Both cytokines and their receptors are either expressed on the surface of cells or are secreted into the extracellular environment, making them prime targets for inhibition via mAbs and soluble receptor-Ig fusion proteins. However, there were several reasons why cytokine therapies might not be effective. Cytokines have redundant and complex actions and their effects include both proinflammatory and anti-inflammatory actions. Cytokines induce other cytokines and in many cases we do not fully understand the hierarchy of cytokine action in human diseases. However, despite these complexities, the success of TNF blocking therapies (etanercept, infliximab, adalimumab) are notable. These agents revolutionized the treatment of RA and other diseases including psoriatic arthritis, ankylosing spondylitis, psoriasis, and CD. Similarly, inhibition of IL-1 with recombinant IL-1 receptor antagonist, anakinra, is highly effective in diseases such as neonatal-onset multisystem inflammatory disease (NOMID) and adult-onset Still's disease, but is only modestly effective in RA.194, 195
Of interest, is that combined therapy with IL-1 and TNFα blockade in RA patients does not enhance efficacy and is associated with an increased incidence of serious infections, and the two treatments are not recommended for co-administration as a result.
Targeting T cell-derived cytokines or cytokines that act on T cells has already shown some measure of success. The mAbs daclizumab and basiliximab bind the IL-2 receptor α-subunit (CD25), blocking IL-2's autocrine growth signal. Both drugs seem to be well tolerated clinically and reduce the frequency of acute rejection in solid-organ transplantation.196, 197
Additionally, daclizumab has been shown to reduce disease severity in animal models of arthritis, and is being tested in uveitis, aplastic anemia and MS.198
However, IL-2's role in promoting differentiation of Treg cells and inhibiting differentiation of Th17 cells complicates matters. In both mice and humans, absence of one of the IL-2 receptor subunits results in autoimmune disease. Consequently, one might expect that interference with IL-2 signaling could precipitate autoimmunity. As of yet, this has not been seen with these drugs, but will need to be monitored in the future as a potential complication.
Also being targeted is the IL-2 receptor-β (CD122), which would ameliorate both IL-2 and IL-15 signaling. In a phase I trial for large granular lymphocyte leukemia, the murine monoclonal antibody for CD122, Mikβ1, did not improve clinical symptoms despite the effective inhibition of both IL-15 and IL-2 signaling in T cells.199
The treatment was not associated with autoimmunity, opening the door for further trials in autoimmune diseases. IL-15's proposed role in the maintenance of memory T cells and in the recruitment of T cells to sites of inflammation makes it a particularly attractive target and clinical trials in RA and psoriasis are underway.
Another important T cell-derived cytokine is IFN-γ. One approach for blunting Th1 mediated pathology has been the targeting of IFN-γ with the mAb fontolizumab. In a phase I–II trial, it was well tolerated and doubled the clinical response rate in patients with CD.200
However, a phase II trial in RA was recently terminated.183
Care is warranted in the blockade of IL-12/IFN-γ signaling however, as human mutations in this pathway escalate opportunistic infections.201
Also being evaluated as potential targets are the type I interferons; there is ample evidence that IFN-α plays a pivotal role in a number of autoimmune diseases, including SLE, thyroiditis, and diabetes.202
Type I IFNs are normally produced by plasmacytoid dendritic cells (pDC) in response to viral infections, but in certain autoimmune conditions such as SLE, these cells are also induced to synthesize IFN-α via Toll-like receptor (TLR) ligation by endogenous derived nucleic acids. Within this context, IFN-α leads to activation of autoreactive T cells.203
Neutralizing mAbs against anti-IFN-α have been developed and a recent phase I clinical trial using a single injection of MEDI-545 in patients with SLE reported a dose-dependent inhibition of type I IFN–inducible genes, as well a reduction in clinical disease activity. No safety problems appeared during this short-term study.204
Phase II studies in SLE are currently underway, as well as phase I studies in dermatomyositis and polymyositis.
