Immunosuppressive Drugs for Kidney Transplantation
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《新英格兰医药杂志》
The central issue in organ transplantation remains suppression of allograft rejection. Thus, development of immunosuppressive drugs is the key to successful allograft function. Immunosuppressive agents are used for induction (intense immunosuppression in the initial days after transplantation), maintenance, and reversal of established rejection. This review focuses on agents that are either approved or in phase 2 or phase 3 trials in kidney transplantation, but many issues covered here are applicable to all organ transplantation. I begin with a model of the alloimmune response to illustrate how these medications act.
Three-Signal Model of Alloimmune Responses
Alloimmune responses involve both naive and memory lymphocytes,1 including lymphocytes previously stimulated by viral antigens cross-reacting with HLA antigens.2 In the graft and the surrounding tissues, dendritic cells of donor and host origin become activated and move to T-cell areas of secondary lymphoid organs. There, antigen-bearing dendritic cells engage alloantigen-reactive naive T cells and central memory T cells that recirculate between lymphoid compartments but cannot enter peripheral tissues3 (Figure 1). Naive T cells are optimally triggered by dendritic cells in secondary lymphoid organs,6,7 but antigen-experienced cells may be activated by other cell types, such as graft endothelium.8
Figure 1. Steps in T-Cell–Mediated Rejection.
Antigen-presenting cells of host or donor origin migrate to T-cell areas of secondary lymphoid organs. These T cells ordinarily circulate between lymphoid tissues, regulated by chemokine and sphingosine-1-phosphate (S-1-P) receptors.4 APCs present donor antigen to naive and central memory T cells. Some presentation of antigen by donor cells in the graft cannot be excluded (e.g., endothelial cells that activate antigen-experienced T cells). T cells are activated and undergo clonal expansion and differentiation to express effector functions. Antigen triggers T-cell receptors (TCRs) (signal 1) and synapse formation.5 CD80 (B7-1) and CD86 (B7-2) on the APC engage CD28 on the T cell to provide signal 2. These signals activate three signal-transduction pathways — the calcium–calcineurin pathway, the mitogen-activated protein (MAP) kinase pathway, and the protein kinase C–nuclear factor-B (NF-B) pathway — which activate transcription factors nuclear factor of activated T cells (NFAT), activating protein 1 (AP-1), and NF-B, respectively. The result is expression of CD154 (which further activates APCs), interleukin-2 receptor chain (CD25), and interleukin-2. Receptors for a number of cytokines (interleukin-2, 4, 7, 15, and 21) share the common chain, which binds Janus kinase 3 (JAK3). Interleukin-2 and interleukin-15 deliver growth signals (signal 3) through the phosphoinositide-3-kinase (PI-3K) pathway and the molecular-target-of-rapamycin (mTOR) pathway, which initiates the cell cycle. Lymphocytes require synthesis of purine and pyrimidine nucleotides for replication, regulated by inosine monophosphate dehydrogenase (IMPDH) and dihydroorotate dehydrogenase (DHODH), respectively. Antigen-experienced T cells home to and infiltrate the graft and engage the parenchyma to create typical rejection lesions such as tubulitis and, in more advanced rejection, endothelial arteritis. However, if the rejection does not destroy the graft, adaptation occurs and is stabilized by immunosuppressive drugs. The photomicrographs of tubulitis and endothelial arteritis are taken from a mouse model in which these lesions are T-cell–dependent but independent of perforin, granzymes, and antibody. IKK denotes inhibitor of nuclear factor-B kinase, CDK cyclin-dependent kinase, and MHC major histocompatibility complex.
An antigen on the surface of dendritic cells that triggers T cells with cognate T-cell receptors constitutes "signal 1," transduced through the CD3 complex. Dendritic cells provide costimulation, or "signal 2," delivered when CD80 and CD86 on the surface of dendritic cells engage CD28 on T cells. Signals 1 and 2 activate three signal transduction pathways: the calcium–calcineurin pathway, the RAS–mitogen-activated protein (MAP) kinase pathway, and the nuclear factor-B pathway.9 These pathways activate transcription factors that trigger the expression of many new molecules, including interleukin-2, CD154, and CD25. Interleukin-2 and other cytokines (e.g., interleukin-15) activate the "target of rapamycin" pathway to provide "signal 3," the trigger for cell proliferation. Lymphocyte proliferation also requires nucleotide synthesis. Proliferation and differentiation lead to a large number of effector T cells. B cells are activated when antigen engages their antigen receptors, usually in lymphoid follicles or in extrafollicular sites, such as red pulp of spleen,10 or possibly in the transplant,11 producing alloantibody against donor HLA antigens. Thus, within days the immune response generates the agents of allograft rejection, effector T cells and alloantibody.
Effectors and Lesions of Rejection
Effector T cells that emerge from lymphoid organs infiltrate the graft and orchestrate an inflammatory response. In T-cell–mediated rejection, the graft is infiltrated by effector T cells, activated macrophages, B cells, and plasma cells and displays interferon- effects, increased chemokine expression, altered capillary permeability and extracellular matrix, and deterioration of parenchymal function. The diagnostic lesions of T-cell–mediated rejection reflect mononuclear cells invading the kidney tubules (tubulitis) and the intima of small arteries (arteritis). Macrophages that are activated by T cells participate through delayed-type hypersensitivity,12 but the injury remains antigen-specific.13 Injury is not simply lysis of target cells, since typical lesions develop in mice lacking cytotoxic T-cell lytic molecules,14 but may involve parenchymal transdifferentiation into mesenchymal cells15 and cell senescence.16
Alloantibody against donor antigens that is produced systemically or locally in the graft targets capillary endothelium.17 Antibody-mediated rejection is diagnosed by clinical,18 immunologic,19 and histologic criteria, including a demonstration of complement factor C4d in capillaries.20
Host–Graft Adaptation
The term "host–graft adaptation" describes the decrease in both donor-specific responsiveness and the risk of rejection in the months after a successful transplantation that is maintained with immunosuppression.21 Changes in the organ — a loss of donor dendritic cells and a resolution of injury — contribute to the adaptation. Regulatory T cells may also be able to control alloimmune responses, by analogy with their ability to suppress autoimmunity,22 although this hypothesis is unproven. The crucial element is that host T cells become less responsive to donor antigens when antigen persists and immunosuppression is maintained. This may be a general characteristic of T-cell responses in vivo, in which antigen persistence with inadequate costimulation triggers adaptations that limit T-cell responsiveness.23 The resulting partial T-cell anergy (known as "adaptive tolerance" or "in vivo anergy") is characterized by decreased tyrosine kinase activation and calcium mobilization (signal 1) and decreased response to interleukin-2 (signal 3). Adaptation in clinical transplantation resembles in vivo anergy — for example, both can occur in the presence of calcineurin inhibitors. The role of maintenance immunosuppression may be to stabilize adaptation by limiting excitation of the immune system and thus antigen presentation. In some experimental models, favorable adaptations are blocked when calcineurin is inhibited,24 leading to suggestions that calcineurin inhibitors prevent adaptations in clinical transplantation. However, the relevance of these models to clinical adaptation, which occurs despite treatment with calcineurin inhibitors, is doubtful.
Immunosuppressive Drugs
Immunosuppression can be achieved by depleting lymphocytes, diverting lymphocyte traffic, or blocking lymphocyte response pathways (Figure 2). Immunosuppressive drugs have three effects: the therapeutic effect (suppressing rejection), undesired consequences of immunodeficiency (infection or cancer), and nonimmune toxicity to other tissues. Immunodeficiency leads to characteristic infections and cancers, such as post-transplantation lymphoproliferative disease,25 which are related more to the intensity of immunosuppression than to the specific agent used.
Figure 2. Individual Immunosuppressive Drugs and Sites of Action in the Three-Signal Model.
