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Abstract

  1. Top of page
  2. Abstract
  3. Small Molecules
  4. Biologic Agents
  5. Antiadhesion Molecules: Alefacept
  6. Conclusions
  7. References

Significant advances have been made in post-kidney transplantation immunosuppression. The introduction of calcineurin inhibitors (CNIs) has led to a reduction in acute rejection and improved 1-year outcomes. However, long-term allograft survival has not improved. This may in part be due to the chronic nephrotoxicity of CNIs and negative effects on the cardiovascular and metabolic risk profile via worsening hypertension, diabetes, and dyslipidemia. New drug development now focuses on maintaining low rejection rates but maximizing long-term allograft survival and modulating the cardio-metabolic side effects seen with CNIs. Two small molecules are currently undergoing Phase 2 investigation. CP-690550 (tasocitinib) is a JAK 3 inhibitor, and AEB-071 (sotrastaurin) is a protein kinase C inhibitor. Two biologic agents are also undergoing development. Belatacept is a humanized antibody that blocks the T-cell co-stimulation pathway and has had promising results in both Phase 2 and 3 investigation. Alefacept is a humanized antibody that inhibits T-cell adhesion and is currently undergoing Phase 2 investigation. This article will review the mechanisms of these drugs and outline the available trial data results.

Changes in post-transplantation immunosuppression, including the introduction of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus in the 1980s and 1990s, respectively, as well as the development of enhanced antiproliferative agents such as mycophenolate mofetil (MMF) and the widespread use of induction agents, have contributed to lower acute rejection rates and improved 1-year outcomes. However, the improved short-term success has not translated into improved long-term renal allograft survival.1 Chronic allograft nephropathy (CAN), whether due to CNI toxicity or chronic antibody-mediated rejection, remains the most common cause of late allograft loss,2 while cardiovascular disease remains the leading cause of death post transplantation.3 Current immunosuppression protocols that rely on CNIs contribute to the progression of CAN and accelerate long-term allograft decline.4 CNIs also exert negative effects on the cardiovascular and metabolic risk profile by worsening hypertension, diabetes, and dyslipidemia.5–7

In this era of reduced acute rejection rates and improved short-term allograft survival, developing new drugs based on these outcome measures has become more difficult to demonstrate and achieve. Consequently, a shift in focus has occurred to the development of new immunosuppressive agents that will maintain similar excellent short-term outcomes but will maximize long-term allograft survival and modulate the cardio-metabolic side effects seen in CNI protocols. Although not a comprehensive review of all drugs currently undergoing clinical trials,8 this review will summarize new agents in Phase 2 or 3 development for rejection prophylaxis in de novo kidney transplant recipients with a focus on drugs that seem promising in terms of minimizing the long-term renal and cardio-metabolic side effects seen with CNIs.

Small Molecules

  1. Top of page
  2. Abstract
  3. Small Molecules
  4. Biologic Agents
  5. Antiadhesion Molecules: Alefacept
  6. Conclusions
  7. References

Small molecules are non-protein drugs that typically target intracellular pathways. Two small molecules, AEB-070 (sotrastaurin) and CP-690550 (tasocitinib) (TableI), are currently are undergoing Phase 2 investigation and will be discussed in detail.

Table I. Small molecules currently undergoing development.
Drug NameTargeted PathwayPhase of Development
CP-690550 (tasocitinib)JAK 3 inhibitor (signal 3)Phase 2a (complete; reported) Phase 2b (ongoing; reported)
AEB-070 (sotrastaurin)Protein kinase C inhibitor (signal 1 and 2)Two initial Phase 2 trials halted due to an increase in acute rejection. Current Phase 2 trial ongoing (AEB + mTOR inhibitor everolimus)

JAK 3 Inhibition: CP-690550 (Tasocitinib)

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that participate in the signaling of a broad range of cell surface receptors, particularly members of the cytokine receptor common gamma (cγ) chain family. There are four mammalian JAKs: JAK 1, 2, 3, and tyrosine kinase 2. Activation of JAK by ligand-receptor interaction results in signaling via the phosphorylation of cytokine receptors and the creation of docking sites for signaling proteins known as signal transducers and activators of transcription (STAT).9 JAKs catalyze STAT phosphorylation, which facilitates STAT dimerization, transport to the nucleus, and ultimately regulation of gene expression.9, 10

