What's Next in the Pipeline

Authors


* Corresponding author: Flavio Vincenti, Flavio.Vincenti@ucsfmedctr.org

Abstract

The first decade of the new millennium has been disappointing for transplant therapeutics: no new immunosuppression agents have been approved. Several high profile drugs and biologics failed the rigors of clinical trials or had disappointing preclinical results (FTY720, FK778, anti-CDI54, anti-IL15, anti-CD28, R3421). Several challenges face the industry and clinical investigators in bringing novel drugs to the clinic including the difficulty in targeting new endpoints for toxicities or chronic allograft disease since acute rejection has been reduced to below 15% as well as the Food and Drug Administration insistence of excluding the use of immunosuppression regimens embraced by the transplant community in control arms of clinical trials. Currently six new agents, 3 small molecules (ISA247, a semisynthetic analogue of cyclosporine; AEB071, a protein kinase C isoforms inhibitor; CP 690,550, a selective Janus kinase inhibitor) are in phase II trials and 3 biologics (belatacept, a second generation CTLA4Ig; efalizumab, a humanized antiCD11a [LFA1] monoclonal antibody; and alefacept, a LFA3-IgG1 fusion receptor protein) are in phase II/III clinical trials. The preclinical pipeline is not only full but promises to address previously neglected targets and fulfill unmet medical needs in transplant therapeutics.

Introduction

This first decade of the new millennium, in contrast to the 1990s, has not witnessed the approval of any new medications specifically indicated for organ transplantation. In a review of the transplantation pipeline published in the American Journal of Transplantation in 2002, several drugs and biologics were identified as promising candidates for clinical investigation which subsequently failed the rigors of clinical development: FTY720, a sphingosine1 phosphate agonist, FK778, a pyrimidine synthesis inhibitor and monoclonal antibodies (mAbs) to CD154 and interleukin (IL) 15, all failed to advance to the clinic as anticipated (1). Another recent casualty is R3421 (previously referred to as BCX 4208), a promising purine nucleoside phosphorylase inhibitor that failed in psoriasis, thus plans for clinical development in transplantation were also shelved.

The lack of novelty in transplant therapeutics is reflected in the trial registry clinicaltrials.gov. Out of the more than 750 clinical studies in transplantation (any organ) listed as actively enrolling patients, only 8 are investigating new drugs (belatacept, AEB071, CP690550, ISA247). Indeed, most are studying antibiotics, complications, or off-label use of existing drugs. This latter category, off-label drug use, refers to the use of a drug approved for one indication but used for another indication based on physician discretion, and represents a substantial trend in transplantation. Many drugs established as efficacious in indications such as oncology (e.g. rituximab and alemtuzumab) or autoimmunity (e.g. leflunomide, alefacept and efalizumab) are now being proposed for use in transplantation. This route has enormous potential with over 400 non-transplant trials investigating agents with potential immune mechanisms of action. However, it presents a significant challenge to the transplant community. Specifically, only a few of these drugs will likely be tested in rigorous trials of the sort mandated to gain FDA approval for a specific indication in transplantation, but their use for conditions such as antibody mediated rejection, desensitization, induction or maintenance therapy, or BK nephropathy will grow based on investigator initiated trials which, while inventive and valuable, invariably remain under-powered to draw firm comparative conclusions. Thus, the short-term pipeline remains rather thin for specific transplant development, but rich in agents targeting relevant cell surface molecules and intracellular pathways. Furthermore, a new wave of agents is emerging in the preclinical pipeline suggesting that the coming decade may see a reversal of the most recent trend of paucity of drugs targeted for transplantation.

Several challenges impede both industry and clinical investigators in bringing novel drugs to the clinic and these were discussed in a recent editorial in this journal (2). The incidence of acute rejection has been reduced to such a low level that it can no longer be used as a yardstick in clinical trials (3). While toxicities associated with current regimens continue to limit long-term outcome, they are difficult to standardize as endpoints per se. There is a consensus that biomarkers should be used in evaluating the impact of novel drugs, however none are yet fully validated. Nonetheless, it is clear that new drugs with novel mechanisms of action and devoid of the toxicities of the current immunosuppressive drugs will be needed to replace the current calcineurin inhibitor (CNI)-based regimens in order to improve both long-term patient and graft survival.

