TOL101 (T10B9, MEDI-500) is a murine IgM k chain mAb directed against the alpha and beta subunits of the TCR and appears to lead to internalization of the TCR rather than T-cell depletion. The predecessor antibodies for TOL101, T10B9, and MEDI-500 have been administered to approximately 135 patients across 13 studies from 1986 to 2000 – over 100 of these patients were recipients of solid organ transplants. The largest of these studies was a 76-patient phase 2 trial investigating T10B9 vs. OKT3 (at that time, considered standard of care) for the treatment of acute renal transplant rejection. Graft survival and subject survival were high (>80 %) over 4 years and similar between the two treatment groups. The incidence and severity of adverse events (including fever, respiratory symptoms, gastrointestinal complaints, and neurological symptoms) were substantially higher in the OKT3 group than in the T10B9 recipients [103]. TOL101 is currently in a phase 1/2 study as part of an immunosuppressive regimen that includes tacrolimus, MMF, and steroids in patients undergoing primary kidney transplantation. Because of the large size of this molecule, the pharmacokinetic profile of this agent may be more favorable (due to longer intravascular retention) in LTX patients, where pharmacokinetics demonstrated higher clearance of IgG preparations.

Sotrastaurin (AEB071) is a novel immunosuppressant that blocks early T-cell activation via inhibition of PKC, integrating in particular signaling pathways downstream of the T-cell receptor (TCR) and the CD28 co-receptor. Sotrastaurin has been shown to specifically inhibit early T-cell activation through signals 1 and 2 but not T-cell proliferation (signal 3) by selectively blocking the calcineurin-independent pathway signaling through NF-kB resulting in inhibition of cytokine gene transcription. AEB071 is being developed for the prevention of acute rejection in solid organ allotransplantation in combination with or without a calcineurin inhibitor (CNI). In contrast to CNIs, AEB071 potently and selectively blocks a calcineurin-independent pathway pointing toward a clear differentiation in mode of action and possibly the side effect profile between AEB071 and CNIs.

Thus far, sotrastaurin has been used in two phase 2 de novo renal transplant trials. In one study, recipients were randomized to sotrastaurin (200 mg b.i.d.) + standard-exposure tacrolimus (SET) or reduced-exposure tacrolimus (RET) (SET: n = 76; RET: n = 66) or control (SET + MPA, 720 mg b.i.d.; n = 74) [104]. In both sotrastaurin groups, patients were converted from tacrolimus to MPA after month 3, achieving CNI-free immunosuppression. The primary endpoint was composite efficacy failure (treated biopsy-proven acute rejection, graft loss, death, or loss to follow-up), while the key secondary endpoint was GFR. Composite efficacy failure rates were 4.1, 5.4, and 1.5 % at month 3 (pre-conversion) and 7.8, 44.8, and 34.1 % at study end in the control, sotrastaurin + SET, and sotrastaurin + RET groups, respectively. In addition, the median GFR at month 6 was 57.0, 53.0, and 60.0 mL/min/1.73 m², respectively. Based on the primary endpoint, the Data Safety Monitoring Board (DSMB) recommended premature study discontinuation. Although the initial sotrastaurin + tacrolimus regimen was efficacious and well tolerated and the postconversion sotrastaurin + MPA regimen showed inadequate efficacy, longer-term evaluation of sotrastaurin + tacrolimus appears warranted.

In another study, de novo renal transplant recipients with immediate graft function were randomized 1:2 to tacrolimus (control, n = 44) or sotrastaurin (300 mg b.i.d.; n = 81) [5]. All patients received anti-IL2-RA, MPA, and steroids. The endpoints were similar to that noted in the previous trial. In this trial, the composite efficacy failure at month 3 was higher for the sotrastaurin versus control regimen (25.7 % vs. 4.5 %, p = 0.001) with rejection rates higher in the sotrastaurin group compared to control, 23.6 % vs. 4.5 %, respectively (p = 0.003), which led to early study termination by the DSMB. Of great interest was the finding that the median estimated GFR was higher for sotrastaurin versus control at month 3: 59.0 vs. 49.5 mL/min/1.73 m² (p = 0.006) [105]. Further follow-up studies have been recently completed showing reasonable efficacy but decreased tolerability compared to current standard regiments [106].