JAK 3, unlike the ubiquitous expression of other JAK subtypes, has a restricted tissue distribution, is found primarily on hematopoietic cells, and uniquely associates with the cγ chain.11 The importance of this signaling pathway is evident when one considers that mice and humans with genetic absence or mutation in either the cγ subunit or JAK 3 express defects in lymphoid development that give rise to a severe combined immunodeficiency syndrome phenotype.12

CP-690550 (CP), or tasocitinib, is a synthetic orally available JAK 3 inhibitor currently undergoing clinical trials. Given the structural similarities between JAK 2 and JAK 3, some cross-reactivity exists between JAK inhibitors. However, despite the intense potency of CP, as demonstrated in vitro with an interleukin-2 (IL-2)-mediated T-cell proliferation assay in which the 50% inhibitory concentration of CP was found to be only 11 ηM,13 it is remarkably specific for JAK 3 inhibition. This is an important feature of the drug as JAK 2 inhibition results in impaired hematopoiesis.14

Early studies utilizing a murine cardiac and a non-human primate (NHP) renal model demonstrated promising results when CP was used either alone or in combination with MMF.13, 15, 16 In one NHP study, 22 animals were treated with varied doses of CP to achieve a median 12-hour trough level ranging from 1 to 147 ng/mL compared with vehicle twice daily.15 The animal survival time was 83.2 ± 6.3 days and 18.8 ± 6.3 days for the highest and lowest levels of CP drug exposure, respectively. The most pronounced side effect was anemia. A follow-up of this trial in 11 NHPs was performed utilizing the combination therapy of CP and MMF twice daily versus MMF alone to target either the high or low CP levels as achieved in the first trial.16 The mean survival time was 23 ± 1 days and 75.2 ± 8.7 days in animals treated with MMF alone versus combination therapy, respectively. Additionally, the high CP level combination therapy animals had a significantly better survival rate. Based on these results, human trials were undertaken utilizing CP in kidney transplant rejection prophylaxis.

The results of a Phase 2a 6-month pilot study comparing two doses of CP (15 and 30 mg twice daily) with tacrolimus in de novo kidney allograft recipients has recently been published.17 All patients received IL-2 receptor antagonist induction, MMF, and corticosteroids. Patients enrolled in the high-dose CP group (CP-30) experienced a significantly higher rate of BK virus nephropathy (20%) as well as cytomegalo-virus (CMV) disease (20%). Consequently, the study protocol was amended after enrollment was complete such that patients receiving CP-30 underwent MMF discontinuation and a more rapid steroid taper. Probably as a result of these changes, the 6-month incidence of biopsyproven acute rejection (BPAR) was 5.3%, 21.1%, and 4.8% for low-dose CP (CP-15), CP-30, and tacrolimus, respectively. The 6-month estimated glomerular filtration rate (eGFR) was similar across the three groups; however, the 12-month extension study eGFR was 83.6, 77.6, and 73.3 mL/min for CP-15, CP-30, and tacrolimus, respectively. By month 12 there was no difference among the three groups with regard to hemoglobin concentration.

Given the promising results with CP-15, an additional Phase 2 trial has begun to evaluate the effectiveness of two different dosing strategies of CP compared with a cyclosporine-based regimen. Group 1 will receive the active comparator cyclosporine. Group 2 will receive CP-15 for months 1-6 and then CP-10 for months 7-12, while group 3 will receive CP-15 for months 1-3 followed by CP-10 for months 4-12. All three groups will also receive MMF and steroids. A 72-month extension study is also planned. Although CP is a promising alternative to a CNI-based regimen, the optimal therapeutic window of CP is yet to be determined. It also remains to be seen whether the cardio-metabolic side effect profile as well as the long-term allograft function achieved with CP will surpass those of CNIs.

PKC Inhibition: AEB-071(Sotrastaurin)

Protein kinase C (PKC) isoforms play an important role in intracellular signaling pathways. Activation of the T-cell receptor (signal 1) plus CD28 (signal 2) results in T-cell activation via PKC signaling and IL-2 production.18 The PKC family consists of at least 10 isoforms, with the isoforms PKC α, β, and θ playing a role in T-or B-cell signaling.19 PKCθ is largely restricted to T lymphocytes and mediates activation of the transcription factors activator protein-1 and nuclear factor (NF) κB, leading to down-stream IL-2 production. The importance of this pathway is illustrated by the fact that PKCθ knockout mice demonstrate impaired T-cell activation.20

AEB-071 (AEB), or sotrastaurin, is an oral low-molecular-weight compound that blocks early T-cell activation by selective inhibition of PKC. In vitro studies have shown that AEB primarily exerts its effects by inhibiting classical and novel PKC isoforms with the resultant blockade of T-cell activation and IL-2 production. Unlike CNIs, AEB exerts minimal effect on nuclear factor of activated T-cells (NFAT) and on cytokine and growth factor-induced cell proliferation.21 With a mechanism for blocking T-cell activation that is distinct from CNIs, there has been tremendous excitement over the possibility that AEB may not possess the toxicities usually associated with inhibition of the calcineurin pathway.

