Costimulation Blockade in Autoimmunity and Transplantation: the CD28 Pathway Andrew B

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Costimulation Blockade in Autoimmunity and Transplantation: The CD28 Pathway Andrew B. Adams, Mandy L. Ford and Christian P. Larsen This information is current as J Immunol 2016; 197:2045-2050; ; of September 26, 2021. doi: 10.4049/jimmunol.1601135 http://www.jimmunol.org/content/197/6/2045 Downloaded from References This article cites 81 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/197/6/2045.full#ref-list-1

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Costimulation Blockade in Autoimmunity and Transplantation: The CD28 Pathway Andrew B. Adams, Mandy L. Ford, and Christian P. Larsen T cell activation is a complex process that requires multiple immune responses. In contrast to what is now a common cell signaling pathways, including a primary recognition practice in pharmaceutical research, early investigations sur- signal and additional costimulatory signals. TCR signaling rounding costimulatory pathways began as a desire to better in the absence of costimulatory signals can lead to an abor- define the molecular mechanisms involved in T cell activation tive attempt at activation and subsequent anergy. One of rather than an aspiration to develop a predefined biologic the best-characterized costimulatory pathways includes the product. In the end, it was the expectation that a heightened Ig superfamily members CD28 and CTLA-4 and their li- understanding of basic immunology might subsequently drive gands CD80 and CD86. The development of the fusion the emergence of a tool with therapeutic usefulness. Downloaded from protein CTLA-4–Ig as an experimental and subsequent therapeutic tool is one of the major success stories in Identifying costimulatory molecules modern immunology. Abatacept and belatacept are clini- In the early 1980s there was an active effort to identify and cally approved agents for the treatment of rheumatoid characterize immune cell surface molecules using mAbs. By the end arthritis and renal transplantation, respectively. Future in- of the decade, dozens of lymphocyte cell surface molecules had been http://www.jimmunol.org/ terventions may include selective CD28 blockade to block identified but, for the most part, little was known about their the costimulatory potential of CD28 while exploiting the function. The search began to determine whether any of these newly coinhibitory effects of CTLA-4. The Journal of Immu- discovered proteins functioned as costimulatory factors. A common nology, 2016, 197: 2045–2050. technique to probe the functions of the newly identified cell surface molecules was to evaluate the impact of specific mAbs in cellular assays. Experiments of this sort revealed that anti-CD28 mAbs had a he concept of T cell costimulation is one of the most profound impact on T cell proliferation (5). Intact Abs generally stimulated proliferation, whereas Fab fragments were inhibitory.

important advances to emerge in the field of immu- by guest on September 26, 2021 T nology in the past few decades. Unlike the discovery Further characterization of CD28 revealed that its extracellular domain was homologous to the Ig V region domains and iden- and characterization of Ig as the putative BCR, elucidation of tified it as an Ig superfamily member. Given its structural similarity the TCR occurred relatively recently. It was not until the early and the results with anti-CD28 Ab treatment, the supposition was 1980s that the TCR was identified as the protein that provided that CD28 on T cells served as a receptor to a yet-uncharacterized specificity to T cell responses (1, 2). Although characterization of ligand or ligands. Indeed, it was subsequently shown that CD28 the TCR helped to elucidate the mechanisms of T cell speci- is constitutively expressed on all naive T cells in mice, as well as 1 1 ficity, it was also apparent that Ag recognition alone was insuf- almost all CD4 T cells and the majority of CD8 Tcellsin ficient for successful T cell activation. Earlier studies by humans. Additional studies demonstrated that the percentage of 2 1 1 Lafferty and Woolnough (3) suggested that additional costim- CD28 CD4 and CD8 T cells increases with aging and with ulatory signals are required to orchestrate an effective immune states of chronic inflammation or infection, suggesting that, response. In fact, Jenkins and Schwartz demonstrated that Ag unlike naive T cells, memory/Ag-experienced T cells down- recognition (signal 1) in the absence of costimulation, or sig- regulate CD28 