Anti-CD154 antibody has been withdrawn from clinical trials but remains of interest. FTY720 engagement of sphingosine-1-phosphate (S-1-P) receptors triggers and internalizes the receptors and alters lymphocyte recirculation, causing lymphopenia. Antagonists of chemokine receptors (not shown) are also being developed in preclinical models. MPA denotes mycophenolic acid.
New immunosuppressive protocols underscored this point by evoking a new infectious complication, BK-related polyomavirus nephropathy.26 This syndrome of tubular injury by a virus that is usually innocuous emerged only with the recent introduction of powerful drug combinations and now contributes to renal injury and graft loss. Fortunately, the newer immunosuppressive agents have resulted in a lower incidence of both infection and cancer than might have been expected, perhaps because preventing rejection reduces the need for powerful agents to reverse it.
Nonimmune toxicity is agent-specific and is often related to the mechanism that is used, because each agent or class of drugs targets molecules with physiologic roles in nonimmune tissues. For example, nephrotoxicity of calcineurin inhibitors may reflect a role of calcineurin within the renal vasculature.
Classification of Immunosuppressive Drugs
Immunosuppressive drugs include small-molecule drugs, depleting and nondepleting protein drugs (polyclonal and monoclonal antibodies), fusion proteins, intravenous immune globulin, and glucocorticoids (Table 1). Because of space limitations, intravenous immune globulin and glucocorticoids cannot be discussed in detail. In brief, intravenous immune globulin has multiple effects27 and is an important component of approaches to suppress alloantibody responses. Glucocorticoids act as agonists of glucocorticoid receptors, but at higher doses they have receptor-independent effects. Receptor-mediated effects are mainly transcriptional through DNA-binding and protein–protein interactions of the steroid-receptor complex, targeting transcription factors such as activator protein 1 and nuclear factor-B.28
Table 1. Classification of Immunosuppressive Therapies Used in Organ Transplantation or in Phase 2–3 Trials.
Most small-molecule immunosuppressive agents are derived from microbial products and target proteins that have been highly conserved in evolution. Small-molecule immunosuppressive drugs at clinically tolerated concentrations probably do not saturate their targets. For example, cyclosporine acts by inhibiting calcineurin but only partially inhibits calcineurin as used clinically.29 Without target saturation, the drug's effects are proportional to the concentration of the drug, which makes dosing and monitoring critical.
Depleting protein immunosuppressive agents are antibodies that destroy T cells, B cells, or both. T-cell depletion is often accompanied by the release of cytokines, which produces severe systemic symptoms, especially after the first dose. The use of depleting antibodies reduces early rejection but increases the risks of infection and post-transplantation lymphoproliferative disease and can be followed by late rejection as the immune system recovers. Recovery from immune depletion takes months to years and may never be complete in older adults. The depletion of antibody-producing cells is better tolerated than T-cell depletion, because it is not usually accompanied by cytokine release and immunoglobulin levels are usually maintained. However, depletion of antibody-producing cells is incomplete because many plasma cells are resistant to the available antibodies that target B cells, such as anti-CD20 antibody.
Nondepleting protein drugs are monoclonal antibodies or fusion proteins that reduce responsiveness without compromising lymphocyte populations. They typically target a semiredundant mechanism such as CD25, which explains their limited efficacy but the absence of immunodeficiency complications. These drugs have low nonimmune toxicity because they target proteins that are expressed only in immune cells and trigger little release of cytokines.
Small-Molecule Drugs
Azathioprine, which is derived from 6-mercaptopurine, was the first immunosuppressive agent to achieve widespread use in organ transplantation30 (Table 2). The developers of azathioprine, Gertrude Elion and George Hitchings, were acknowledged by a share of the 1988 Nobel Prize. Azathioprine is thought to act by releasing 6-mercaptopurine, which interferes with DNA synthesis. Other possible mechanisms include converting costimulation into an apoptotic signal.41 After cyclosporine was introduced, azathioprine became a second-line drug.
Table 2. Characteristics of Small-Molecule Immunosuppressive Drugs Used in Organ Transplantation or in Phase 2–3 Trials.
Calcineurin Inhibitors
Cyclosporine, a cornerstone of immunosuppression in transplantation for two decades, is in effect a prodrug that engages cyclophilin, an intracellular protein of the immunophilin family, forming a complex that then engages calcineurin.42 The adverse effects of cyclosporine, which are related to the concentration of the drug, include nephrotoxicity, hypertension, hyperlipidemia, gingival hyperplasia, hirsutism, and tremor. Cyclosporine can also induce the hemolytic–uremic syndrome and post-transplantation diabetes mellitus. Recent developments include monitoring of the peak cyclosporine levels two hours after administration to better reflect exposure to the drug.43,44A chemically modified cyclosporine, ISA(TX)247, is under development.45
Tacrolimus engages another immunophilin, FK506-binding protein 12 (FKBP12), to create a complex that inhibits calcineurin with greater molar potency than does cyclosporine. Initial trials indicated that there was less rejection with tacrolimus than with cyclosporine,46,47 but recent analyses suggest that in the current dosing strategies, the efficacy of cyclosporine is similar to that of tacrolimus.48,49 Tacrolimus resembles cyclosporine in that it can result in nephrotoxicity and the hemolytic–uremic syndrome, but it is less likely to cause hyperlipidemia, hypertension, and cosmetic problems and more likely to induce post-transplantation diabetes. Tacrolimus has been suspected of inducing more BK-related polyomavirus nephropathy than has cyclosporine in patients who have undergone kidney transplantation, especially when used with mycophenolate mofetil, but renal function may be better with tacrolimus.49 New developments include a preparation of modified-release tacrolimus to permit once-daily dosing.
The use of tacrolimus has increased steadily, and the drug is now the dominant calcineurin inhibitor, but most transplantation programs exploit the strengths of both tacrolimus and cyclosporine, depending on the risks in individual patients. Hypertension, hyperlipidemia, and the risk of rejection argue for tacrolimus, whereas a high risk of diabetes (e.g., older age or obesity) argues for cyclosporine.
Inosine Monophosphate Dehydrogenase Inhibitors
The use of inhibitors of purine synthesis for immunosuppression was based on the observation that inborn errors in this pathway produce immunodeficiency without damaging other organs, in contrast to errors in the purine salvage pathway.50,51 Mycophenolic acid inhibits inosine monophosphate dehydrogenase, a key enzyme in purine synthesis. Mycophenolate mofetil is a prodrug that releases mycophenolic acid, and in large-scale trials with cyclosporine, it was superior to azathioprine in preventing rejection of kidney transplants.52,53,54,55 Protocols using mycophenolate mofetil and calcineurin inhibitors improved patient survival and graft survival and reduced early and late allograft rejection.56,57 Mycophenolate mofetil has also been evaluated in heart transplantation.58 The drug has largely replaced azathioprine and is widely used because it is effective in combination with many other agents, simple to use without monitoring, and free from organ toxicity and cardiovascular risk. Its principal nonimmune toxic effects are gastrointestinal (mainly diarrhea) and hematologic (anemia, leukopenia). Mycophenolate mofetil may increase cytomegalovirus disease but in vitro manifests antipneumocystis activity.59 Enteric-coated mycophenolic acid has been introduced as an alternative to mycophenolate mofetil.60
Target-of-Rapamycin Inhibitors
Sirolimus61 and everolimus engage FKBP12 to create complexes that engage and inhibit the target of rapamycin but cannot inhibit calcineurin (Figure 2). Inhibition of the target of rapamycin blocks signal 3 by preventing cytokine receptors from activating the cell cycle. The principal nonimmune toxic effects of sirolimus and everolimus include hyperlipidemia, thrombocytopenia, and impaired wound healing. Other reported effects include delayed recovery from acute tubular necrosis in kidney transplants, reduced testosterone concentrations,62 aggravation of proteinuria, mouth ulcers, skin lesions, and pneumonitis. However, sirolimus and everolimus may reduce cytomegalovirus disease.63 Sirolimus and everolimus were developed for use with cyclosporine,64,65 but the combination increased nephrotoxicity, the hemolytic–uremic syndrome, and hypertension. Sirolimus has been combined with tacrolimus (e.g., the Edmonton protocol for pancreatic islet transplantation) to avoid the toxicity of sirolimus–cyclosporine combinations.66,67 However, a controlled trial in renal transplantation showed that sirolimus plus tacrolimus produced more renal dysfunction and hypertension than did mycophenolate mofetil plus tacrolimus,68 which indicates that sirolimus potentiates tacrolimus nephrotoxicity. Practitioners can reduce the toxicity of the combination of a target-of-rapamycin inhibitor and a calcineurin inhibitor by withdrawing one of the drugs. For example, withdrawing cyclosporine in patients in stable condition who are receiving the sirolimus–cyclosporine combination reduces renal dysfunction and hypertension, with a small increase in rejection episodes,69 which suggests a strategy for avoiding the toxic effects of calcineurin inhibitors (Table 3).