As opposed to blocking discrete cytokines or their receptors, an alternative approach has been to target select functional components of their intracellular signaling pathways. Binding of interferons and many interleukins results in phosporylation of the Janus kinases (Jak1, Jak2, Jak3 and Tyk2) and the cytokine receptor subunits; this phosphorylation step is critical for the initiation of downstream signaling.205, 206
Of the Jaks, Jak3 seems to have the most discrete function, as it associates with only one cytokine receptor - the common gamma chain or γc
. This is a shared receptor subunit that pairs with other ligand-specific subunits to form the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Severe combined immunodeficiency, characterized by the absence of T cells and NK cells and impaired B cell function, can be caused by mutation of the gene encoding either γc
Notably, no non-lymphoid tissue or organ is affected by the defect, suggesting that actions are restricted to hematopoietic cells. All of this very compelling data led to the development of an orally available, Jak3 antagonist, CP690,550, with nanomolar potency.208
The therapeutic effects of CP 690,550 have been evaluated in clinical trials in CD, psoriasis, RA and kidney transplantation.209
Overall, the results show that the compound appears to be an effective immunosuppressant. The toxicities that appear to be related to this compound include anemia and neutropenia, which might be the result of inhibiting other members of the Jak family.210
The discovery of a subset of myeloproliferative disorders (MPDs) associated with Jak2 mutations has stimulated the consideration of Jak2 as a reasonable therapeutic target. At present, there are at least fifteen clinical trials in MPDs using various inhibitors that target Jak2, only some of them selectively. One potent Jak1/2 inhibitor, INCB18424 (Incyte) has been studied in Phase I and II trials in patients with myeloproliferative disorders, but is also being tested in RA and psoriasis. Generation of a selective Tyk2 inhibitor is also of potential interest, given its importance in IL-12 and IL-23 signaling. In fact, mice with a mutation in Tyk2 have been shown to possess considerable resistance to the development of collagen-induced arthritis.211
One potential downfall of such a scheme is that Tyk2 is known to be important in IFN signaling and its blockade could lead to problems with viral infection, as observed in patients with Tyk2 deficiency.212
Just downstream of the Jaks, the STAT family of proteins might also serve as good potential targets and could theoretically be inhibited by phosphorylated peptidomimetics and “decoy” oligonucleotides.213
Additionally, the endogenous feedback inhibitors of the STATs, the SOCS family of proteins have been considered as a potential target.
mTOR (mammalian target of rapamycin) is a critical link in coupling cell growth stimuli with cell cycle progression downstream of cytokine receptors.214
Rapamycin, also known as sirolimus, is approved for use in allograft rejection, but in addition, there are currently three rapamycin derivatives undergoing clinical trials: temsirolimus, everolimus, and AP23573. Beyond benefits in transplant rejection, temsirolimus has shown effectiveness in RA and phase II MS trials.215
Some of these effects may be mediated by the recent finding that mTOR blockade promotes the differentiation of regulatory T cells while inhibiting the Th17 population.216
However, the ubiquitous nature of mTor means that targeting this molecule can result in a host of side-effects.
Tregs as a therapeutic modality
In both humans and mice, the importance of Foxp3-expressing Treg cells is well-established.61–64
Furthermore, transfer of isolated murine Treg cells routinely inhibits disease in models of autoimmune disease including: IBD, RA, MS, diabetes mellitus, SLE, and IPEX.217, 218
While administration of Tregs is straightforward in mice, there are a number of issues that must be addressed in human trials. The first is how to select pure populations of human Tregs. Tregs are defined as CD4+
cells; however, CD25 is also a marker of activated T cells, making it difficult to ascertain whether one is enriching Treg or effector cells. The second issue is that Foxp3 expression correlates less well with immunosuppressive activity in human cells than it does in the mouse, as activated human T cells also express Foxp3. Much effort at present is being devoted to finding other markers that reliably enrich Treg vs effector cells. One candidate is the IL-7 receptor (CD127) which has been proposed as a negative marker for Treg cells.219
In principle, one could also imagine inducing Treg cells from naïve precursors with the appropriate cytokine cocktail (e.g. TGFβ-1 and IL-2); however, this raises the issue of the stability of the Treg cell phenotype. Recent studies show that Treg cells have the capacity to differentiate into pro-inflammatory Th17 cells.220
Furthermore, their ability to produce TGFβ-1 can promote naïve cells to become either Treg or Th17 cells depending upon the cytokine milieu.27
The potential for flexibility and plasticity within the Treg and Th17 lineages is a fascinating issue, but certainly complicates the therapeutic use of Tregs. Such findings raise the disturbing possibility that administration of Tregs into a host with inflammation and high levels of IL-6 levels could backfire and enhance rather than alleviate pathology.