on their cell surface, reflective of a diminished nal 2, leads to the induction of an active process of non- reliance on costimulatory signals (6, 7). responsiveness, termed anergy (4). This characteristic of the Subsequent studies set out to identify the natural ligand(s) immune system is now thought to be one of the major mech- for CD28 through a series of interesting experiments. Aruffo anisms in tolerance to self-antigens in the periphery, crucial in and Seed (8) cloned CD28 and overexpressed it in a trans- the prevention of autoimmunity. Although these concepts were fected cell line as a tool to decipher which cell types and, evolving in generalities, there were no specific molecules yet ultimately which cell surface protein(s), served as ligand(s) to identified. Subsequent work was fueled by an intense interest to CD28. Further studies narrowed the search to those cells better define which pathways were essential for T cell–dependent capable of Ag presentation and identified the cell surface

Emory Transplant Center, Department of Surgery, Emory University School of Med- Address correspondence and reprint requests to Dr. Christian P. Larsen, Emory Trans- icine, Atlanta, GA 30322 plant Center, Department of Surgery, Emory University School of Medicine, 101 Woodruff Circle, 5105 WMB, Atlanta, GA 30322. E-mail address: [email protected] This work was supported by National Institutes of Health Grants R37 AI040519 (to C.P.L.), U19 AI051731 (to C.P.L. and A.B.A.), and R01 AI104699 and R01 AI073707 Abbreviations used in this article: CTLA-4, CTL Ag 4; RA, rheumatoid arthritis; Teff, (to M.L.F.). effector T cell; Treg, regulatory T cell.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601135 2046 TRANSLATING IMMUNOLOGY: CD28 PATHWAY IN AUTOIMMUNITY AND TRANSPLANT protein B7/BB-1 or B7-1 (named after the Ab clones that were T cell response rather than its initiation. Finally, a pairing of used to identify it but later renamed as CD80) as the purported cell surface molecules was identified, providing legitimacy to ligand of CD28 (9, 10) (Fig. 1A). Immediate efforts were made the theory of costimulation, which had been described almost a to evaluate the biologic effect of anti–B7-1 Abs (11). Intrigu- decade earlier. It was the start of an expanding area of research ingly, in several systems Ab therapy directed at CD80 alone was that continues today. ineffective, prompting further studies that eventually identified a second ligand, B7-2 (CD86), as an alternative receptor to CTLA-4 and therapeutic potential CD28 (12, 13). Interestingly, unlike CD80, the expression of With the pathway identified, it was only a matter of time before which is inducible upon activation of APCs (dendritic cells, molecules with therapeutic potential were developed and tested activated B cells, monocytes/macrophages) and some T cells in various experimental models of disease. The technology for following exposure to inflammatory signals (e.g., IFN-g, generating Ig-fusion proteins using recombinant engineering TNF, pathogen- or danger- associated molecular patterns, techniques had been recently perfected, providing a method to alarmins), CD86 is constitutively expressed on resting cells, readily generate therapeutics with prolonged half-lives. Peach including interdigitating and peripheral blood dendritic cells/ and colleagues (14) developed a soluble CD28 fusion protein, monocytes, Langerhans cells, and memory/germinal center CD28-Ig, presuming that it would block the costimulatory B cells. CD86 expression is further upregulated in the context interactions between CD28 and its ligands, CD80 and CD86. of inflammation. Thus, CD86 seemed likely to play a more Although studies with it and B7-Ig molecules confirmed the critical role in providing early and sustained costimulatory costimulatory properties of this pathway, further studies de- Downloaded from signals, whereas CD80-derived signals modulate the fate of the scribed a relatively weak affinity of CD28 for its ligands, suggesting that an unreasonable concentration of fusion protein would be required in vivo to achieve biologic activity. Fortu- itously in the midst of this work, Linsley and colleagues made a serendipitous discovery. Several years earlier, anotherIgsuperfamilymemberhadbeen http://www.jimmunol.org/ cloned as a part of a series of experiments designed to describe the cell surface molecules upregulated during T cell activation. CTL Ag 4 (CTLA-4), also designated CD152, was shown to resemble CD28, specifically a shared series of cysteine residues in the extracellular domain (15–17). Further investigations into the function of CTLA-4 were stymied, primarily as a result of the lack of reagents to probe its function. Following the same ap-

