in the pancreas, and to preserve beta cell function in type 1 diabetes (T1D).The safety and efficacy of treatment was demonstrated in NOD mice as assessed by delayed disease progression, necessity of both antigen and IL-2 for increased efficacy and robustness, and tolerability of chronic dosing [165].However, no pharmacodynamicrelated measurements such as T reg activity or cytokine expression were performed.The phase 1 trial in adults with recent-onset T1D is currently recruiting and will evaluate safety, tolerability, and pharmacokinetics of the therapy (NCT04279613).
Adeno-associated viral (AAV) vector-mediated gene transfer for systemic and continuous IL-2 production has been investigated using a single administration of an AAV-IL-2 vector in mice.The treatment enabled sustained stimulation and expansion of T reg without inducing effector T cell activation while preventing diabetes in NOD mice [109] or alleviating Alzheimer's disease in APP/PS1ΔE9 mice with established pathology [166].Moreover, the long-term IL-2 expression did not impair immune responses to infections, vaccination or cancer [109].However, this approach does not allow to interrupt or stop the treatment in case of adverse events.A tissue-specific gene-delivery approach of IL-2 for the treatment of neuroinflammatory pathologies has been developed by Yshii et al.T reg constitute a small resident cell population in the brain, where low levels of IL-2 are thought to limit the natural anti-inflammatory processes.Tissue-specific IL-2 expression targeted to astrocytes via an AAV vector induced a local and transient expansion of the T reg cell population in the mouse brain, which led to beneficial effects in mouse models of traumatic brain injury, multiple sclerosis and stroke [167].Both the tissue-specific IL-2 delivery system as well as the ability to control the encoded protein expression are promising approaches to improve the clinical translation of gene therapy.

IL-2/anti-IL-2 antibody complexes
Coupling of IL-2 to specific monoclonal antibodies can modify the interaction of IL-2 with its receptor leading to a targeted and longer-lasting in vivo biological activity compared with soluble IL-2 [93,119].Depending on the antibody-binding site on IL-2, the IL-2:antibody complex (IL-2c) can preferentially activate either the cells expressing high levels of CD122, such as memory CD8 T cells and NK cells, or CD25-expressing cells such as T reg .A prominent and well-studied example is the complex of mouse IL-2 bound to the anti-mouse IL-2 antibody JES6-1.JES6-1 binding sterically obstructs mouse IL-2 interaction with the IL-2Rβγ heterodimer to block the signalling on IL-2Rα low effector cells.Thereupon, IL-2 is preferentially delivered to the trimeric receptor via a unique allosteric exchange mechanism, where the IL-2Rα subunit displaces the JES6-1 antibody allowing IL-2 to initiate signalling via the IL-2βγ subunits.This complex prolonged the in vivo halflife of IL-2 and led to selective expansion of murine T reg in a murine dextran sodium sulphate colitis model [119,168].The efficacy of this approach has been further demonstrated in various experimental models of autoimmune diseases or other inflammatory settings as exemplified below: • enhanced allograft survival in a murine model of islet transplantation and experimental autoimmune encephalomyelitis (EAE) prevention in combination with rapamycin [169], • markedly attenuated acute GvHD while preserving graftversus-leukaemia activity after allo-hematopoietic cell transplantation at higher efficacy than tacrolimus treatment [170], • survival of fully MHC-mismatched skin allograft: IL-2c failed to augment the survival of skin allografts as monotherapy but initial treatment with anti-IL-6 monoclonal antibody followed by supplementation with rapamycin led to graft survival and elevated intra-graft T reg levels [171], • attenuation of CNS inflammation and neurological deficits in EAE [172], • suppression of experimental myasthenia gravis [173], • inhibition of collagen-induced arthritis [174], • attenuation of atherosclerosis in apolipoprotein E-deficient mice [175], • decreased myofiber injury in murine muscular dystrophy model [176].
These results motivate the investigation of IL-2-based therapies in inflammatory diseases or conditions that are not caused by autoimmune or alloimmune reactions.
A fully human anti-IL-2 antibody F5111.2 that resembles the exchange mechanism observed for the anti-mouse IL-2 antibody JES6-1, was developed by Trotta et al. [120].Comparison of the crystal structure of IL-2c with the IL-2/IL-2R quaternary structure revealed that F5111.2 sterically obstructs the binding of human IL-2 to IL-2Rβ and allosterically reduces the affinity of the cytokine to IL-2Rα.Administration of F5111.2-hIL-2complex results in the preferential STAT5 phosphorylation of T reg in vitro and selective expansion of T reg in vivo.When complexed with human IL-2, F5111.2 induced remission of T1D in the NOD mouse model, reduced disease severity in a model of EAE and protected mice against xenogeneic GvHD [120].
Another anti-human IL-2Rα-biased IL-2 antibody, UFKA-20, uses a similar mechanism to selectively target T reg [121].The IL-2 bound to UFKA-20 fails to induce cell activation via the dimeric IL-2R unless the cells also express CD25.Once the IL-2/UFKA-20 complex is bound to CD25, the antibody dissociates from IL-2 and allows the formation of high affinity quaternary IL-2/IL-2R structure that leads to intracellular signalling.Consequently, the IL-2/UFKA-20 complexes efficiently and preferentially stimulated CD4 pos T reg in freshly isolated human T cells ex vivo and in mice and rhesus macaques in vivo [121].
The clinical translation of the IL-2/antibody complex approach is complicated by the instability of the cytokine/antibody complex and the need to optimise dosing ratios, as dissociation would lead to off-target effects and rapid clearance.Genetically fusing IL-2 and the antibody should circumvent these drawbacks [168,177].A single-chain hIL-2/F5111 antibody-fusion protein has been engineered that demonstrated selective T reg bias and showed efficacy in mouse models of colitis and checkpoint inhibitor-induced diabetes mellitus [178].