Initial Phase 2 trials evaluating the effectiveness of AEB were disappointing and were stopped early due to an increase in acute rejection in AEB-treated groups. The complete results of one of these Phase 2 trials have recently been published.22 In this trial patients were initially placed on AEB and steroids plus either standard exposure tacrolimus (SET) or reduced exposure tacrolimus (RET). At 3 months patients were eligible for conversion to mycophenolic acid (MPA) in place of tacrolimus. The control group consisted of a standard regimen of tacrolimus, MPA, and steroids. During the 3-month precon-version period, both AEB groups demonstrated comparable efficacy to the control group in regard to the composite endpoint of BPAR, graft loss, or death. However, post conversion there was a significant increase in the composite endpoint in AEB-treated patients compared with the control group, driven primarily by an increase in BPAR. The Kaplan-Meier estimate demonstrated a 4.6%, 40.2%, and 32.4% rate of BPAR by study end in the control, SET, and RET groups, respectively. The incidence of new-onset diabetes in the control group was 14.9% compared with 6.7% and 7.7% in the SET and RET groups, respectively. However, the median eGFR was not significantly different for the two study groups compared with the control group at any time point.

A second Phase 2 study utilized a de novo CNI-free regimen of AEB, MPA, and steroids and was compared with the control group of tacrolimus, MPA, and steroids. Again, a higher acute rejection rate was noted in the AEB group, and the trial was stopped early. Given the disappointing results in the conversion trial and in the CNI-free trial, interest has shifted to utilizing AEB in combination with everolimus (an mTOR inhibitor). A Phase 2 trial evaluating AEB in combination with everolimus is under way. The study hypothesis suggests that mTOR inhibition in addition to AEB will provide more effective immunosuppression compared with MPA in the absence of a CNI.

Biologic Agents

  1. Top of page
  2. Abstract
  3. Small Molecules
  4. Biologic Agents
  5. Antiadhesion Molecules: Alefacept
  6. Conclusions
  7. References

New biologic agents in development are likely to usher in a paradigm shift in the design and delivery of immunosuppression for organ transplantation. Thus far the use of biologic agents has been limited to perioperative induction as well as the treatment of rejection. In contrast, new biologic agents are being developed expressly for maintenance therapy in an attempt to improve the specificity of long-term immunosuppression without the requirement for and toxicity of daily agents such as CNIs. Chronic biologic therapy has been made possible largely through the perfection of protein humanization and the virtual elimination of long-term immunogenicity.23 At least two reasons underlie the shift in focus in the use of biologic agents. First, the targets of new biologics are typically non-depletional, thus lending themselves to chronic therapeutic use without undue global immunodeficiency. Second, biologics are expensive to produce, making economic models that rely on short-term use difficult to develop. Long-term use allows for the development and implementation of a financially viable model for production and distribution in small patient populations. The biologic that best epitomizes this paradigm shift is belatacept.24 Another biologic that appears promising is alefacept25, 26 (TableII).

Table II. Biologic agents currently undergoing development.
Drug NameTargeted ReceptorPhase of Development
BelataceptCo-stimulation blockade (CD28/CD80-86)Phase 2 (complete; reported) Phase 3 (complete; reported)
AlefaceptAnti-CD2Phase 2 (complete; not reported)

Co-Stimulation Blockade: Belatacept

The CD28/B7 (CD80 and 86) co-stimulation pathway is an essential signal for T-cell activation. After 25 years of research the fusion receptor protein CTLA4-Ig (abatacept), a competitive antagonist for CD28 blocking CD80/CD86 binding, was approved for human use in the treatment of rheumatoid arthritis.27 Early experiments with co-stimulation blockade in transplantation were mixed. Prolongation of graft survival using co-stimulation blockade in rodent transplantation experiments could not be reproduced in NHPs.24, 28 CTLA4-Ig did not achieve as good affinity to CD86 compared with CD80 and was the likely cause of failure in a more stringent animal model.24