(Disclosure: the authors have participated in preclinical and clinical trials with the following agents: CP690,550, AEB071, anti-CD154L, belatacept, Gen2 21955, efalizumab, alefacept and alemtuzumab.)

Small Molecules

Non-protein drugs targeting intracellular pathways are typically referred to as small molecules. Three small molecules, ISA247, AEB247 and CP690, 550 are currently in various stages of clinical development in renal transplantation (Table 1).

Table 1.  Small molecules in clinical trials
DrugPathwayPhase of StudyMaintenance RegimenResults/1° Endpoint
  1. CS = corticosteroids; Tac = tacrolimus; MPS = mycophenolic sodium; MMF = mycophenolate mofetil.

ISA247 (Isotechnika)Calcineurin inhibitor (signal one)Phase IIThree dose levels of ISA247 versus Tac. All patients are treated with MMF + CS1° Endpoint: similar efficacy and renal function in all treatment groups
AEB07 (Novartis)Protein kinase C (signal one and two)Phase IIAEB + Everolimus + CS1° Endpoint: acute rejection
Phase IIAEB + Tac + CS with Tac withdrawal at 3 months versus Tac + MPS + CSStudy halted because of an increase in acute rejection after Tac withdrawal.
Phase IIAEB + MPS + CS versus Tac + MPS + CSStudy halted due to increase in acute rejection
CP 690,550 (Pfizer)Janus kinase 3 (signal three)Phase IiaCP 690,550 15 mg or 30 mg bid + MMF + CS versus Tac + MMF + CSComparable efficacy between all treatment groups. More infections in high CP690, 550 group.
Phase IIbClinical trial in progress with 2 doses CP 690,550 15 mg and 10 mg bid with MMF + CS1° Endpoint: acute rejection

ISA247

ISA247 is a novel oral semisynthetic structural analogue of cyclosporine that has been modified at the first amino acid residue of the molecule (4). ISA247 has been shown to be more potent than cyclosporine in an in vitro calcineurin inhibition assay and in vivo in rat heterotopic heart transplantation. Furthermore, studies in rats, rabbits, dogs and cynomolgus monkeys suggest that ISA247 has no nephrotoxicity (5). Thus, enhanced efficacy and decreased nephrotoxicity are stimulating the clinical development of ISA247.

A 6-month phase II trial in renal transplant recipients has randomized patients to three dose levels of ISA247 (0.4, 0.6, or 0.8 mg/kg twice daily) versus tacrolimus in combination with an interleukin-2 receptor (IL-2R) antagonist, mycophenolate mofetil (MMF) and corticosteroids (6). Results from 334 patients have shown a low incidence of rejection in all groups (10.7%, 9.1% and 2.3% in the ISA247-treated patients by dose vs. 5.8% in the tacrolimus-treated patients). Renal function was similar in all treated patient groups with new onset diabetes observed less frequently in ISA247 treated patients. Based on the promising phase II results, ISA247 appears likely to advance to phase III trials.

Janus kinase inhibitors/CP-690550

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. Mammals have four JAKs: JAK1, JAK2, JAK3 and tyrosine kinase 2. Ligand-receptor-induced activation of JAKs initiates signaling by phosphorylating cytokine receptors and creating docking sites for signaling proteins known as signal transducers and activators of transcription (STATs) (Figure 1) (7). JAKs catalyze STAT phosphorylation that facilitates STAT dimerization, transport to the nucleus and gene regulation (7).

Figure 1.

Novel biologics and small molecules targeting cell surface receptors and intracellular pathways of the T cell. PKC = Protein Kinase C, CN = Calcineurin.