Table 3. Characteristics of Protein Immunosuppressive Drugs Used in Organ Transplantation or in Phase 2–3 Trials.
Sirolimus and everolimus may have antineoplastic and arterial protective effects. Since these agents slow the growth of established experimental tumors,70 they have potential applications in oncology. The possibility that sirolimus and everolimus can protect arteries is suggested by two observations: target-of-rapamycin inhibitors that are incorporated into coronary stents inhibit restenosis,71 and target-of-rapamycin inhibitors plus calcineurin inhibitors reduce the incidence of graft coronary artery disease associated with heart transplantation.63 But alternative explanations exist for both observations. Target-of-rapamycin inhibitors may suppress restenosis of mechanically dilated arteries by suppressing wound healing72 rather than by atherogenesis and may prevent graft coronary artery disease simply by preventing rejection. Potential arterial protective effects of sirolimus and everolimus must be weighed against the effects of the hyperlipidemia these drugs induce.73
Dihydroorotate Dehydrogenase Inhibitors
Dihydroorotate dehydrogenase is a key enzyme in pyrimidine synthesis. Leflunomide is a dihydroorotate dehydrogenase inhibitor that is approved for rheumatoid arthritis but is not widely used in transplantation.74 Its active metabolite, A77 1726, was modified to create FK778, which is in phase 2 trials in kidney transplantation. FK778 may have activity against BK-related polyomavirus and have a lower incidence of gastrointestinal effects than does mycophenolate mofetil, but its nonimmune toxic effects such as anemia must be evaluated.
FTY720
FTY720 is derived from myriocin, a fungus-derived sphingosine analogue. After phosphorylation, FTY720 engages lymphocyte sphingosine-1-phosphate receptors and profoundly alters lymphocyte traffic, acting as a functional sphingosine-1-phosphate antagonist.75 FTY720 drives T cells into lymphoid tissues and prevents them from leaving and homing to the graft. Despite low overall toxicity, FTY720 induces reversible bradycardia during the first doses,76 arousing concern about the potential for cardiac arrest when combined with the influences of other agents (e.g., general anesthetics or beta-blockers). FTY720 in combination with cyclosporine has completed phase 2 trials77 and entered phase 3 trials in renal transplantation.
Depleting Antibodies
Polyclonal antithymocyte globulin is produced by immunizing horses or rabbits with human lymphoid cells, harvesting the IgG, and absorbing out toxic antibodies (e.g., those against platelets and erythrocytes) (Table 3). As an induction agent, polyclonal antithymocyte globulin is usually used for 3 to 10 days to produce "profound and durable" lymphopenia that lasts beyond one year.78 In addition to immunodeficiency complications, toxic effects of polyclonal antithymocyte globulin include thrombocytopenia, the cytokine-release syndrome, and occasional serum sickness or allergic reactions. Rabbit preparations of polyclonal antithymocyte globulin (such as Thymoglobulin and ATG-Fresenius) are favored over horse polyclonal antithymocyte globulin because of greater potency.
Muromonab-CD3, a mouse monoclonal antibody against CD3, has been used for 20 years to treat rejection79 and for induction.80 Muromonab-CD3 binds to T-cell-receptor–associated CD3 complex and triggers a massive cytokine-release syndrome before both depleting and functionally altering T cells. Humans can make neutralizing antibodies against muromonab-CD3 that terminate its effect and limit its reuse. Prolonged courses of muromonab-CD3 increase the risk of post-transplantation lymphoproliferative disease.81 The use of muromonab-CD3 declined when newer small-molecule immunosuppressive drugs reduced rejection episodes. A trial of a humanized anti-CD3 monoclonal antibody in kidney transplantation82 was stopped. (A nonactivating humanized anti-CD3 monoclonal antibody is being developed to suppress beta-cell injury in patients with autoimmune diabetes mellitus of recent onset83 but is not currently used for transplantation.)
Alemtuzumab, a humanized monoclonal antibody against CD52, massively depletes lymphocyte populations. It is approved for treating refractory B-cell chronic lymphocytic leukemia but is not approved for immunosuppression in transplantation. A small study in renal transplantation that concluded that alemtuzumab induced "prope tolerance" (meaning near-tolerance)84 was not confirmed in later studies.85 Predictions that target-of-rapamycin inhibitors plus alemtuzumab would induce tolerance were also not confirmed. This combination is associated with rejection episodes, including antibody-mediated rejection.86 Side effects of alemtuzumab include first-dose reactions, neutropenia, anemia, and (rarely) pancytopenia and autoimmunity (e.g., hemolytic anemia, thrombocytopenia, and hyperthyroidism).87 The risks of immunodeficiency complications (infections and cancer) with alemtuzumab are unknown. Alemtuzumab is used off-label for induction in some centers, but controlled trials are needed to establish dosing, safety, and efficacy.
Rituximab (anti-CD20 monoclonal antibody) eliminates most B cells and is approved for treating refractory non-Hodgkin's B-cell lymphomas, including some post-transplantation lymphoproliferative disease in organ-transplant recipients. Rituximab is used off-label in combination with maintenance immunosuppressive drugs, plasmapheresis, and intravenous immune globulin to suppress deleterious alloantibody responses in transplant recipients. Although plasma cells are usually CD20-negative, many are short-lived and require replacement from CD20-positive precursors. Thus, depletion of CD20-positive cells does reduce some antibody responses. CD20-positive B cells can act as secondary antigen-presenting cells, which raises the possibility that rituximab can ameliorate T-cell responses. Off-label applications for rituximab include treatment of antibody-mediated rejection and possibly severe T-cell–mediated rejection88 and suppression of preformed alloantibody before transplantation. Again, controlled trials are needed.
Nondepleting Antibodies and Fusion Proteins
Daclizumab and Basiliximab
The anti-CD25 monoclonal antibodies daclizumab and basiliximab are widely used in transplantation for induction in patients who have a low-to-moderate risk of rejection. Because expression of CD25 (interleukin-2 receptor chain) requires T-cell activation, anti-CD25 antibody causes little depletion of T cells. Anti-CD25 antibody is moderately effective since it reduces rejection by about one third when used with calcineurin inhibitors and has minimal toxic effects.89,90,91,92
LEA29Y
LEA29Y is a second-generation cytotoxic-T-lymphocyte–associated antigen 4 (CTLA-4) immune globulin that is a fusion protein combining CTLA-4 (which engages CD80 and CD86) with the Fc portion of IgG. Results of a phase 2 trial in patients undergoing renal transplantation who are receiving mycophenolate mofetil, glucocorticoids, and anti-CD25 antibody are available in abstract form (www.atcmeeting.org/2004). In this trial with six months of follow-up, the effect of repeated administration of LEA29Y was similar to that of cyclosporine in preventing rejection. The LEA29Y trial introduces the concept of long-term use of nondepleting protein immunosuppressive agents to reduce reliance on toxic small-molecule immunosuppressive drugs.