Belatacept, a re-engineered CTLA4Ig with two amino acid substitutions in the CTLA4 binding domains, binds CD80 2-fold better and CD86 4-fold better than CTLA4-Ig and has a 10-fold more potent inhibition of T-cell activation in vitro versus CTLA4-Ig.29 The in vitro superiority of belatacept in blocking T-cell responses was confirmed by better survival of renal allografts in an NHP model.24 In these experiments, a CNI-free regimen with belatacept and a combination of an anti-IL-2 receptor antibody and maintenance therapy with MMF and steroids resulted in marked prolongation of the survival of renal allografts.24

These findings led to the design of a Phase 2 multicenter clinical trial comparing the safety and efficacy of a more intensive (MI) and a less intensive (LI) regimen of belatacept compared with cyclosporine.30 In this trial belatacept performed with equivalent efficacy to cyclosporine and was associated with better renal function and histology. Lipid levels and blood-pressure values were similar or slightly lower in the belatacept groups. However, the development of post-transplant lympho-proliferative disorder (PTLD) arose as a safety concern. Three cases of PTLD were identified in the MI group versus none in the LI or cyclosporine arms. Recently, the results of the Phase 2 long-term extension trial have been published.31 Continued follow-up of 128 of the original 218 patients demonstrated that no patients who were treated with belatacept and 1 patient who was treated with cyclosporine developed PTLD during the extension follow-up period. Additionally, renal function was superior in belatacept-treated patients. There was an average calculated GFR of 77.2 ± 22.7 mL/min per 1.73 m2 versus 59.3 ± 15.3 mL/min per 1.73 m2 at 60 months in the belatacept- versus the cyclosporine-treated patients, respectively.

Given these promising results, two Phase 3 trials were undertaken, and data are available. The first trial, Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial (BENEFIT)32 is a 3-year, randomized, Phase 3 trial. Adult patients receiving a living-donor kidney or standard criteria deceased-donor kidney were eligible. Patients received one of three regimens: MI belatacept, LI belatacept, or cyclosporine. Patients in all treatment arms received basiliximab induction and were maintained on MMF and cortico-steroids. At 1 year patients enrolled in the MI, LI, and cyclosporine treatment groups had 95%, 97%, and 93% patient and graft survival, respectively. The mean measured GFR was 65, 63.4, and 50.4 mL/min/1.73 m2 in the MI, LI-, and cyclosporine-treated patients, respectively (p < 0.0001 for both MI and LI versus cyclosporine).

The prevalence of chronic allograft nephropathy on protocol biopsies was lower in belatacept-treated patients compared with cyclosporine-treated patients. There was a higher incidence of acute rejection at 12 months in the belatacept-treated groups compared with the cyclosporine-treated group (22% MI; 17% LI; 7% cyclosporine). The incidence of acute rejection met the non-inferiority cutoff for LI versus cyclosporine groups but not for MI versus cyclosporine groups. Almost 100% of rejections occurred within the first 6 months post transplantation. Interestingly, the mean measured GFR at month 12 was higher in belatacept-treated patients with acute rejection compared with cyclosporine-treated patients without acute rejection. Belatacept-treated patients had a significantly lower mean blood pressure (MI 133/79 mmHg; LI 131/79 mmHg) compared with cyclosporine-treated patients (139/82 mmHg). By 12 months, 1, 2, and 1 patient in the MI, LI, and cyclosporine groups developed PTLD, respectively. Additionally, after month 12 two additional patients in the MI group developed central nervous system (CNS) PTLD. Four of the 6 patients who developed PTLD had known risk factors. One patient had Epstein-Barr virus (EBV) negative serology pre-transplant, 1 patient received lymphocyte-depleting therapy as treatment for an acute rejection, and 2 patients had both EBV-negative serology and received lymphocyte-depleting therapy. Lastly, 2 patients with EBV-negative serology received transplants from EBV-seropositive donors.

The second Phase 3 trial, Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial—EXTended criteria donors (BENEFIT—EXT)33 is a 3-year, randomized trial in patients who received a kidney transplant from an extended-criteria donor. Patients were treated with basiliximab induction, MMF, and corticosteroids. Patients were randomized to receive MI, LI, or cyclosporine. Both belatacept regimens were non-inferior to cyclosporine on the primary endpoint of patient and graft survival. The prevalence of biopsy-proven CAN was similar between the three groups. The mean measured GFR at 12 months was 52.1, 49.5, and 45.2 mL/min/1.73 m2 for the MI-, LI-, and cyclosporine-treated groups, respectively and was significantly better in the MI-treated patients versus the cyclosporine-treated patients (p = 0.0083) but not for the LI group compared with cyclosporine (P = 0.1039). There was no difference in the incidence of acute rejection among the three groups. Mean systolic and diastolic blood pressure was lower for both belatacept groups compared with the cyclosporine-treated group.