proach used with CD28, Linsley’s group generated a fusion by guest on September 26, 2021 protein, CTLA-4–Ig, and tested its ability to bind the B7 molecules.Totheirsurprise,CTLA-4-IgnotonlyboundtotheB7 proteins, but it did so much more effectively than CD28 (18). Given the higher affinity of CTLA-4 for the B7 molecules, .20- fold higher than CD28-Ig, it was recognized that CTLA-4–Ig could be an effective therapeutic to block CD28–B7 molecular interactions and, thus, inhibit T cell costimulation and resultant activation and effector function (Fig. 1B, left panel). Although the function of CTLA-4 itself was not clearly defined at that time, future studies went on to define a coinhibitory role (19). It soon became apparent that CTLA-4 was a negative regulator of CD28-mediated T cell costimulation (20). Ligation of constitutively expressed CD28 by the B7 molecules results in a positive costimulatory signal leading to activation, cytokine production, proliferation, and effector function (Fig. 1A). 1 CTLA-4, in contrast, is upregulated on activated CD4 and 1 CD8 T cells and, as a result of its higher affinity for the B7 molecules, outcompetes CD28 for CD80 and CD86 ligation. This results in an effective local blockade of positive signaling via CD28, and CTLA-4 itself also delivers a negative sig- FIGURE 1. Impact of CTLA-4 Ig versus anti-CD28 domain Abs on nal, inhibiting TCR- and CD28-mediated signal transduction alloreactive T cell responses. (A) Alloreactive T cells become activated and and tempering the overall immune response. Internalization of differentiate into cytokine-secreting effector T cells (Teffs) upon TCR rec- cell surface CTLA-4 is rapid, with .80% of surface CTLA-4 ognition of alloantigen. Tregs can dampen this response by CTLA-4–mediated being internalized within 5 min (21). Interestingly, more recent competition for CD28 ligands (CD80 and CD86) and by secretion of anti- data support a role for trans-endocytosis of CD80 and CD86 inflammatory cytokines IL-10 and TGF-b.(B) CTLA-4–Ig inhibits Teff function but also impairs Treg survival and suppressive capacity, whereas from APCs as a means to effectively remove the ligands for selective CD28 blockade better inhibits Teff accumulation and function and CD28 and reduce costimulatory capacity (22). Thus, CTLA-4 better maintains Treg suppressor function by preserving CTLA-4–mediated seems to function in both a cell-intrinsic and a cell-extrinsic coinhibitory signals. manner to regulate T cell responses (23). The Journal of Immunology 2047

1 1 1 In contrast to conventional T cells, CD4 CD25 FOXP3 of targeting not only CD28 costimulatory activity, but also the regulatory T cells (Tregs) constitutively express CTLA-4, and the coinhibitory effects of CTLA-4, suggesting that certain dis- activation of these cells results in further upregulation of CTLA- ease processes were differentially dependent on CD28 and 4 (24, 25). It is now clear that the presence of CTLA-4 and CTLA-4 signaling and perhaps not all T cell subsets were Tregsiscriticalforcontrolofnormalimmuneresponses,because equally reliant on CD28 signaling, a point that we address mice genetically engineered to lack CTLA-4 develop lympho- later in this review. proliferative disorders and die early of autoimmune disease (26, Further studies in a rat model of collagen-induced arthritis 27). Additional studies associated various polymorphisms of the demonstrated that prophylactic treatment (CTLA-4–Ig treat- CTLA-4 gene in humans with several common autoimmune ment prior to immunization with bovine type II collagen) ef- conditions (28–30). In addition to the critical role of CTLA-4 in fectively prevented the onset of disease (46). Joints from treated modulating Treg suppressive function, CD28 plays an essential animals showed no abnormalities in contrast to control animals role in the thymic development and peripheral homeostasis of in which there was complete erosion of the articular cartilage 1 Foxp3 Tregs. Tang et al. (31) showed that CD28 signaling is and bone. Subsequent studies confirmed that blockade of both required for the maintenance of a stable pool of Tregs in the B7 molecules was required for the clinical effect because treat- periphery by enhancing their survival and promoting their self- ment with anti-CD80 or anti-CD86 alone failed to prevent renewalinanIL-2–dependentfashion.Inparticular,CD28 disease (47). The promise shown in these experimental models engagement functions to increase