Abatacept is a chimeric fusion protein that consists of the extracellular domain of CTLA-4, and the Fc domain of IgG blocks the B7 (CD80, CD86)/CD28 pathway. This agent is approved for use in moderate to severe psoriasis. Belatacept is a molecular mutation of abatacept, differing from abatacept in two amino acid positions in the binding domain to B7, associated with a higher binding avidity and slower dissociation rate, with resultant inhibition of T-cell activation greater than that of abatacept. With both molecules, blockade of the B7/CD28 interaction leads to inhibition of T-cell proliferation. Belatacept was investigated in a phase 2 de novo kidney transplant trial with a CsA regimen as control. This trial consisted of belatacept injections every 2 weeks for 1 year. There was an improvement in the GFR in the belatacept-treated group compared to the CsA group, but there was no difference in biopsy-proven acute rejection [107]. In follow-up pivotal phase 3 trials, belatacept demonstrated 1-year subject and graft survival that was comparable to CsA, with improved renal function and less metabolic complications such as incidence of new-onset diabetes mellitus, blood pressure, and abnormal lipid profile [108]. An increased risk of posttransplant lymphoproliferative disorder (PTLD), particularly among Epstein-Barr virus-negative recipients, was a notable adverse event. However, belatacept was associated with an increased frequency of early acute rejection compared with CsA. With longer-term follow-up, the impact of these rejections appeared to be limited. Extended follow-up over 3 years demonstrated evidence of ongoing efficacy, which did not differ between the two belatacept dose regimens (LI and MI) evaluated, and in particular, GFR was better preserved in both belatacept groups, even in those that experienced an acute rejection, compared to CsA-treated recipients. In addition, the incidence of chronic allograft nephropathy was also significantly lower in the belatacept-treated patients [109, 110]. Based on this data, belatacept was recently approved by the FDA for kidney transplantation. The additional long-term benefits that accrue to patients on belatacept relating to improvements in metabolic parameters have been fully assessed.

Belatacept was next investigated in a phase 2b multicenter prospective partially blind clinical trial in LTX [111]. Five treatment groups were utilized: Group 1, anti-IL2RA + belatacept more intensive (MI) + MMF; Group 2, belatacept (MI) + MMF; Group 3, belatacept less intensive (LI) + MMF; Group 4, tacrolimus + MMF; and Group 5, tacrolimus. The primary objective was to evaluate the effects of belatacept relative to TAC on the triple composite endpoint of the incidence of acute rejection (AR), death, and graft loss by 6 months after receiving a deceased donor transplant. An imbalance in deaths in the belatacept treatment arms relative to the tacrolimus + MMF arm was noted. The frequencies of death were noted as 12, 21, and 22 % in the anti-IL2RA + belatacept MI + MMF, belatacept MI + MMF, and belatacept LI + MMF arms, respectively, in comparison to 6 % in the TAC + MMF arm and 14 % in the TAC arm. Of note was the marked difference in GFR in the belatacept groups compared to the control groups. There were two reports (one fatal) of PTLD and one report of fatal progressive multifocal leukoencephalopathy (PML). In addition there was an increase in viral and fungal infections in the combined belatacept groups versus the tacrolimus groups, potentially due to the degree of immunosuppression.

Future trials with belatacept may possibly include a short period of CNI exposure in the perioperative period, and these considerations are ongoing at this time.

Efalizumab is a humanized IgG1 mAb directed against the leukocyte function-associated antigen-1 (LFA-1, CD11a). CD11a plays an important role in adhesion of leukocytes to endothelial cells and also serves as a costimulatory molecule. Approved for treatment of moderate to severe psoriasis, a pilot study was performed in 38 primary kidney transplant recipients [11]. Patients were randomized to receive efalizumab 0.5 or 2 mg/kg weekly subcutaneously for 12 weeks. Patients were maintained on full-dose CsA, MMF, and steroids (n = 10 0.5 mg/kg efalizumab, n = 10 2.0 mg/kg efalizumab) or half-dose CsA, sirolimus, and prednisone (n = 9 0.5 mg/kg efalizumab, n = 9 2.0 mg/kg efalizumab). At 6 months following transplant, patient survival was 97 % and graft survival was 95 %. Clinical biopsy-proven acute rejection in the first 6 months after transplantation was confirmed in one of each of the immunosuppressive combination (e.g., 4/38, 11 %). Three patients (8 %) developed PTLD, all in the highest dose efalizumab with full-dose CsA [112]. Although this drug appeared promising, subsequent reports of the development of PML in patients treated for extended periods with efalizumab for psoriasis resulted in withdrawal of this agent from the market [113]. Nevertheless, interference of this pathway is seemingly a novel approach for future trials.