Compared to other members of the JAK family, JAK3 has special features that make it a potentially attractive target for immunosuppression. First, JAK3 has a restricted tissue distribution and is found primarily on hematopoetic cells. Second, JAK3 associates specifically with the cγ chain, which is shared by tissue receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Moreover, mice and human beings with the genetic absence or mutation of JAK3 express defects in lymphoid cell development that give rise to a severe combined immunodeficiency syndrome phenotype (8). Therefore, blocking JAK3 is likely to depress the immune response and afford some degree of selectivity. In contrast, JAK2 is associated with downstream signaling of the erythropoietin receptor and inhibition at this site evokes impaired hematopoiesis, leading to anemia. Given the structural similarities between JAK2 and JAK3, cross-reactivity exists to some extent with most JAK inhibitors. The side effect profiles thus largely depend on the degree of JAK3 selectivity. Another related intracellular kinase, Syk, facilitates Fc receptor signaling and has been suggested to be a potential target for B cell or macrophage inhibition. Regardless, this class of agents targets T-cell activation without interruption of T-cell signaling.

CP-690550, the only JAK3 inhibitor currently in clinical trials, has demonstrated improved renal allograft survival in murine and non-human primate (NHP) studies (9,10). CP-690550 was well tolerated, with no signs of nephrotoxicity, malignancy, or delay in wound healing.

In a 6-month phase II trial, 61 adult renal transplant patients were randomized to one or two dose levels of CP-690550 (15 mg or 30 mg twice daily) versus tacrolimus in combination with an IL-2R antagonist, MMF and corticosteroids. The incidence of biopsy-proven acute rejection and renal function at 6 months was comparable in all three groups, with no graft loss, deaths, or malignancies (11). Patients in the high-dose CP-690550 group developed more infections, cytomegalovirus (CMV) disease and BK-associated nephropathy. A new 12-month phase II study, randomizing patients to one or two lower dose levels of CP-690550 versus cyclosporine is ongoing (Table 1).

Given the preliminary success of CP-690550, other JAK inhibitors are being developed in various stages of preclinical study. In general, variations on this theme are being pursued with an eye on improved JAK2 avoidance to decrease hematological side effects.

PKC inhibition/AEB071

Protein kinase (PKC) isoforms play a key role in signaling pathways downstream of the T-cell receptor (signal 1) and CD28 (signal 2), and thereby block early T-cell activation (12). The PKC family can be divided into three categories (conventional or classical, novel and atypical isoforms) on the basis of their cofactor requirements. Based on in vivo studies of PKC isoenzyme-selective knockout mice, three of the isoforms, PKC α, β and θ, have been shown to be important in T- and B-cell signaling. The best characterized is the PKC member, PKCθ, which is largely restricted to T lymphocytes and mediates activation of the transcription factors activator protein-1 and nuclear factor (NF) κB, leading to downstream IL-2 production. The PKCθ knockout mice demonstrate impaired T-cell activation (13).

AEB071 (AEB) is a new oral low molecular weight compound that effectively blocks early T-cell activation by selective inhibition of PKC and therefore has a different mechanism of action from that of the CNIs. AEB exerts its immunosuppressive effect by inhibiting classical (α, β) and novel (δ, ε, η, θ) PKC isoforms. In vitro studies have shown that AEB blocks T-cell activation and IL-2 production with minimal effect on nuclear factor of activated T-cells (NFAT) and on cytokine and growth factor-induced cell proliferation (14). Therefore, AEB has a mechanism for blockade of T-cell activation that is independent of that of CNIs and thus may lack the toxicities associated with inhibition of the calcineurin pathway (Figure 1).

Preclinical studies have shown that monotherapy with AEB, or in combinations with a variety of adjunct immunosuppression agents, prolongs rat heterotopic heart transplant survival and cynomolgus monkey renal allograft survival (15–18). Preliminary results from a 14-day multiple-dose study in psoriasis patients demonstrated a dose-dependent improvement in the severity of psoriatic plaques, with good tolerability (19).

Three separate phase II dose-finding trials with AEB were launched in the past year (Table 1). The first study attempted CNI withdrawal (tacrolimus and AEB for 3 months followed by discontinuation of tacrolimus and institution of mycophenolic sodium [MPS]) while the second study was designed to be CNI free (AEB with MPS). Both trials were halted because of an increase in acute rejection (in the first trial rejections occurred after tacrolimus was discontinued). The third trial, conducted only in European centers, AEB was paired with everolimus and steroids and was compared to a control arm consisting of tacrolimus, MPS and steroids. This study is ongoing. Since in the first trial the combination of CNI and AEB (prior to withdrawal of CNI) appeared to be as effective as the CNI and MPS control arm, it is possible that AEB will be developed as an adjunct to CNI based regimens.