IL-2 production by conven- spurred the clinical development of CTLA-4–Ig in human trials tional T cells, as well as the expression of CD25 on Tregs. Work of autoimmunity and, eventually, transplant. Downloaded from from Turka and colleagues (32) clarified the cell-intrinsic role of CD28 in Treg function by demonstrating that Treg- Clinical trials in autoimmunity and transplant specific CD28 conditional-knockout animals possessed a Approximately 5% of the population of the United States has an highly nonfunctional Treg compartment and developed autoimmune disease. The majority of these diseases (psoriasis, severe autoimmunity. These mechanistic findings are cor- rheumatoid arthritis [RA], multiple sclerosis, lupus) are chronic roborated by data showing that CTLA-4–Ig treatment of mice and, as such, require lifelong medication after diagnosis; thus, the http://www.jimmunol.org/ and humans results in a decrease in peripheral Treg frequencies potential market for disease-modifying medications is significant. (33, 34). The impact of reduced Treg number and function- By far, the most significant effort to evaluate the clinical efficacy ality on the efficacy of CD28 costimulation blockers is an area of CTLA-4–Ig has been in patients with RA. RA is an immune- of active investigation. mediated disease that is characterized by pain, swelling, and Early investigations had already shown that interactions destruction of joints, with resultant disability. The disease is between CD28 and B7-1 provided costimulation that drove driven, in part, by the development of autoantigen-specific allogeneic proliferation and cytotoxicity of human and T cells that mediate pathologic effects. Within the inflamed murine lymphocytes in vitro (35–38). In the same issue of joint there are lymphoid aggregates with clonally expanded by guest on September 26, 2021 Science, Linsley et al. (39) and Bluestone and colleagues (40) T cells expressing phenotypic markers of activation, including were the first to report the impact of CTLA-4–Ig treatment in CD28 and CTLA-4; thus, it seemed reasonable to examine vivo. Not only did CTLA-4–Ig effectively attenuate T-cell– the impact of CTLA-4–Ig in these patients (48). The clinical dependent Ab responses, it inhibited rejection of transplanted benefits of costimulation modulation in RA were confirmed xenogeneic islets and seemed to promote long-term donor- through an extensive clinical trial program, with .10 y of specific tolerance. Subsequent studies tempered the claim that follow-up. CTLA-4–Ig was assessed in several double-blinded, CTLA-4–Ig can induce lasting tolerance, because results have randomized, placebo-controlled phase II and phase III trials. been quite mixed (41–43). However, it was shown quite clearly There were statistically significant and clinically meaningful that administration of CTLA-4–Ig can be an effective tool to improvements in signs and symptoms of disease, physical modulate CD28 costimulation, resulting in prolonged allograft function, and health-related quality of life in those patients survival in a myriad of transplant models using various organ/ treated with CTLA-4–Ig compared with placebo (49, 50). tissue types. Subsequent trials revealed that CTLA-4–Ig provided additional Additional studies sought to observe the impact of CTLA-4–Ig benefit in patients whose disease was not adequately controlled on various autoimmune conditions. In a mouse model of lupus, with other treatments, such as methotrexate or anti-TNF CTLA-4–Ig was able to halt disease progression and reduce the therapies (51–53). As a result of the benefit documented in severity of established disease (44). Treatment completely sup- these trials, CTLA-4–Ig (abatacept [ORENCIA]) was approved pressed the development of anti-dsDNA Abs and prolonged by the U.S. Food and Drug Administration in December 2005 survival, even when therapy was delayed until the most ad- for use in patients with RA, becoming the first selective co- vanced stage of clinical disease. In contrast to the results seen in stimulation modulator to be introduced into clinical practice. the lupus model, the results achieved with CTLA-4–Ig in ani- CTLA-4–Ig was tested in several other autoimmune condi- mal models of multiple sclerosis have varied. In a mouse model tions with mixed results. The clinical efficacy of CTLA-4–Ig in of experimental allergic encephalitis, a dose of CTLA-4–Ig autoimmunity was actually first assessed in patients with the repressed the first occurrence of disease but not subsequent inflammatory skin disorder psoriasis vulgaris (54). In a small relapses (45). Unexpectedly, multiple treatments with CTLA- phase I dose-escalation study, nearly half of treated patients 4–Ig prior to disease onset resulted in worsening of disease achieved a .50% sustained improvement in clinical disease compared with control animals. Additional experiments activity, with greater response rates in patients receiving the revealed that blocking CD80 resulted in protection from highest doses. The study documented a dose-dependent re- disease, whereas selective blockade of CD86 resulted in duction in the number of infiltrating T cells when comparing disease exacerbation. This called into question the impact pre- and posttreatment skin biopsies, as well as normalization 2048 TRANSLATING IMMUNOLOGY: CD28 PATHWAY IN AUTOIMMUNITY AND TRANSPLANT of keratinocyte proliferation and maturation, and reduced ex- patients treated with calcineurin inhibitor–based immunosuppression of CD80, CD86, CD40, MHC class II, and CD83 pression. In addition, there was a significantly higher in- on lesional dendritic cells (55). Treatment with CTLA-4–Ig cidence of posttransplant lymphoproliferative disorder in was also assessed in patients with multiple sclerosis in a phase I EBV-seronegative recipients, prompting a warning to avoid single-dose trial with limited follow-up. Although the treat- its use in these individuals (69). Based on the earlier results, ment appeared safe, and patients who received CTLA-4–Ig belatacept was given U.S. Food and Drug Administration showed a reduction in myelin basic protein–specific T cell approval in June 2011 for use in kidney transplant recipients responses, there was no significant change in clinical parame- and became the first new immunosuppressant approved for ters (magnetic resonance imaging or neurologic symptoms) use in transplantation in over a decade. (56). Abatacept was also found to slow, but not eliminate, progression of disease in patients with the recent onset of type Conclusions I diabetes mellitus (57). Additional trials were conducted in We now know that T cell activation is a much more complex patients with asthma, lupus, and ulcerative colitis, but none of and dynamic event than first described, with signal strength, these revealed a significant improvement in disease burden cytokine microenvironment, and various costimulatory and when patients were treated with CTLA-4–Ig (58–60). Given coinhibitory molecules contributing to the ultimate path of these results, clinical development in autoimmunity has been T cell activation and differentiation. The CD28/CTLA-4 largely confined to patients with RA. In addition to the sig- pathway was proved to be one of the most critical events in nificant interest in autoimmunity, there continued to be a this process. The discovery and description of T cell costim- Downloaded from drive toward evaluating CTLA-4–Ig treatment in the field of ulation and the subsequent work to develop fusion proteins transplantation. that specifically block these signals represents one of the most T cells play a key role in the rejection of transplanted organs; important advances in modern immunology. Although we thus, it seemed like an ideal indication to test the efficacy of have reviewed the beneficial effects of abatacept and belatacept costimulation blockade. Numerous studies in murine models in autoimmunity and transplant, there remain a significant had shown significant promise (40, 41, 61). Surprisingly, when number of questions relating to their mechanisms of action. It http://www.jimmunol.org/ 1 preclinical studies were performed in nonhuman primates to is now apparent that certain T cell subsets, such as CD8 assess the effects of CTLA-4–Ig on transplant rejection, the memory T cells or Th17 cells, may downregulate CD28 and/ results were quite disappointing (62, 63). These data suggested or are less dependent on signaling through this pathway and, that more effective blockade of CD28 signaling might be re- therefore, are less susceptible to treatment with CTLA-4–Ig (70, quired in the more rigorous nonhuman primate model. Given 71). These Ag-experienced or memory T cells and specific T cell that the affinity of CTLA-4–Ig is ∼100-fold higher for CD80 subsets (e.g., Th17) likely play an important role in the compared with CD86, Peach et al. (14) reasoned that a mole- breakthrough rejection responses seen in the belatacept-