Additional Drugs
Many of the critical steps that are depicted in Figure 1 can be targeted by small molecules or proteins to create new drugs.93 Potential targets for small-molecule drugs include those previously discussed (e.g., calcineurin) as well as others (e.g., tyrosine kinases, protein kinase C, MAP kinases such as Jun N-terminal kinase, phosphoinositide-3-kinase, and chemokine receptors). Potential targets for protein drugs include many membrane proteins.
Janus kinase 3 (JAK3) inhibitors39,40 illustrate how small-molecule immunosuppressive drugs are developed. JAK3, a tyrosine kinase associated with the cytokine receptor chain, participates in the signaling of many cytokine receptors (interleukin-2, 4, 7, 9, 15, and 21) (Figure 1). JAK3 inhibitor CP-690,55039 was developed by screening a chemical library and modifying candidate compounds to produce an oral agent that is immunosuppressive in rodents and nonhuman primates. One adverse effect is anemia, perhaps reflecting activity against Janus kinase 2, which is needed for erythropoietin action.
Protocol Development and Emerging Issues
For two decades, the options for immunosuppressive drugs were initial induction with the use of protein immunosuppressive therapy; preadaptation maintenance therapy with three drugs — a calcineurin inhibitor, a second line of drugs (azathioprine and now mycophenolate mofetil), and glucocorticoids; and postadaptation therapy with the same combination of drugs at lower doses. Rejection was reversed with high-dose steroids or depleting antibodies. Now hundreds of potential combinations exist, and many new protocols have emerged, often including a reduced reliance on glucocorticoids94 and calcineurin inhibitors. Some examples are listed in Table 4. Developing evidence-based approaches to this confusing choice of protocols presents a challenge.
Table 4. Examples of Current and Experimental Immunosuppressive Drug Protocols.
Progress in the control of early and late rejection and in managing infections such as cytomegalovirus has improved both the survival of patients and the function of grafts.57,100,101 For example, in kidney transplantation, the estimated glomerular filtration rate has improved102 and is stable in many patients, rather than slowly deteriorating, as in the past.57 This raises the hope that many organ transplantations that are performed today represent a permanent cure for end-stage organ failure.
But concerns temper this optimism. Outcomes are not continuing to improve,103 and the rate of late graft loss remains excessive. For example, in the United States each year, end-stage kidney failure develops in 4500 patients who have undergone kidney transplantation, a finding that highlights transplant failure as a major cause of end-stage renal disease.104 Patients who have undergone liver transplantation have an excessive recurrence rate of hepatitis; coronary artery disease develops in some patients with transplanted hearts; and bronchiolitis obliterans often develops in patients with transplanted lungs.105,106 The rate of premature death with functioning allografts remains excessive, in part because of cardiovascular and other complications of immunosuppression.
Nonimmune and immunodeficiency complications of transplant immunosuppression should be reduced. The major nonimmune toxic effects are nephrotoxicity, hypertension, hyperlipidemia, diabetes mellitus, and anemia. Five years after surgery, serious renal injury is present in 7 to 21 percent of patients who have undergone nonrenal transplantation,107 and end-stage kidney failure develops in many patients. The toxic effect of calcineurin inhibitors is an important contributor to the problem of renal failure. Post-transplantation diabetes mellitus develops after three years in 24 percent of patients who have undergone renal transplantation.108 Hyperlipidemia109 and anemia110 are common and undertreated. Options for reducing toxicity include choosing more selective drugs, avoiding toxic combinations, and maintaining vigilance for toxic effects.
Cancers111 and infections that are induced by transplantation remain frequent, with infections now exceeding rejection in pediatric transplant recipients.112 Choosing more selective drugs can reduce these risks. For example, anti-CD25 antibody has little effect on the risk of infection and post-transplantation lymphoproliferative disease.25 New protocols must emphasize reducing the rates of cancer and infection rather than simply lowering the rate of rejection.
New immunosuppressive drug applications and protocols113 are emerging without adequate trials to establish dosing, safety, and efficacy. Examples are the regimens of induction with alemtuzumab or radical minimization of maintenance immunosuppression. Moreover, the quality of transplantation trials is suboptimal.114 One problem is that the decline in the incidence of rejection, the end point in most trials, now limits the evaluation of new agents.115 New composite end points could incorporate organ function and drug toxicity or emerging laboratory measurements of immune mechanisms.
Optimizing outcomes requires long-term follow-up by knowledgeable caregivers who recognize and react to changes. Allografts with deteriorating function should not be dismissed as instances of "chronic rejection"; instead, the source of injury should be diagnosed (e.g., rejection that is T-cell–mediated or antibody-mediated, recurrent disease, drug toxicity, or infection).116 The assumption must be that new deterioration reflects new injury, not an inexorable consequence of an earlier injury. The identification of mechanisms of injury may be rewarded by the arresting of further deterioration.
Robust tests for rejection that is T-cell–mediated or antibody-mediated would change clinical management and clinical trials (e.g., microarray analysis of gene expression in biopsy specimens).11 Measurement of immune responses could guide transplantation management in the same way that measurement of disease activity guides other fields (e.g., the measurement of lipid levels in the management of hyperlipidemia).
Interest in suppressing alloantibody responses is growing. Emerging evidence links alloantibody to late graft deterioration,19 and transplantation is increasingly offered to patients who have previously been excluded by existing alloantibody, including ABO blood-group barriers.117 Options include the optimization of baseline immunosuppression, the administration of rituximab or intravenous immune globulin, and plasmapheresis, but new strategies are needed.
Pharmacogenomics offers possibilities for individualizing immunosuppression, an important goal with respect to toxic drugs with narrow therapeutic indexes.118,119 For example, CYP3A (cytochrome P-450-3A) allele CYP3A5*1, which is associated with increased CYP3A5 levels, is present in 70 to 80 percent of blacks but in only 5 to 10 percent of whites.120 Since CYP3A5 genotyping can be used to predict slower achievement of target tacrolimus levels and earlier rejection,121 it could help reduce rejection in black patients.
For most patients, no practical method of achieving true tolerance to HLA-incompatible organ transplants is at hand. True tolerance is durable antigen-specific unresponsiveness in an immunocompetent host that is induced by previous exposure to the antigen. The only clinical strategy that currently meets this definition is stem-cell transplantation.122 The stability of the adaptation usually depends on immunosuppression or damage to the immune tissues. At some point, most immunosuppressive agents are billed as tolerogenic, an assertion that is typically followed by the realization that, among at least some patients, the immunologic tolerance is not durable after withdrawal of the drug therapy and recovery from its effects. Indeed, the first report of an immunosuppressive drug was entitled "Drug-Induced Immunological Tolerance."123 Many "tolerance trials"124 in fact use immunosuppression and are probably based on host–graft adaptation. Excellent immunosuppression with long-term clinical surveillance remains the best prospect for achieving the potential of transplantation to restore and maintain health.
Dr. Halloran reports having received lecture fees from Roche and Fujisawa.
I am indebted to Dr. Deborah James for her help in preparing the text and tables; to Drs. Karl Brinker, Arthur Matas, Sita Gourishankar, and Attapong Vongwiwatana for reviewing the manuscript; and to Pam Publicover for her assistance in the preparation of the manuscript.
Source Information
From the Division of Nephrology and Transplantation Immunology, University of Alberta, Edmonton, Canada.
Address reprint requests to Dr. Halloran at 250 Heritage Medical Research Centre, Edmonton, AB T6G 2S2, Canada, or at phil.halloran@ualberta.ca.
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Oz HS, Hughes WT. Novel anti-Pneumocystis carinii effects of the immunosuppressant mycophenolate mofetil in contrast to provocative effects of tacrolimus, sirolimus, and dexamethasone. J Infect Dis 1997;175:901-904.