Biologic Agents

The new biologic agents in clinical development are likely to usher in a paradigm shift in the design and delivery of immunosuppressive regimens for organ transplantation. Traditionally, the use of biologics has been limited to perioperative induction involving either nonspecific depletion of T lymphocytes with agents including OKT3, polyclonal antilymphocyte preparations and more recently alemtuzumab, or non-depletional immunomodulation with the anti-interleukin 2 receptor mAbs. In contrast, the new generation of biologics 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 oral agents (especially CNIs and steroids) (20). Chronic biologic therapy has been made possible largely through the perfection of protein humanization and the virtual elimination of long-term immunogenicity (21). Two reasons underlie this shift. First, the targets of the new biologics are in general, non-depletional thus lending themselves to chronic therapeutic use without undue global immunodeficiency. Second, biologics are expensive to manufacture and it is difficult to develop a financially viable model for their short-term use. Long-term use allows the potential to recoup manufacturing expense in small patient populations. The prototype of the paradigm shift in biologic immunosuppression is belatacept (22). Two other biologics that have been approved for use in psoriasis are now being developed for chronic use in transplantation are efalizumab, a humanized anti-LFA1 mAb and alefacept, an LFA3-Ig fusion protein (Table 2) (23,24).

Table 2.  Biologics in clinical trials
AgentTargetPhase of StudyProtocolResults/1° Endpoint
  1. SCD = standard criteria donor; ECD = extended criteria donor; CNI = calcineurin inhibitor; MMF = mycophenolate mofetil; Tac = tacrolimus; CS = cyclosporine.

Belatacept (Bristol-Myers-Squibb)CostimulationPhase III SCDBelatacept with MMF + CS1° Endpoint: measured GFR
Phase III ECDBelatacept with MMF + CS1° Endpoint: measured GFR
CNI to Belatacept ConversionRandomization to continue CNI or convert to belatacept1° Endpoint: calculated GFR
Rapid steroid withdrawal (at day 5)Thymoglobulin induction followed by belatacept with either MMF or sirolimus versus Tac + MMF1° Endpoint: acute rejection
Efalizumab (Genentech)Anti-adhesionPhase IIEfalizumab and MMF + CS versus CsA and MMF + CS1° Endpoint: patient and graft survival at 12 months
Alefacept (Astellas)Anti-CD2Phase IIAlefacept and tacrolimus + MMF + CS1° Endpoint: acute rejection

Costimulation Blockade/Belatacept

The costimulation or signal 2 pathways have been shown to be critical for optimal and sustained naive T-cell activation, and have been one of the most intensively investigated areas in immunology particularly when considering therapeutic intervention (25). Specifically, the CD28/B7 (CD80 and 86) pathway has proven itself to be relevant in multiple models of transplantation and autoimmunity. After a quarter century of research, the fusion receptor protein CTLA4-Ig (abatacept), a competitive antagonist for CD28 for CD80/CD86 binding, was approved for the therapy of rheumatoid arthritis and is today available for human use (26). Belatacept, a higher affinity and more potent second generation abatacept was developed specifically for use in organ transplantation where more robust immunosuppression is required (22,26). The therapeutic activity of both abatacept and belatacept is primarily due to their binding to CD80/CD86 with greater affinity than the CD28 receptor and not through activities mediated by the Fc domain such as complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity (26).

Belatacept has been shown to prolong graft survival alone and in combination therapies with basiliximab or MMFand prednisone compared to abatacept in NHP renal transplant studies (22). These findings were used to design a phase II multicenter clinical trial comparing the safety and efficacy of 2 dosing regimens of belatacept, one lower and one higher intensity, compared to cyclosporine (27). In this trial belatacept performed with equivalent efficacy to cyclosporine but was associated with better renal function and histology. A potentially important finding from the phase II trial was the occurrence of three cases of post-transplant lymphoproliferative disease (PTLD) in the more intense regimen of belatacept and a higher rate of subclinical rejection in the treatment arm using the less intensity regimen. Reassuringly, in the extension study from the phase II trial no additional cases of PTLD were reported in 102 patients treated with belatacept for 48 months.