To avoid the issues of cell transfer, one might speculate that a logical approach to expand Tregs in vivo would be the administration of IL-2, given the role of IL-2 in Treg differentiation and the fact that IL-2 knockout mice develop severe autoimmunity221
. However, it needs to be borne in mind that IL-2 is the prototypic T cell growth factor. It is a critical positive regulator of immune responses, having an essential role in T cell memory.222
It also is a potent inducer of proinflammatory cytokines. Based on this activity, IL-2 is an FDA approved drug for use in patients with renal cell carcinoma; however its use is limited by a sepsis-like, cytokine release syndrome, not exactly what one would expect to be beneficial for the treatment of autoimmune disease. Whether it will be possible to balance the pro- vs. anti-inflammatory actions of IL-2 in a therapeutically useful manner remains to be determined. Administration of TGFβ-1 has also been considered, but like IL-2, it too has pro-inflammatory actions, as well as profibrotic effects.223
As discussed below, retinoic acid and arylhydrocarbon receptor agonists can also regulate Foxp3 expression and affect the balance between Treg and Th17 differentiation.46, 55, 224, 225
As indicated, modified anti-CD3 antibodies may also exert their effect by promoting the production of Tregs and IL-10 from CD4+
T cell precursors.226, 227
The suppressive action of these cells is thought to be TGF-β1 dependant and they express low levels of Foxp3.89
However, all of these therapies hinge on the assumption that an increase in the number of Treg cells would be beneficial for the treatment of autoimmune disease. The preclinical studies discussed above certainly suggest that Tregs can prevent autoimmunity, but few have shown that an enhancement of Treg numbers can be used in a therapeutic setting. In fact, Tregs have been shown to be present in high numbers at sites of local inflammation, suggesting that simply increasing their numbers may not prove therapeutic.228
Additionally, nonspecific immunosuppression and predisposition to infection or malignant disease is a huge concern with Treg therapies, especially if antigen specificity cannot be assured. This holds particularly true in autoimmunity due to the chronic nature of therapies for these diseases.
Given the complexities associated with administration or expansion of Tregs, it might be argued that it would be beneficial to simply administer Treg products. Recombinant human IL-10 was tried initially in patients with psoriasis, with some benefit.229
However, when used in patients with RA in a small study, it failed to show any clinical improvement.230
Studies in Wegener's granulomatosus patients (phase I) and psoriasis patients (phase II) have been completed, but the results have yet to be reported. Recently, it has been reported that IL-35, a heterodimeric cytokine comprising p35 and EBI3, is preferentially produced by Treg cells and has immunosuppressive actions.65
Interestingly, administration of IL-35 in an arthritis model expanded Tregs and decreased disease severity.231
However, because the suppressive effects of Tregs are thought to be multifactorial, involving both contact-dependant and –independent mechanisms, recapitulation of all of the effects of Treg suppression by administration of a single cytokine is likely to prove difficult.
Strategies for targeting Th17 cells
Because of the critical role of IL-17 in autoimmunity, much thought is presently being given to targeting Th17 cells. Ironically, before we even understood the differentiation of Th17 cells, an apparently effective therapy had been generated. Specifically, targeting the p40 subunit of IL-12 has been shown to be effective in the treatment of IBD and psoriasis.11, 12
However, we now know that p40 is a subunit of IL-12 and IL-23 and the salutary effects of this drug are more likely to be due to interference with the latter. Two recent phase III trials with an anti-p40 mAb, ustekinumab, showed significant improvement in psoriasis symptoms, with side-effects comparable to placebo treated groups.232, 233
Ustekinumab also provided significant improvement in a phase II psoriatic arthritis trial, but failed to demonstrate efficacy in an MS trial.234, 235
Clearly the utility of anti-p40 mAb in the treatment of a variety of other autoimmune diseases will be an intense area of investigation. In addition, it will be important to ascertain the relative benefit of targeting IL-23 selectively (anti-p19 mab) versus IL-12 and IL-23 jointly (anti-p40).
IL-6 is one of the most prominent cytokines found within inflamed joints. It has long been recognized as a critical proinflammatory cytokine and inducer of the acute phase response. More recently, we have learned that IL-6 is also a critical differentiation factor for Th17 cells. This new data has increased excitement over tocilizumab, a humanized mAb against IL-6 receptor α subunit. Very recent studies indicate that tocilizumab appears to be as efficacious as anti-TNF therapies in RA, both in terms of reducing symptoms and in inhibiting joint destruction.236–239
Additionally, this treatment has also been shown to be quite useful in the treatment of systemic-onset juvenile arthritis.240
This drug is approved for use in Japan and Europe in adult patients with moderate to severe RA who respond inadequately to other treatments.241
. Because of the seemingly critical role of Th17 cells in murine models, MS would seem to be a very logical target for further study with this agent; however, the lack of efficacy of ustekinumab this setting gives one pause. It will also be interesting to see if tocilizumab has efficacy in SLE. Phase II trials of a subcutaneously administered mAb against IL-6 (CNTO 136
) have also been initiated.183
With the demonstrated utility of tocilizumab in Th17 associated diseases, IL-21 might also be a good target, given its role as an autocrine positive regulator of Th17 differentiation.34, 42, 43, 242
In addition, IL-21 has critical functions in regulating B cells.243
Because Jak3 is essential for IL-21 signaling, capitalizing on this one aspect of Jak3 inhibitors might be the logical first choice for IL-21 signaling blockade.