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Kirk AD, Hale DA, Mannon RB, et al. Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (CAMPATH-1H). Transplantation 2003;76:120-129.
Knechtle SJ, Pirsch JD, Fechner J Jr, et al. Campath-1H induction plus rapamycin monotherapy for renal transplantation: results of a pilot study. Am J Transplant 2003;3:722-730.
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Becker YT, Becker BN, Pirsch JD, Sollinger HW. Rituximab as treatment for refractory kidney transplant rejection. Am J Transplant 2004;4:996-1001.
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Cole E, Landsberg D, Russell D, et al. A pilot study of steroid-free immunosuppression in the prevention of acute rejection in renal allograft recipients. Transplantation 2001;72:845-850.
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Three-Signal Model of Alloimmune Responses
Alloimmune responses involve both naive and memory lymphocytes,1 including lymphocytes previously stimulated by viral antigens cross-reacting with HLA antigens.2 In the graft and the surrounding tissues, dendritic cells of donor and host origin become activated and move to T-cell areas of secondary lymphoid organs. There, antigen-bearing dendritic cells engage alloantigen-reactive naive T cells and central memory T cells that recirculate between lymphoid compartments but cannot enter peripheral tissues3 (Figure 1). Naive T cells are optimally triggered by dendritic cells in secondary lymphoid organs,6,7 but antigen-experienced cells may be activated by other cell types, such as graft endothelium.8
Figure 1. Steps in T-Cell–Mediated Rejection.
Antigen-presenting cells of host or donor origin migrate to T-cell areas of secondary lymphoid organs. These T cells ordinarily circulate between lymphoid tissues, regulated by chemokine and sphingosine-1-phosphate (S-1-P) receptors.4 APCs present donor antigen to naive and central memory T cells. Some presentation of antigen by donor cells in the graft cannot be excluded (e.g., endothelial cells that activate antigen-experienced T cells). T cells are activated and undergo clonal expansion and differentiation to express effector functions. Antigen triggers T-cell receptors (TCRs) (signal 1) and synapse formation.5 CD80 (B7-1) and CD86 (B7-2) on the APC engage CD28 on the T cell to provide signal 2. These signals activate three signal-transduction pathways — the calcium–calcineurin pathway, the mitogen-activated protein (MAP) kinase pathway, and the protein kinase C–nuclear factor-B (NF-B) pathway — which activate transcription factors nuclear factor of activated T cells (NFAT), activating protein 1 (AP-1), and NF-B, respectively. The result is expression of CD154 (which further activates APCs), interleukin-2 receptor chain (CD25), and interleukin-2. Receptors for a number of cytokines (interleukin-2, 4, 7, 15, and 21) share the common chain, which binds Janus kinase 3 (JAK3). Interleukin-2 and interleukin-15 deliver growth signals (signal 3) through the phosphoinositide-3-kinase (PI-3K) pathway and the molecular-target-of-rapamycin (mTOR) pathway, which initiates the cell cycle. Lymphocytes require synthesis of purine and pyrimidine nucleotides for replication, regulated by inosine monophosphate dehydrogenase (IMPDH) and dihydroorotate dehydrogenase (DHODH), respectively. Antigen-experienced T cells home to and infiltrate the graft and engage the parenchyma to create typical rejection lesions such as tubulitis and, in more advanced rejection, endothelial arteritis. However, if the rejection does not destroy the graft, adaptation occurs and is stabilized by immunosuppressive drugs. The photomicrographs of tubulitis and endothelial arteritis are taken from a mouse model in which these lesions are T-cell–dependent but independent of perforin, granzymes, and antibody. IKK denotes inhibitor of nuclear factor-B kinase, CDK cyclin-dependent kinase, and MHC major histocompatibility complex.
An antigen on the surface of dendritic cells that triggers T cells with cognate T-cell receptors constitutes "signal 1," transduced through the CD3 complex. Dendritic cells provide costimulation, or "signal 2," delivered when CD80 and CD86 on the surface of dendritic cells engage CD28 on T cells. Signals 1 and 2 activate three signal transduction pathways: the calcium–calcineurin pathway, the RAS–mitogen-activated protein (MAP) kinase pathway, and the nuclear factor-B pathway.9 These pathways activate transcription factors that trigger the expression of many new molecules, including interleukin-2, CD154, and CD25. Interleukin-2 and other cytokines (e.g., interleukin-15) activate the "target of rapamycin" pathway to provide "signal 3," the trigger for cell proliferation. Lymphocyte proliferation also requires nucleotide synthesis. Proliferation and differentiation lead to a large number of effector T cells. B cells are activated when antigen engages their antigen receptors, usually in lymphoid follicles or in extrafollicular sites, such as red pulp of spleen,10 or possibly in the transplant,11 producing alloantibody against donor HLA antigens. Thus, within days the immune response generates the agents of allograft rejection, effector T cells and alloantibody.
Effectors and Lesions of Rejection
Effector T cells that emerge from lymphoid organs infiltrate the graft and orchestrate an inflammatory response. In T-cell–mediated rejection, the graft is infiltrated by effector T cells, activated macrophages, B cells, and plasma cells and displays interferon- effects, increased chemokine expression, altered capillary permeability and extracellular matrix, and deterioration of parenchymal function. The diagnostic lesions of T-cell–mediated rejection reflect mononuclear cells invading the kidney tubules (tubulitis) and the intima of small arteries (arteritis). Macrophages that are activated by T cells participate through delayed-type hypersensitivity,12 but the injury remains antigen-specific.13 Injury is not simply lysis of target cells, since typical lesions develop in mice lacking cytotoxic T-cell lytic molecules,14 but may involve parenchymal transdifferentiation into mesenchymal cells15 and cell senescence.16
Alloantibody against donor antigens that is produced systemically or locally in the graft targets capillary endothelium.17 Antibody-mediated rejection is diagnosed by clinical,18 immunologic,19 and histologic criteria, including a demonstration of complement factor C4d in capillaries.20
Host–Graft Adaptation
The term "host–graft adaptation" describes the decrease in both donor-specific responsiveness and the risk of rejection in the months after a successful transplantation that is maintained with immunosuppression.21 Changes in the organ — a loss of donor dendritic cells and a resolution of injury — contribute to the adaptation. Regulatory T cells may also be able to control alloimmune responses, by analogy with their ability to suppress autoimmunity,22 although this hypothesis is unproven. The crucial element is that host T cells become less responsive to donor antigens when antigen persists and immunosuppression is maintained. This may be a general characteristic of T-cell responses in vivo, in which antigen persistence with inadequate costimulation triggers adaptations that limit T-cell responsiveness.23 The resulting partial T-cell anergy (known as "adaptive tolerance" or "in vivo anergy") is characterized by decreased tyrosine kinase activation and calcium mobilization (signal 1) and decreased response to interleukin-2 (signal 3). Adaptation in clinical transplantation resembles in vivo anergy — for example, both can occur in the presence of calcineurin inhibitors. The role of maintenance immunosuppression may be to stabilize adaptation by limiting excitation of the immune system and thus antigen presentation. In some experimental models, favorable adaptations are blocked when calcineurin is inhibited,24 leading to suggestions that calcineurin inhibitors prevent adaptations in clinical transplantation. However, the relevance of these models to clinical adaptation, which occurs despite treatment with calcineurin inhibitors, is doubtful.
Immunosuppressive Drugs
Immunosuppression can be achieved by depleting lymphocytes, diverting lymphocyte traffic, or blocking lymphocyte response pathways (Figure 2). Immunosuppressive drugs have three effects: the therapeutic effect (suppressing rejection), undesired consequences of immunodeficiency (infection or cancer), and nonimmune toxicity to other tissues. Immunodeficiency leads to characteristic infections and cancers, such as post-transplantation lymphoproliferative disease,25 which are related more to the intensity of immunosuppression than to the specific agent used.