Alefacept is a lymphocyte function-associated molecule 3/immunoglobulin G (LFA3-IgG1) fusion receptor protein, which functions by interfering with the CD2 receptor on T cells, causing apoptosis of effector memory T cells. By blocking LFA-3/CD2 interactions, alefacept can inhibit T-cell activation and proliferation. It has been approved for moderate to severe chronic plaque psoriasis and has no known nephrotoxicity. In addition, it has been used for the treatment of graft-versus-host disease in bone marrow transplantation [114]. In transplantation, a phase 2, multicenter, randomized, double-blind, placebo-controlled study in primary adult kidney transplant patients comparing alefacept, tacrolimus, and MMF to placebo, tacrolimus, and MMF was conducted. The primary endpoint was an incidence of biopsy-proven acute rejection at 6 months. No statistical differences between treatment arms were observed for the primary endpoint, patient or graft survival, as well as renal function. Alefacept was associated with statistically significant reduction in T memory lymphocyte subsets. Given that alefacept appears to react with a different population of T cells, i.e., effector memory T cells, rather than naïve T cells, it would seem that the results of this study are not surprising. There was an increased rate of malignancy in the alefacept group [115]. Alefacept may be better in models where memory T cells have a pathophysiologic role, e.g., sensitized or retransplant patients, GVHD after LTX. In fact, alefacept has been used successfully in such a case [116].

Tasocitinib (CP-690,550, tofacitinib) is an orally active immunosuppressant currently being tested for a variety of immune-mediated disorders, including prevention of transplant rejection. Tasocitinib specifically inhibits Janus-activated kinase 3 (JAK3), which is a hematopoietic cell-restricted tyrosine kinase involved in cytokine signal transduction associated with lymphocyte proliferation, specifically interfering with IL-2-mediated STAT5 activation in CD4+ T cells. A randomized pilot study compared two dosages of tasocitinib (15 mg BID and 30 mg BID, n = 20 each) with tacrolimus (n = 21) in de novo kidney allograft recipients [117]. Patients received anti-IL-2RA, MMF, and corticosteroids. The 6-month biopsy-proven acute rejection rates were 5, 20, and 4.8 % for low- and high-dose tasocitinib and tacrolimus groups, respectively. The infectious complications were most frequent in the high-dose tasocitinib/MMF group with BK virus infection developing in 20 % and cytomegalovirus infection in 20 % of patients. Other side effects of tasocitinib included hyperlipidemia, anemia, and neutropenia. A larger study of tasocitinib in primary kidney transplantation utilized 15 mg BID for either 3 or 6 months followed by 10 mg BID thereafter and compared to a CsA control group – all groups were induced with anti-IL-2RA and given maintenance MPA and corticosteroids [118]. Of the 109 CsA patients, the ACR rate at 12 months was 18.8 % compared to longer high-dose tasocitinib (n = 106) at 17.4 % and shorter high-dose tasocitinib (n = 107) at 15.4 %. The finding that tasocitinib preserves regulatory T-cell function may be particularly important in LTX, as this may help to explain the immunologically privilege of LTX related to lack of chronic rejection and possible tolerance.

The CD40 molecule is expressed on antigen-presenting cells and serves as a costimulatory molecule through its interaction with CD154. Activation of CD40/CD154 has been shown to promote T-cell activation, B-cell proliferation and class switching, macrophage function, and a variety of other immunological processes. Page et al. have demonstrated that CD40- or CD40L-specific mAb could prevent and even reverse acute allograft rejection leading to prolongation of MHC-mismatched renal allografts in primates without the need of chronic maintenance immunosuppression [119]. Early studies with humanized anti-CD154 mAb were hampered by unexpected thromboembolic complications [120]. Further studies suggested that this was a function of the effects on integrin-binding sites on CD154 which are believed to aid in arterial plaque stabilization [121]. More recently, fully human anti-CD40 monoclonal antibodies, 4D11/ASKP1240, have been tested in a primate model with marked suppression of T-cell responses and prolongation of kidney allograft survival [122]. ASKP 1240 has recently undergone phase 1 evaluation, and currently phase 2a study is underway to assess the utility of ASKP1240 in MMF and CNI avoidant regiments (Basilixumab induction+ASKP1240+steroids+MMF vs Basilixumab+steroids+MMF+tacrolimus vs Basilixumab+ASKP1240+steroids+Tacrolimus) [123].

“Immunological tolerance,” the state whereby the immune system fails to respond to a stimulus that would normally elicit an immunological response, is one of the “Holy Grails” of clinical transplantation. The ability to induce tolerance would obviate the need for maintenance immunosuppression and its long-term risks and associated complications as well as mitigate allograft rejection. The field of transplant tolerance was born in 1953 with the landmark report of Billingham, Brent, and Medawar where exposure during the fetal life of mice and chickens to homologous antigens leads to immunological tolerance. This was manifested by a lack of response to skin grafting from the organism that was used in the inoculation process [124]. Subsequently, this neonatal tolerance has been demonstrated to be mediated through negative selection via mechanism(s) of thymic deletion of reactive T cells, also referred to as “central tolerance” [125]. However, similar approaches in adult recipients could not reproduce acceptance of donor tissues, unless the recipient had been cytoablated, in this early period, accomplished through lethal irradiation and reconstitution with donor bone marrow (radiation-induced chimerism) [126]. Chimerism refers to the development of an immune constitution that is comprised of cells of both donor and recipient lineages. In contrast to neonatally tolerant animals, the mechanism(s) of tolerance in these adult recipients involves not only clonal deletion but also active suppression [127].