Belatacept is now in two phase III trials: one study for patients receiving kidneys from extended criteria donors, the second study enrolling recipients of kidneys from living donors and standard criteria kidneys from deceased donors. The regimen in the low intensity group has been modified by adding an additional infusion of belatacept on day 4. Also, unlike the phase II trial, long-term administration of belatacept was maintained every 4 weeks (in the phase II study patients were randomized to receive belatacept at 4 or 8 week intervals but the latter regimen was associated with increased subclinical rejection). Two additional studies with belatacept are ongoing: a conversion study from CNIs to belatacept and a rapid steroid withdrawal trial following induction with Thymoglobulin and maintenance therapy with belatacept with either MMF or sirolimus. A trial supported by the Immune Tolerance Network will evaluate the effectiveness of a regimen consisting of belatacept and sirolimus to induce tolerance in recipients of kidneys from living donors (University of California, San Francisco and Emory University). An additional trial is ongoing pairing belatacept with alemtuzumab (Emory University). A phase II liver transplant study with belatacept also is currently enrolling patients. If and when belatacept is approved, more experimental designs will likely emerge for it use.

Costimulation blockade/other targets

The failure of the anti-CD154 mAbs (Biogen Idec, Cambrigde, MA) due to thromboembolic complications has shifted interest to several CD40-specific antibodies that have been shown to have clear efficacy in NHP models and appear poised for clinical development (28,29). Most notable among these is 4D11 (Astellas, Deerfield, IL) (30). This is a CD40-specific antibody developed using a mouse strain engineered to express human IgG4 immunoglobulin genes. The resulting antibody is thus a fully human IgG4 rather than a humanized (partially murine) molecule. It is essentially non-immunogenic and does not support complement-mediated lysis. This molecule has been shown in NHP studies to markedly delay allograft rejection and appears on track for clinical testing in the near future (30).

Many other agents, including mAbs specific for the B7 molecules have failed for a variety of reasons. In theory, the ideal inhibition of costimulation is the direct blockade of the CD28 receptor, which would inhibit T-cell activation by depriving the cell of its needed costimulation while simultaneously leaving its counter receptor CD152 (CTLA4) free to ligate CD80/CD86 on APCs and transduce an inhibitory signal. However, CD28-specific antibodies have been shown to have agonistic effects resulting in cytokine release and unacceptable complications (31,32). Specifically, TGN1412 (TeGeneroAG, Würzburg, Germany), a super-agonist anti-CD28 mAb induced a cytokine storm in 6 healthy volunteers leading to multi-organ failure (31). A second anti-CD28, FK734 (Astellas), which appeared preclinically to be safe and effective has been dropped from clinical development because of unacceptable adverse events (32). From these and other trials, it is clear that direct manipulation of T-cell costimulation molecules can impact the immune system in autoimmune disease and organ transplantation.

Anti-adhesion Molecules

Lymphocyte-associated function-1 (LFA-1) is a member of the heterodimeric beta-2 integrin family (33). It consists of a non-covalently linked unique alpha chain (CD11a) and a beta chain (CD18) that is common to other beta-2 integrins. LFA-1 is expressed by neutrophils, monocytes, macrophages and lymphocytes and binds to intercellular adhesion molecules (ICAMs), members of the Ig superfamily that are expressed on antigen presenting cells (APCs) and endothelial cells. Interaction 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 APCs, and providing co-activation signals (33). Experimental models of transplantation have shown that inhibitors of LFA-1 are immunosuppressive and prolong graft survival (33).

Efalizumab is a humanized CD11a-specific IgG1 that was approved for the treatment of psoriasis in 2003. It is now being developed specifically for a transplant indication as a subcutaneously administered maintenance immunosuppressive agent. A Phase II trial in renal transplantation with efalizumab provided promising results but patients treated with the higher dose of efalizumab (2 mg/kg) with full dose cyclosporine had a high incidence of PTLD (23). A second and larger phase II trial in renal transplantation is currently being designed and will likely use an intermediate weekly dose of efalizumab (1 mg/kg) in the context of a CNI-free regimen. The potential of efalizumab as a novel immunosuppression agent was recently demonstrated in a single center trial at University of California, San Francisco in islet transplant recipients who achieved euglycemia with a regimen consisting of thymoglobulin induction and maintenance therapy with efalizumab and sirolimus (34). Efalizumab's mechanism of action does not antagonize costimulation-based agents and, in fact, may act synergistically with agents like belatacept.