cule engineered with a higher affinity to CD86 might be more treated kidney transplant patients or in the patients with by guest on September 26, 2021 efficacious. Subsequent efforts resulted in the development of a autoimmune diseases in whom CTLA-4–Ig treatment is inef- mutant version of CTLA-4–Ig, designated LEA29Y for the two fective. Other disease processes are highly dependent on the amino acid substitutions that significantly increased the binding coinhibitory signals transmitted by CTLA-4 and, thus, treatment avidity to CD80 and CD86. This increase in binding avidity with CTLA-4–Ig may actually exacerbate disease. This may be resulted in increased in vitro activity and improved efficacy in due, in part, to Tregs, which have the ability to suppress auto- vivo, because LEA29Y treatment significantly prolonged allo- and alloimmune responses and may be dependent on cell surface graft survival compared with CTLA-4–Ig (62, 64). In addition, expression of CTLA-4 for their mechanism of action, suggesting when LEA29Y was combined with conventional immunosup- that treatment with CTLA-4–Ig or belatacept may inhibit Treg pressants in a multidrug regimen, there was significant synergy function. Indeed, coinhibitory signals were shown to be critical resulting in enhanced protection from rejection. These studies in antitumor T cell responses where blocking Abs directed formed the basis for a clinical trial in renal transplant recipients. against coinhibitory receptors, such as CTLA-4, demonstrated The improved form of CTLA-4–Ig, now termed belatacept extreme promise in patients with advanced stage melanoma and (NULOJIX), was evaluated in a phase II clinical trial involving lung cancer; however, a significant number of these patients, not ∼200 patients that compared belatacept with the calcineurin unexpectedly, developed autoimmune-like syndromes while re- inhibitor cyclosporine. The results were encouraging; belatacept ceiving anti–CTLA-4 Ab therapy, highlighting the importance was equally effective at preventing rejection and demonstrated of this molecule in normal immune homeostasis (72, 73). substantially improved renal function, as well as reduced chronic Given the importance of intact CTLA-4 signaling in the allograft nephropathy, at 1 y, suggesting likely improvement in control of normal immune responses, more recent efforts have long-term outcomes (65). A subsequent phase III trial, the directed the focus toward CD28-specific therapy. Conceptually Belatacept Evaluation of Nephroprotection and Efficacy as selective CD28 blockade would interrupt the costimulatory signals First-line Immunosuppression Trial (BENEFIT), confirmed the transmitted through CD28 while preserving the inhibitory impact improvements in renal function and, in additional follow-up of intact CTLA-4 ligation (Fig. 1B). More than a decade ago, studies, documented reduced metabolic and cardiovascular the CD28-specific mAb TGN1412 was evaluated for safety in toxicities (66, 67). The most recent 7-y follow-up data from this a phase I trial (74). Unfortunately, there were disastrous results; trial indicate that those patients treated with belatacept enjoy a all six healthy trial participants developed life-threatening com- survival advantage, as well as a 43% risk reduction for the plications from massive T cell activation and cytokine storm (75). combined end point of death and graft loss (68). Intriguingly, Despite reassuring preclinical data, the intact Ab cross-linked despite improved renal function, the groups treated with CD28 molecules and led to nonspecific, polyclonal T cell acti- belatacept had higher rates and grades of rejection compared with vation. Results from this trial likely tempered any additional The Journal of Immunology 2049 efforts to develop CD28-directed therapies at that time. More terization of CTLA-4 structure and expression on human T cells. J. Immunol. 151: 3489–3499. recently, there has been a renewed interest in developing CD28- 16. Brunet, J. F., F. Denizot, M. F. Luciani, M. Roux-Dosseto, M. Suzan, specific therapies. Two groups, Effimune and Bristol-Myers M. G. Mattei, and P. Golstein. 1987. A new member of the immunoglobulin superfamily–CTLA-4. Nature 328: 267–270. Squibb, are moving forward with clinical trials of Abs engi- 17. Harper, K., C. Balzano, E. Rouvier, M. G. Matteí, M. F. Luciani, and P. Golstein. neered to selectively target CD28 in patients with autoimmune 1991. CTLA-4 and CD28 activated lymphocyte molecules are closely related in conditions. Both reagents are single-chain anti-CD28–specific both mouse and human as to sequence, message expression, gene structure, and chromosomal location. J. Immunol. 147: 1037–1044. molecules: a pegylated humanized Fab9 fragment in the case of 18. Linsley, P. S., W. Brady, M. Urnes, L. S. 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