Figure 2. Individual Immunosuppressive Drugs and Sites of Action in the Three-Signal Model.
Anti-CD154 antibody has been withdrawn from clinical trials but remains of interest. FTY720 engagement of sphingosine-1-phosphate (S-1-P) receptors triggers and internalizes the receptors and alters lymphocyte recirculation, causing lymphopenia. Antagonists of chemokine receptors (not shown) are also being developed in preclinical models. MPA denotes mycophenolic acid.
New immunosuppressive protocols underscored this point by evoking a new infectious complication, BK-related polyomavirus nephropathy.26 This syndrome of tubular injury by a virus that is usually innocuous emerged only with the recent introduction of powerful drug combinations and now contributes to renal injury and graft loss. Fortunately, the newer immunosuppressive agents have resulted in a lower incidence of both infection and cancer than might have been expected, perhaps because preventing rejection reduces the need for powerful agents to reverse it.
Nonimmune toxicity is agent-specific and is often related to the mechanism that is used, because each agent or class of drugs targets molecules with physiologic roles in nonimmune tissues. For example, nephrotoxicity of calcineurin inhibitors may reflect a role of calcineurin within the renal vasculature.
Classification of Immunosuppressive Drugs
Immunosuppressive drugs include small-molecule drugs, depleting and nondepleting protein drugs (polyclonal and monoclonal antibodies), fusion proteins, intravenous immune globulin, and glucocorticoids (Table 1). Because of space limitations, intravenous immune globulin and glucocorticoids cannot be discussed in detail. In brief, intravenous immune globulin has multiple effects27 and is an important component of approaches to suppress alloantibody responses. Glucocorticoids act as agonists of glucocorticoid receptors, but at higher doses they have receptor-independent effects. Receptor-mediated effects are mainly transcriptional through DNA-binding and protein–protein interactions of the steroid-receptor complex, targeting transcription factors such as activator protein 1 and nuclear factor-B.28
Table 1. Classification of Immunosuppressive Therapies Used in Organ Transplantation or in Phase 2–3 Trials.
Most small-molecule immunosuppressive agents are derived from microbial products and target proteins that have been highly conserved in evolution. Small-molecule immunosuppressive drugs at clinically tolerated concentrations probably do not saturate their targets. For example, cyclosporine acts by inhibiting calcineurin but only partially inhibits calcineurin as used clinically.29 Without target saturation, the drug's effects are proportional to the concentration of the drug, which makes dosing and monitoring critical.
Depleting protein immunosuppressive agents are antibodies that destroy T cells, B cells, or both. T-cell depletion is often accompanied by the release of cytokines, which produces severe systemic symptoms, especially after the first dose. The use of depleting antibodies reduces early rejection but increases the risks of infection and post-transplantation lymphoproliferative disease and can be followed by late rejection as the immune system recovers. Recovery from immune depletion takes months to years and may never be complete in older adults. The depletion of antibody-producing cells is better tolerated than T-cell depletion, because it is not usually accompanied by cytokine release and immunoglobulin levels are usually maintained. However, depletion of antibody-producing cells is incomplete because many plasma cells are resistant to the available antibodies that target B cells, such as anti-CD20 antibody.
Nondepleting protein drugs are monoclonal antibodies or fusion proteins that reduce responsiveness without compromising lymphocyte populations. They typically target a semiredundant mechanism such as CD25, which explains their limited efficacy but the absence of immunodeficiency complications. These drugs have low nonimmune toxicity because they target proteins that are expressed only in immune cells and trigger little release of cytokines.
Small-Molecule Drugs
Azathioprine, which is derived from 6-mercaptopurine, was the first immunosuppressive agent to achieve widespread use in organ transplantation30 (Table 2). The developers of azathioprine, Gertrude Elion and George Hitchings, were acknowledged by a share of the 1988 Nobel Prize. Azathioprine is thought to act by releasing 6-mercaptopurine, which interferes with DNA synthesis. Other possible mechanisms include converting costimulation into an apoptotic signal.41 After cyclosporine was introduced, azathioprine became a second-line drug.
Table 2. Characteristics of Small-Molecule Immunosuppressive Drugs Used in Organ Transplantation or in Phase 2–3 Trials.
Calcineurin Inhibitors
Cyclosporine, a cornerstone of immunosuppression in transplantation for two decades, is in effect a prodrug that engages cyclophilin, an intracellular protein of the immunophilin family, forming a complex that then engages calcineurin.42 The adverse effects of cyclosporine, which are related to the concentration of the drug, include nephrotoxicity, hypertension, hyperlipidemia, gingival hyperplasia, hirsutism, and tremor. Cyclosporine can also induce the hemolytic–uremic syndrome and post-transplantation diabetes mellitus. Recent developments include monitoring of the peak cyclosporine levels two hours after administration to better reflect exposure to the drug.43,44A chemically modified cyclosporine, ISA(TX)247, is under development.45
Tacrolimus engages another immunophilin, FK506-binding protein 12 (FKBP12), to create a complex that inhibits calcineurin with greater molar potency than does cyclosporine. Initial trials indicated that there was less rejection with tacrolimus than with cyclosporine,46,47 but recent analyses suggest that in the current dosing strategies, the efficacy of cyclosporine is similar to that of tacrolimus.48,49 Tacrolimus resembles cyclosporine in that it can result in nephrotoxicity and the hemolytic–uremic syndrome, but it is less likely to cause hyperlipidemia, hypertension, and cosmetic problems and more likely to induce post-transplantation diabetes. Tacrolimus has been suspected of inducing more BK-related polyomavirus nephropathy than has cyclosporine in patients who have undergone kidney transplantation, especially when used with mycophenolate mofetil, but renal function may be better with tacrolimus.49 New developments include a preparation of modified-release tacrolimus to permit once-daily dosing.
The use of tacrolimus has increased steadily, and the drug is now the dominant calcineurin inhibitor, but most transplantation programs exploit the strengths of both tacrolimus and cyclosporine, depending on the risks in individual patients. Hypertension, hyperlipidemia, and the risk of rejection argue for tacrolimus, whereas a high risk of diabetes (e.g., older age or obesity) argues for cyclosporine.
Inosine Monophosphate Dehydrogenase Inhibitors
The use of inhibitors of purine synthesis for immunosuppression was based on the observation that inborn errors in this pathway produce immunodeficiency without damaging other organs, in contrast to errors in the purine salvage pathway.50,51 Mycophenolic acid inhibits inosine monophosphate dehydrogenase, a key enzyme in purine synthesis. Mycophenolate mofetil is a prodrug that releases mycophenolic acid, and in large-scale trials with cyclosporine, it was superior to azathioprine in preventing rejection of kidney transplants.52,53,54,55 Protocols using mycophenolate mofetil and calcineurin inhibitors improved patient survival and graft survival and reduced early and late allograft rejection.56,57 Mycophenolate mofetil has also been evaluated in heart transplantation.58 The drug has largely replaced azathioprine and is widely used because it is effective in combination with many other agents, simple to use without monitoring, and free from organ toxicity and cardiovascular risk. Its principal nonimmune toxic effects are gastrointestinal (mainly diarrhea) and hematologic (anemia, leukopenia). Mycophenolate mofetil may increase cytomegalovirus disease but in vitro manifests antipneumocystis activity.59 Enteric-coated mycophenolic acid has been introduced as an alternative to mycophenolate mofetil.60
Target-of-Rapamycin Inhibitors
Sirolimus61 and everolimus engage FKBP12 to create complexes that engage and inhibit the target of rapamycin but cannot inhibit calcineurin (Figure 2). Inhibition of the target of rapamycin blocks signal 3 by preventing cytokine receptors from activating the cell cycle. The principal nonimmune toxic effects of sirolimus and everolimus include hyperlipidemia, thrombocytopenia, and impaired wound healing. Other reported effects include delayed recovery from acute tubular necrosis in kidney transplants, reduced testosterone concentrations,62 aggravation of proteinuria, mouth ulcers, skin lesions, and pneumonitis. However, sirolimus and everolimus may reduce cytomegalovirus disease.63 Sirolimus and everolimus were developed for use with cyclosporine,64,65 but the combination increased nephrotoxicity, the hemolytic–uremic syndrome, and hypertension. Sirolimus has been combined with tacrolimus (e.g., the Edmonton protocol for pancreatic islet transplantation) to avoid the toxicity of sirolimus–cyclosporine combinations.66,67 However, a controlled trial in renal transplantation showed that sirolimus plus tacrolimus produced more renal dysfunction and hypertension than did mycophenolate mofetil plus tacrolimus,68 which indicates that sirolimus potentiates tacrolimus nephrotoxicity. Practitioners can reduce the toxicity of the combination of a target-of-rapamycin inhibitor and a calcineurin inhibitor by withdrawing one of the drugs. For example, withdrawing cyclosporine in patients in stable condition who are receiving the sirolimus–cyclosporine combination reduces renal dysfunction and hypertension, with a small increase in rejection episodes,69 which suggests a strategy for avoiding the toxic effects of calcineurin inhibitors (Table 3).