Alefacept is a human LFA-3-IgG1 fusion protein (24). The LFA-3 portion of alefacept binds to CD2 on T lymphocytes, blocking the interaction between LFA-3 and CD2 and interfering with T-cell activation. In 2003, alefacept was approved by the FDA for use in psoriasis (35). Treatment with alefacept has been shown to produce a dose-dependent reduction in T-effector memory cells (CD45 RO+) but not in naïve cells (CD45 RA+). This effect has been related to its efficacy in psoriatic disease, and of significant interest in transplantation since T effector memory cells have been associated with costimulation blockade resistant and depletional induction resistant rejection (36). Alefacept has been shown to delay rejection in NHP cardiac transplantation and has recently been shown to have synergistic potential when used with costimulation blockade and/or sirolimus based regimens in NHPs (37,38). A phase II randomized open label parallel group multicenter study to assess the safety and efficacy of maintenance therapy with alefacept in kidney transplant recipients is currently underway.

Natalizumab (Biogen Idec) is a humanized anti-integrin (VLA4) mAb that is approved for use in multiple sclerosis and Crohn's disease. After being pulled from the marked over fears of progressive multifocal leukoencephalopathy, it has emerged as a potentially useful anti-adhesion agent that could be useful in transplantation. To date, no transplant trials have been planned.

Targeting reperfusion injury

With a growing interest in extending the donor pool to suboptimal organs, there has been increasing interest in preventing the initial inflammatory response evoked by ischemia and reperfusion injury. Several adhesion molecules and related compounds are being developed for a specific indication in extended criteria organs and avoidance of early graft injury.

rPSGL-Ig (Thios Pharmaceuticals, Emeryville, CA) is a novel fusion protein resulting in the combination of the binding portion of the high-affinity P-selectin glycoprotein ligand-1 (PSGL-1, CD62) and the Fc domain of human IgG1 (39). P-selectin along with E-selectin and l-selectin, mediate adhesion of leukocytes and platelets, endothelial cells and lymphocytes, respectively. PSGL-1 is found on the surface of most leukocytes. rPSGL-Ig inhibits all selectin molecule interactions and thus has the potential to disrupt many proinflammatory events related to thrombosis and pathologic cell adhesion. rPSGL-Ig was found to be effective in ischemia reperfusion injury in experimental studies and was tested in a phase II study in patients with acute myocardial infarction. rPSGL-Ig is currently in a phase I-II clinical trial to prevent ischemic reperfusion injury for delayed graft function.

Reparixin (Dompé Pharma, Milan, Italy) is a small molecular weight allosteric inhibitor of CXCR1 and CXCR2, the receptors for CXCL8 (IL-8). IL-8 is released by activated PBMCs and binds to CXCR1 and CXCR2, facilitating the recruitment and granular exocytosis of neutrophils and has been implicated in ischemic reperfusion injury and antibody mediated rejection. Preclinical studies in rodent cerebral and liver ischemica-reperfusion models suggest a salutary role for blockade of this pathway. Reparixin has been investigated in a phase I-II trial for delayed graft function (DGF) in kidney transplantation.

Annexin V is a surface molecule expressed on apoptotic cells that binds to phosphatidylserine receptors and plays a fundamental role in the identification and clearance of apoptotic cells by monocytes and macrophages. Diannexin (Alavita) is a dimerized form of Annexin V that has an extended half-life and has been shown to substantially improve hepatic function in mouse models of warm ischemia reperfusion injury. The molecule is now being studied in a phase 2 trial in renal transplantation investigating its potential efficacy in preventing delayed graft function, particularly in extended criteria donors.

Small interfering RNA strands (siRNA) are emerging as an exciting new therapeutic modality to treat several diseases. AKI-5 is a siRNA (Quark Biotech, Fremont, CA) that inhibits p53 and has been shown experimentally to prevent ischemia reperfusion injury. A study to test its effect on DGF in renal transplantation is planned.