CONCLUSIONS
IL-2 is central in the biology of T reg during development, functional maturation, lineage stability, peripheral homoeostasis, and function.The consequence of the dependency of T reg on IL-2 is the development of autoimmunity in the absence of IL-2 signalling.T reg compensate for the dependency with an exceptional IL-2 capture sensitivity that outcompetes that of other cell types.The necessity for IL-2 signalling and the high expression of the high-affinity trimeric IL-2R make the IL-2 signalling pathway a prime-candidate for T reg -targeting therapeutic approaches in autoimmune and inflammatory diseases as well as in the prevention of transplant rejection.
Despite its high efficacy, given the limitations of low-dose IL-2, numerous approaches have been developed to increase the targeting specificity of IL-2 and hence to avoid binding of the new IL-2-based biologicals to non-T reg cells.Informed by structural and empirical studies, modified IL-2-based molecules are being tested in pre-clinical studies as well as in clinical trials.Yet, informed design may not entirely predict therapeutic success as illustrated by insufficient efficacy and incomplete translation of pre-clinical data in clinical trials for some candidates.However, despite the requirement for thorough clinical assessment of therapeutic benefit in each disease, recent successes in clinical trials for several modified IL-2-based molecules in various autoimmune contexts are representative of the promising therapeutic perspective of IL-2-based immunotherapeutics.
The further possibility to target (modified) IL-2 to T reg subsets of particular prevalence in a disease context by the use of additional moieties may expand the drug development toolbox in the future.Similarly, combinatorial therapy, such as with rapamycin, may prove beneficial but will require assessment in clinical trials.Finally, and undoubtedly, an increasing understanding of structural modifications and their functional consequences will further the design of IL-2-based molecules to increase targeting efficiency as well as to minimise risk for off-target activity and hence maximise safety and efficacy.

Table 1 .
IL-2-based biologics evaluated for the treatment of autoimmune and inflammatory diseases.