Table 3. Characteristics of Protein Immunosuppressive Drugs Used in Organ Transplantation or in Phase 2–3 Trials.
Sirolimus and everolimus may have antineoplastic and arterial protective effects. Since these agents slow the growth of established experimental tumors,70 they have potential applications in oncology. The possibility that sirolimus and everolimus can protect arteries is suggested by two observations: target-of-rapamycin inhibitors that are incorporated into coronary stents inhibit restenosis,71 and target-of-rapamycin inhibitors plus calcineurin inhibitors reduce the incidence of graft coronary artery disease associated with heart transplantation.63 But alternative explanations exist for both observations. Target-of-rapamycin inhibitors may suppress restenosis of mechanically dilated arteries by suppressing wound healing72 rather than by atherogenesis and may prevent graft coronary artery disease simply by preventing rejection. Potential arterial protective effects of sirolimus and everolimus must be weighed against the effects of the hyperlipidemia these drugs induce.73
Dihydroorotate Dehydrogenase Inhibitors
Dihydroorotate dehydrogenase is a key enzyme in pyrimidine synthesis. Leflunomide is a dihydroorotate dehydrogenase inhibitor that is approved for rheumatoid arthritis but is not widely used in transplantation.74 Its active metabolite, A77 1726, was modified to create FK778, which is in phase 2 trials in kidney transplantation. FK778 may have activity against BK-related polyomavirus and have a lower incidence of gastrointestinal effects than does mycophenolate mofetil, but its nonimmune toxic effects such as anemia must be evaluated.
FTY720
FTY720 is derived from myriocin, a fungus-derived sphingosine analogue. After phosphorylation, FTY720 engages lymphocyte sphingosine-1-phosphate receptors and profoundly alters lymphocyte traffic, acting as a functional sphingosine-1-phosphate antagonist.75 FTY720 drives T cells into lymphoid tissues and prevents them from leaving and homing to the graft. Despite low overall toxicity, FTY720 induces reversible bradycardia during the first doses,76 arousing concern about the potential for cardiac arrest when combined with the influences of other agents (e.g., general anesthetics or beta-blockers). FTY720 in combination with cyclosporine has completed phase 2 trials77 and entered phase 3 trials in renal transplantation.
Depleting Antibodies
Polyclonal antithymocyte globulin is produced by immunizing horses or rabbits with human lymphoid cells, harvesting the IgG, and absorbing out toxic antibodies (e.g., those against platelets and erythrocytes) (Table 3). As an induction agent, polyclonal antithymocyte globulin is usually used for 3 to 10 days to produce "profound and durable" lymphopenia that lasts beyond one year.78 In addition to immunodeficiency complications, toxic effects of polyclonal antithymocyte globulin include thrombocytopenia, the cytokine-release syndrome, and occasional serum sickness or allergic reactions. Rabbit preparations of polyclonal antithymocyte globulin (such as Thymoglobulin and ATG-Fresenius) are favored over horse polyclonal antithymocyte globulin because of greater potency.
Muromonab-CD3, a mouse monoclonal antibody against CD3, has been used for 20 years to treat rejection79 and for induction.80 Muromonab-CD3 binds to T-cell-receptor–associated CD3 complex and triggers a massive cytokine-release syndrome before both depleting and functionally altering T cells. Humans can make neutralizing antibodies against muromonab-CD3 that terminate its effect and limit its reuse. Prolonged courses of muromonab-CD3 increase the risk of post-transplantation lymphoproliferative disease.81 The use of muromonab-CD3 declined when newer small-molecule immunosuppressive drugs reduced rejection episodes. A trial of a humanized anti-CD3 monoclonal antibody in kidney transplantation82 was stopped. (A nonactivating humanized anti-CD3 monoclonal antibody is being developed to suppress beta-cell injury in patients with autoimmune diabetes mellitus of recent onset83 but is not currently used for transplantation.)
Alemtuzumab, a humanized monoclonal antibody against CD52, massively depletes lymphocyte populations. It is approved for treating refractory B-cell chronic lymphocytic leukemia but is not approved for immunosuppression in transplantation. A small study in renal transplantation that concluded that alemtuzumab induced "prope tolerance" (meaning near-tolerance)84 was not confirmed in later studies.85 Predictions that target-of-rapamycin inhibitors plus alemtuzumab would induce tolerance were also not confirmed. This combination is associated with rejection episodes, including antibody-mediated rejection.86 Side effects of alemtuzumab include first-dose reactions, neutropenia, anemia, and (rarely) pancytopenia and autoimmunity (e.g., hemolytic anemia, thrombocytopenia, and hyperthyroidism).87 The risks of immunodeficiency complications (infections and cancer) with alemtuzumab are unknown. Alemtuzumab is used off-label for induction in some centers, but controlled trials are needed to establish dosing, safety, and efficacy.
Rituximab (anti-CD20 monoclonal antibody) eliminates most B cells and is approved for treating refractory non-Hodgkin's B-cell lymphomas, including some post-transplantation lymphoproliferative disease in organ-transplant recipients. Rituximab is used off-label in combination with maintenance immunosuppressive drugs, plasmapheresis, and intravenous immune globulin to suppress deleterious alloantibody responses in transplant recipients. Although plasma cells are usually CD20-negative, many are short-lived and require replacement from CD20-positive precursors. Thus, depletion of CD20-positive cells does reduce some antibody responses. CD20-positive B cells can act as secondary antigen-presenting cells, which raises the possibility that rituximab can ameliorate T-cell responses. Off-label applications for rituximab include treatment of antibody-mediated rejection and possibly severe T-cell–mediated rejection88 and suppression of preformed alloantibody before transplantation. Again, controlled trials are needed.
Nondepleting Antibodies and Fusion Proteins
Daclizumab and Basiliximab
The anti-CD25 monoclonal antibodies daclizumab and basiliximab are widely used in transplantation for induction in patients who have a low-to-moderate risk of rejection. Because expression of CD25 (interleukin-2 receptor chain) requires T-cell activation, anti-CD25 antibody causes little depletion of T cells. Anti-CD25 antibody is moderately effective since it reduces rejection by about one third when used with calcineurin inhibitors and has minimal toxic effects.89,90,91,92
LEA29Y
LEA29Y is a second-generation cytotoxic-T-lymphocyte–associated antigen 4 (CTLA-4) immune globulin that is a fusion protein combining CTLA-4 (which engages CD80 and CD86) with the Fc portion of IgG. Results of a phase 2 trial in patients undergoing renal transplantation who are receiving mycophenolate mofetil, glucocorticoids, and anti-CD25 antibody are available in abstract form (www.atcmeeting.org/2004). In this trial with six months of follow-up, the effect of repeated administration of LEA29Y was similar to that of cyclosporine in preventing rejection. The LEA29Y trial introduces the concept of long-term use of nondepleting protein immunosuppressive agents to reduce reliance on toxic small-molecule immunosuppressive drugs.