Complement inhibition

Several recent studies have suggested that there is a fundamental role played by complement cascade components in determining the initiation and persistence of an alloimmune response. As a clinical example, it has been shown that C3 isoforms segregate with long-term renal allograft outcome (40). For this reason, interest in complement inhibition as a therapeutic strategy in transplantation has persisted, although its promise has yet to be fulfilled in the clinic.

Eculizumab (Alexion, Cheshire, CT) is a humanized mAb specific for C5a. It inhibits membrane attack complex formation and was FDA approved for the treatment of paroxysmal nocturnal hemoglobinuria in 2006. It has been shown to facilitate transplantation in preclinical models of alloantibody sensitization (41) and off-label trials for antibody-mediated rejection will likely be forthcoming.

TP-10 (sCR3) (Avant Biotech, Needham, MA) is a recombinant form of soluble complement receptor type 1 that has been show in multiple preclinical models of xenotransplantation to prevent hyperacute rejection (42). Phase 2 studies designed to test its effectiveness in cardiopulmonary by-pass associated cardiac ischemic injury showed that it was clearly effective in inhibiting complement activation in humans, but it had no effect on outcome in primary analysis. (42) A secondary analysis suggested a salutary effect in males. Evaluation in sensitized transplant recipients would be logical, but as this agent is not approved for any indication, progress will depend on corporate development strategy.

Targeting the B Cell

Most of the advances in transplantation can be attributed to drugs designed to inhibit T-cell responses. As a result, T-cell mediated acute rejection has become less problematic while B-cell mediated responses such as antibody mediated rejection and other effects of donor specific antibody have become more evident. Thus, several agents, both biologics and small molecules with B cell specific effects are now being considered for development in transplantation (Figure 2).

Figure 2.

Novel monoclonal antibodies and fusion receptor proteins that deplete and/or block activation of B cells.

An approved agent for the treatment of B cell malignancies, rituximab is being used off label in transplantation and has been shown to effectively mediate depletion of CD20 expressing B cells (43). However, antibody-producing plasma cells do not express CD20 and rituximab does not have any direct effects on antibody production. Use of rituximab has been suggested to interrupt B cell functions such as antigen presentation. Several humanized or human anti-CD20 are being developed for lymphoma and autoimmune disease. AME-133 (Lilly) is a second generation CD20 with higher affinity binding and improved ADCC effects compared to rituximab. Similarly, Ocrelizumab (Genentech), a humanized anti-CD20 mAb and Ofatumumab (Genmab), a fully human CD20-specific mAb are currently in trials in rheumatoid arthritis, chronic lymphoblastic leukemia and non-Hodgkin's lymphoma. For largely economic reasons, it is unlikely that these second generation mAbs will be developed for FDA approval in organ transplantation.

Epratuzumab (Immunomedics) is a humanized CD22-specific mAb. CD22 is expressed on B cells with increasing density as the B cell progresses through its maturation. It may thus selectively target B cells that have committed to alloantibody formation or memory without depleting all B cells.

A newly targeted pathway relevant to B-cell specific immunosuppression involves the molecule B lymphocyte Stimulator (BLyS). BLyS (also known as B-cell activating factor or BAFF) is a secreted tumor necrosis factor (TNF) family member that along with the TNF family member APRIL, serves as a ligand for the cell surface receptors BCMA, TACI and BAFF-R (BLyS only). BLyS and APRIL are secreted by a variety of APCs in response to interferon-γ and are thought to amplify de novo B cell response. These receptor ligand pairs are essential development factors for B cell and plasma cell development (44–47).

At least three agents are in development targeting this pathway. Belimumab (Human Genome Sciences, Rockville, MD) is a new BLyS-specific fully human mAb that is in the development pipeline for systemic lupus erythematosis (SLE) and other autoimmune diseases associated with autoantibodies. It reduces B cell counts, likely by BLyS deprivation and induction of apoptosis. Atacicept (ZymoGenetics, Seattle, WA) is a fusion protein that combines the extracellular ligand-binding portion of TACI with an IgG Fc region. It blocks activation of TACI by April and BLyS, and has been shown to produce B cell depletion. It is in early stage development for the treatment of multiple myeloma, SLE and rheumatoid arthritis. BR3-Fc (Genentech, South San Francisco, CA / Biogen Idec) is a recombinant fusion protein utilizing the ligand-binding portion of BAFF-R preventing the binding of BLyS (47). It is in early stage development for rheumatoid arthritis.