The incidence of NODAT was significantly lower in the MI group compared with the cyclosporine group; however, there was not a significant difference in NODAT in the LI group compared with the cyclosporine group. One patient in the MI group and 2 patients in the LI group developed PTLD during the 12-month follow-up period. One additional patient in each of the belatacept groups developed PTLD after month 12. No patients in the cyclosporine group developed PTLD. Three of the 5 PTLD patients had negative EBV serology pre-transplant. None of the patients who developed PTLD were exposed to lymphocyte-depleting therapy.

Thus far belatacept is the only agent in clinical development that has demonstrated an advantage in renal preservation and a trend toward an improved cardio-metabolic profile when compared with a CNI. So far the optimal use of belatacept seems to be in a lower intensity regimen utilized in low immunologic risk patients who are EBV positive. It is becoming clearer that when used in the right subset of patients, belatacept seems to confer improved long-term renal allograft survival compared with CNIs without additional safety concerns.

Antiadhesion Molecules: Alefacept

  1. Top of page
  2. Abstract
  3. Small Molecules
  4. Biologic Agents
  5. Antiadhesion Molecules: Alefacept
  6. Conclusions
  7. References

The upregulation of T-cell activation is affected not only by CD28/CD80-86-mediated co-stimulation but also by the interactions between the CD2 receptor, present on natural killer (NK) and T cells, and the more ubiquitously expressed cell surface glycoprotein lymphocyte-associated function-1 (LFA-1). The interactions between LFA-1 and its ligands have been shown to be important in the recruitment of leukocytes to the site of inflammation, in stabilizing the interaction between T cells and antigen presenting cells, and in providing co-activation signals.34 Experimental models of transplantation have shown that inhibitors of LFA-1 are immunosuppressive and prolong graft survival.34

Alefacept is a human LFA-3-IgG1 fusion protein that binds to CD-2 receptors on T lymphocytes, thereby blocking the interaction between LFA-3 and CD-2 and interfering with T-cell activation.35, 36 In 2003, alefacept was approved by the Food and Drug Administration (FDA) for the treatment of psoriasis.37 Based on its effectiveness in modulating the immune response in psoriasis, studies were undertaken to determine its potential role as an adjunctive agent in organ transplantation. Alefacept has been shown to delay rejection in non-human primate cardiac transplantation and has recently been shown to have synergistic potential when used with co-stimulation blockade and/or sirolimus-based regimens in non-human primates.38, 39 A Phase 2, four-arm parallel group, multi-center, open-label trial to determine the safety and efficacy of alefacept in de novo kidney transplant recipients is currently under way. The treatment regimens for the four groups are listed in TableIII. Whether alefacept has the potential to be part of a CNI-free immunosuppression regimen remains to be determined.

Table III. Phase 2 trial of alefacept: 4 study arms.
Study ArmsAssigned Interventions
  1. Abbreviations: CNI, calcineurin inhibitor; MMF, mycophenolate mofetil.

1. Comparator regimen (active comparator: tacrolimus)Tacrolimus, basiliximab, MMF, and steroids
2. CNI reductionAlefacept, tacrolimus, MMF, and steroids
3. MMF replacementAlefacept, tacrolimus, and steroids
4. Alternative alefacept dosingAlefacept, tacrolimus, MMF, and steroids

Conclusions

  1. Top of page
  2. Abstract
  3. Small Molecules
  4. Biologic Agents
  5. Antiadhesion Molecules: Alefacept
  6. Conclusions
  7. References

The success and pitfalls of CNIs have led to the development of new immunosuppressive agents that can duplicate the low rejection rates of CNIs and minimize their untoward renal and cardio-metabolic side effects. Multiple targets exist on the immune cell surface as well as in down-stream intracellular mechanisms to develop novel ways to promote immunosuppression. Several promising drugs are undergoing Phase 2 and 3 clinical trials, with more on the horizon still in early development. The story of immunosuppression development is still being told. As a community, the challenge will be to encourage rigorous trial conduct and insist upon data to drive the regimens of the future.

References

  1. Top of page
  2. Abstract
  3. Small Molecules
  4. Biologic Agents
  5. Antiadhesion Molecules: Alefacept
  6. Conclusions
  7. References