Additional Drugs
Many of the critical steps that are depicted in Figure 1 can be targeted by small molecules or proteins to create new drugs.93 Potential targets for small-molecule drugs include those previously discussed (e.g., calcineurin) as well as others (e.g., tyrosine kinases, protein kinase C, MAP kinases such as Jun N-terminal kinase, phosphoinositide-3-kinase, and chemokine receptors). Potential targets for protein drugs include many membrane proteins.
Janus kinase 3 (JAK3) inhibitors39,40 illustrate how small-molecule immunosuppressive drugs are developed. JAK3, a tyrosine kinase associated with the cytokine receptor chain, participates in the signaling of many cytokine receptors (interleukin-2, 4, 7, 9, 15, and 21) (Figure 1). JAK3 inhibitor CP-690,55039 was developed by screening a chemical library and modifying candidate compounds to produce an oral agent that is immunosuppressive in rodents and nonhuman primates. One adverse effect is anemia, perhaps reflecting activity against Janus kinase 2, which is needed for erythropoietin action.
Protocol Development and Emerging Issues
For two decades, the options for immunosuppressive drugs were initial induction with the use of protein immunosuppressive therapy; preadaptation maintenance therapy with three drugs — a calcineurin inhibitor, a second line of drugs (azathioprine and now mycophenolate mofetil), and glucocorticoids; and postadaptation therapy with the same combination of drugs at lower doses. Rejection was reversed with high-dose steroids or depleting antibodies. Now hundreds of potential combinations exist, and many new protocols have emerged, often including a reduced reliance on glucocorticoids94 and calcineurin inhibitors. Some examples are listed in Table 4. Developing evidence-based approaches to this confusing choice of protocols presents a challenge.
Table 4. Examples of Current and Experimental Immunosuppressive Drug Protocols.
Progress in the control of early and late rejection and in managing infections such as cytomegalovirus has improved both the survival of patients and the function of grafts.57,100,101 For example, in kidney transplantation, the estimated glomerular filtration rate has improved102 and is stable in many patients, rather than slowly deteriorating, as in the past.57 This raises the hope that many organ transplantations that are performed today represent a permanent cure for end-stage organ failure.
But concerns temper this optimism. Outcomes are not continuing to improve,103 and the rate of late graft loss remains excessive. For example, in the United States each year, end-stage kidney failure develops in 4500 patients who have undergone kidney transplantation, a finding that highlights transplant failure as a major cause of end-stage renal disease.104 Patients who have undergone liver transplantation have an excessive recurrence rate of hepatitis; coronary artery disease develops in some patients with transplanted hearts; and bronchiolitis obliterans often develops in patients with transplanted lungs.105,106 The rate of premature death with functioning allografts remains excessive, in part because of cardiovascular and other complications of immunosuppression.
Nonimmune and immunodeficiency complications of transplant immunosuppression should be reduced. The major nonimmune toxic effects are nephrotoxicity, hypertension, hyperlipidemia, diabetes mellitus, and anemia. Five years after surgery, serious renal injury is present in 7 to 21 percent of patients who have undergone nonrenal transplantation,107 and end-stage kidney failure develops in many patients. The toxic effect of calcineurin inhibitors is an important contributor to the problem of renal failure. Post-transplantation diabetes mellitus develops after three years in 24 percent of patients who have undergone renal transplantation.108 Hyperlipidemia109 and anemia110 are common and undertreated. Options for reducing toxicity include choosing more selective drugs, avoiding toxic combinations, and maintaining vigilance for toxic effects.
Cancers111 and infections that are induced by transplantation remain frequent, with infections now exceeding rejection in pediatric transplant recipients.112 Choosing more selective drugs can reduce these risks. For example, anti-CD25 antibody has little effect on the risk of infection and post-transplantation lymphoproliferative disease.25 New protocols must emphasize reducing the rates of cancer and infection rather than simply lowering the rate of rejection.
New immunosuppressive drug applications and protocols113 are emerging without adequate trials to establish dosing, safety, and efficacy. Examples are the regimens of induction with alemtuzumab or radical minimization of maintenance immunosuppression. Moreover, the quality of transplantation trials is suboptimal.114 One problem is that the decline in the incidence of rejection, the end point in most trials, now limits the evaluation of new agents.115 New composite end points could incorporate organ function and drug toxicity or emerging laboratory measurements of immune mechanisms.
Optimizing outcomes requires long-term follow-up by knowledgeable caregivers who recognize and react to changes. Allografts with deteriorating function should not be dismissed as instances of "chronic rejection"; instead, the source of injury should be diagnosed (e.g., rejection that is T-cell–mediated or antibody-mediated, recurrent disease, drug toxicity, or infection).116 The assumption must be that new deterioration reflects new injury, not an inexorable consequence of an earlier injury. The identification of mechanisms of injury may be rewarded by the arresting of further deterioration.
Robust tests for rejection that is T-cell–mediated or antibody-mediated would change clinical management and clinical trials (e.g., microarray analysis of gene expression in biopsy specimens).11 Measurement of immune responses could guide transplantation management in the same way that measurement of disease activity guides other fields (e.g., the measurement of lipid levels in the management of hyperlipidemia).
Interest in suppressing alloantibody responses is growing. Emerging evidence links alloantibody to late graft deterioration,19 and transplantation is increasingly offered to patients who have previously been excluded by existing alloantibody, including ABO blood-group barriers.117 Options include the optimization of baseline immunosuppression, the administration of rituximab or intravenous immune globulin, and plasmapheresis, but new strategies are needed.
Pharmacogenomics offers possibilities for individualizing immunosuppression, an important goal with respect to toxic drugs with narrow therapeutic indexes.118,119 For example, CYP3A (cytochrome P-450-3A) allele CYP3A5*1, which is associated with increased CYP3A5 levels, is present in 70 to 80 percent of blacks but in only 5 to 10 percent of whites.120 Since CYP3A5 genotyping can be used to predict slower achievement of target tacrolimus levels and earlier rejection,121 it could help reduce rejection in black patients.
For most patients, no practical method of achieving true tolerance to HLA-incompatible organ transplants is at hand. True tolerance is durable antigen-specific unresponsiveness in an immunocompetent host that is induced by previous exposure to the antigen. The only clinical strategy that currently meets this definition is stem-cell transplantation.122 The stability of the adaptation usually depends on immunosuppression or damage to the immune tissues. At some point, most immunosuppressive agents are billed as tolerogenic, an assertion that is typically followed by the realization that, among at least some patients, the immunologic tolerance is not durable after withdrawal of the drug therapy and recovery from its effects. Indeed, the first report of an immunosuppressive drug was entitled "Drug-Induced Immunological Tolerance."123 Many "tolerance trials"124 in fact use immunosuppression and are probably based on host–graft adaptation. Excellent immunosuppression with long-term clinical surveillance remains the best prospect for achieving the potential of transplantation to restore and maintain health.
Dr. Halloran reports having received lecture fees from Roche and Fujisawa.
I am indebted to Dr. Deborah James for her help in preparing the text and tables; to Drs. Karl Brinker, Arthur Matas, Sita Gourishankar, and Attapong Vongwiwatana for reviewing the manuscript; and to Pam Publicover for her assistance in the preparation of the manuscript.
Source Information
From the Division of Nephrology and Transplantation Immunology, University of Alberta, Edmonton, Canada.
Address reprint requests to Dr. Halloran at 250 Heritage Medical Research Centre, Edmonton, AB T6G 2S2, Canada, or at phil.halloran@ualberta.ca.
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