Bortezomib (Millennium Pharmaceutical) is a proteasome inhibitor that was approved for the use of multiple myeloma in 2005 (48). It is also under investigation in other hematologic cancers, particularly certain types of lymphoma and in a variety of solid tumors, including prostate, lung, breast and ovarian cancer. Bortezomib targets the 26s proteosome complex, which is involved in the processing and degradation of excess proteins in highly metabolically active cells. This pathway is particularly important in cells with very high protein production, and its inhibition in cells with excessive production, e.g. myeloma cells producing antibody components, results in apoptosis. This is conceivably true of all active plasma cells. A single center study with bortezomib is beginning an off-label trial in antibody-mediated rejection (Mayo Clinic, Rochester, MN). Bortezomib is also thought to inhibit NFκB, a key mediator in leukocyte activation. Given the central role of the NFκB pathway in cytokine secretion, adhesion molecule expression and antigen presentation, its inhibition may have important, albeit broad, immunosuppressive properties.

Cytokine Pathways

Cytokines are ubiquitous components of an alloimmune response and they have been successfully targeted for a variety of immune-mediated diseases. Most notably, the IL2 receptor antagonists have made their way into the standard armamentarium of the transplant clinician. Several other cytokine pathways are now being investigated with an eye on transplantation.

IL-6 is produced by a variety of cells, including renal tubular epithelial cells and has been described as both a pro- and anti-inflammatory cytokine (49). A key feature in the regulation of IL-6 responses has been the identification of a soluble IL-6R receptor (sIL-6R) that is capable of stimulating a variety of cellular responses. IL-6 is increased in the serum and urine of transplant recipients. Tocilizumab (Roche) is a humanized anti-IL6R mAb that was used in clinical trials in rheumatoid arthritis (50). It is waiting for FDA approval for the treatment of arthritis but its development strategy in transplantation remains unclear.

Several biologics (infliximab, golimumab and etanercept) have been developed to sequester or inhibit the effects of TNF-α. These drugs have been shown to have salutary effects predominantly in inflammatory bowel disease, rheumatoid arthritis and other autoimmune disorders, but have attracted attention in off-label pilot trials in transplantation. While TNF sequestration is a highly specific way to inhibit TNF-α associated actions, the intracellular effects of TNF-α are diverse and can broadly be divided into activating and pro-apoptotic effects. In an effort to screen compounds for intracellular modulating activity, a novel agent Genz29155 (Genzyme), was identified that inhibited TNF-α induced apoptosis (51). Genz29155 has an unknown intracellular target, but has been shown to have salutary effects in several in vivo models including mouse models of lupus nephritis and experimental autoimmune encephalitis. Genz29155 has been shown to extend murine heterotopic cardiac allograft survival in a manner that is highly synergistic with sirolimus, but neither additive or synergistic with cyclosporine. Based on these results, preliminary NHP studies have been initiated showing prolonged renal allograft survival in rhesus monkeys treated with Genz-29155 and sirolimus compared to sirolimus alone. More extensive NHP studies are now underway.

Conclusion

The dominant forces propelling transplant drug development are now the long-term toxicities associated with current regimens, and immune targets that have not been adequately addressed by the current drugs. There are a gratifyingly high number of new agents available for study both as primary transplant therapeutics or off-label candidates for investigation. The opportunities for pilot trials will certainly grow as clinicians seize opportunities to pair mechanistically promising drugs. As a community, our challenge will be to encourage rigorous trial conduct and insist upon data to drive the regimens of the future.

Acknowledgment

The authors would like to acknowledge Peggy Millar for the preparation of the manuscript and for designing Figures 1 and 2. Dr. Vincenti received research grants from Bristol Myers Squibb, Pfizer, Wyeth, Novartis Roche, Genzyme, Astellas and Genentech. He has been a consultant for Pfizer, Bristol Myers Squibb, Novartis, Genentech and Roche, but has not received consulting fees from any of these companies. Dr. Kirk has received research grants from Genzyme. He has received consulting fees from Astellas and Alavita.

Ancillary