More recently, retinoic acids have been shown to regulate Th17/Treg differentiation. Specifically, the production of endogenous retinoic acids by gut dendritic cells, or macrophages, promotes the expression of Foxp3 and inhibits IL-17 production.55, 224
All-trans retinoic acid (ATRA), commonly used topically for the treatment of acne vulgaris and keratosis pilaris, as well as systemically for the treatment of acute promyelocytic leukemia (APML), has effects both in vitro and in vivo – limiting IL-17 production and enhancing Foxp3 expression.54
However, it is a known teratogen and it is unknown whether ATRA has effects on fully polarized IL-17 producing memory cells or just undifferentiated effectors. At present, it is uncertain whether selective blockade of the lineage specific transcription factor, RORγt, or activators of retinoic acid receptors will be better clinical candidates for Th17 inhibition.
Another exciting development in the reciprocal regulation of Th17 and Treg cells has been the very recent finding that AHR ligands selectively influence the differentiation of both fates. Two separate studies showed that administration of 6-formylindolo[3,2-b]carbazole (FICZ), an endogenous ligand for AHR, enhanced Th17 differentiation in vitro and enhanced the disease severity in mouse models of MS.46, 225
However, the administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), another AHR ligand derived from Agent Orange, instead induced Foxp3 and suppressed disease in the same models. It should be noted that these differences were certainly not “black and white” but more moderate in nature, with AHR knock-out mice still being susceptible to disease. Clinical translation of these findings could thus be quite challenging, especially considering that compounds like TCDD are extremely toxic. Blockade of the endogenous FICZ could be efficacious, but the effect is not likely to be dramatic and all of the downstream targets of FICZ are not known. Additionally, direct targeting of AHR itself is likely to be even less efficacious, as evidenced by the knock-out studies discussed above. Despite these potential therapeutic pitfalls, these findings are also of interest in that they provide a direct link between environmental toxins and immunoregulation.
Rather than inhibiting Th17 differentiation, another obvious strategy is the direct blockade of the Th17 signature cytokine, IL-17, through the use of a mAb. AIN457, an IL-17A mAb, has completed a phase I/II trial for psoriasis and phase I/II trials in RA and CD are underway.183
One complicating factor to this strategy is that two forms of IL-17 are secreted from T cells, e.g. IL-17A and IL-17F, and their respective functions have not yet been fully elucidated. Such complications can perhaps be avoided through the alternative targeting of the common receptor for both IL-17A and IL-17F, IL-17R. In fact, AMG 827, a fully human monoclonal antibody that binds to and blocks signaling via IL-17R, is currently undergoing phase I trials in psoriasis and phase I/II trials in RA are soon to recruit.183
However, all IL-17 signaling blocking strategies ignore the possibility that other Th17-associated cytokines may also be critical for autoimmunity, a hypothesis that is supported by the susceptibility of mice in certain disease models when IL-17 signaling is blocked.14, 244–247
IL-22 is one such Th17-associated cytokine, its blockade being shown to be curative in mouse models of psoriasis.75
While targeting IL-22 seems logical, this is another cytokine with complex and sometimes contradictory actions. In some settings, IL-22 has critical anti-inflammatory properties. Additionally, IL-22 plays a critical role in host-defense within the gut.74, 77
One final proposed target for the inhibition of Th17 cells is the chemokine receptor(s) selectively expressed by this subset. Chemokine receptor expression identifies subgroups of human memory CD4+
T cells: specifically, CCR6 and CCR4 positive cells are thought to be IL-17 producers.248
Joint targeting of these receptors has been given some consideration. Unfortunately, CCR6 is not a very selective marker, being expressed on approximately 50% of CD4+
Additionally, CCR6 and CCR4 coexpression has been recently reported to also occur in Treg subsets.121
Interestingly, the ligand for CCR6, CCL20, has also been shown to be produced by Th17 cells and the CCR6/CCL20 signaling axis has been proposed as a paracrine signaling mechanism for recruitment of Th17 cells.31, 250
In support of this hypothesis, CCR6 deficiency or blockade protected against the development of arthritis and experimental autoimmune encephalomyelitis in mouse models.251
Especially in the CNS, CCR6+
T cells serve as “pioneers” that initiate inflammation, allowing other pathogenic T cells to follow.252
Taken together, the current data suggests that more work and characterization needs to be completed before definitive therapeutic targets can be developed.