Over the next 50 years, attempts were made in various animal models to induce tolerance with varying degrees of success, including early attempts of whole body and total lymphoid irradiation, shown to be necessary for the induction of immunological tolerance in bone marrow transplant patients. However, the development of potent immunosuppressive agents became the pathway to successful clinical solid organ transplantation. Because neither bone marrow nor any other kind of donor hematolymphopoietic cells were given adjunctively in solid organ transplantation, the enigmatic mechanisms of organ engraftment were assumed to be independent of leukocyte chimerism. However, there were clues that organ engraftment was a state of variable tolerance that in some cases became immunosuppression independent. Tolerance was inferred from a rapidly declining need for maintenance immunosuppression following the successful treatment of rejection. In addition, Starzl and coworkers demonstrated that long surviving allografts could be weaned from immunosuppression in a significant proportion of kidney and liver transplant recipients [128]. The finding that low-level multilineage donor leukocyte chimerism (microchimerism) was found in all tolerant patients and in one or more locations that included the skin, lymph nodes, heart, lungs, spleen, intestine, kidneys, bone marrow, and thymus emphasizes the importance of antigen migration and tolerance, as advocated by Starzl and Zinkernagel [129]. At any given site, the donor leukocytes were present in larger numbers in liver recipients than in kidney recipients studied at comparable posttransplant times. With the persistence of donor cells for as long as 30 years, it was inferred that the passenger leukocyte population of organ grafts was critical in establishing clinically operational tolerance. The migration of donor antigens, either as living cells or by shed alloantigens from the allograft, initiates a recipient immune response via direct or indirect antigen presentation pathways, respectively. Effector mechanisms include generation of cytotoxic T cells (CD8+) as well as downstream alloantibody as a result of CD4+ cytokines. As noted before, T-cell activation is essential for subsequent tolerance generation [130], and the use of potent immunosuppression is likely to delay or prevent the deletion of CD8+ effector T cells and the regulation of CD4+ helper T cells, likely mediated through apoptotic inducing clonal exhaustion as well as other peripheral tolerogenic pathways mediated through active suppressive regulation, such as T-regulatory cells (Treg) or myeloid-derived suppressor cells (MDSC). The liver allograft is naturally endowed with high levels of hepatic stellate cells, Tregs, and MDSCs, which may facilitate the evolution to clinically operational tolerance [131].

The mechanism(s) by which tolerance is induced and maintained in clinical transplant recipients has been an area of ongoing investigation. A European consortium of liver transplant centers presented preliminary results of a prospective trial on 102 adult LTX recipients who were enrolled in a prospective immunosuppressive drug weaning study. A total of 41 recipients (40 %) were successfully weaned off immunosuppression a median to 11 years after LTX [132]. Similarly, in a pediatric recipient cohort receiving living donor liver allografts, a median of 7 years prior to enrollment in a prospective weaning protocol, 60 % of patients were successfully weaned [133], validating the earlier reports from the Kyoto University group of a 40 % success rate of weaning pediatric recipients of partial livers from parental donors from a steroid-sparing tacrolimus-based regimen [20]. While a variety of biomarkers have been associated with the development of clinical operational tolerance following LTX, including increased expression of hepatic iron homeostatic genes [21]; increased circulating CD4+, CD25+, CD127-, and FoxP3+ T-cell subsets [21]; and alterations in γδ1 T cells with an increased γδ1/γδ2 ratio [22], these preliminary results suggest that although the mechanism(s) associated with liver allograft tolerance are still being elucidated, obtaining success in clinically operational tolerance in liver transplantation is strictly related to the careful selection of the candidates for long-term weaning and follow-up [133–135].

The following are preliminary results of current tolerance studies, primarily being conducted in kidney transplants because of the requirement for preconditioning that is inherent with the requirement for donor-specific activation and deletion. Unfortunately, extrapolation of findings in the living donor scenario to deceased donors may prove to be a considerable barrier. To this point all successful attempts at tolerance have been accomplished by co-induction of hematopoietic chimerism. Induction of persistent mixed chimerism has been difficult to achieve in humans. Despite this, several studies have suggested that persistence of chimerism may not be necessary for the development of allograft tolerance. Scandling et al. published their cohort of 16 patients who underwent kidney transplantation with an induction protocol including ten doses of 80 cGy TBI each and five doses of rabbit ATG to human recipients of combined HLA-matched G-CSF “mobilized” blood mononuclear cell and kidney transplants from HLA-matched sibling donors. The hematopoietic grafts in the latter protocol were selected CD34+ cells with 1 × 10⁶ CD3+ T cells/kg added back to the hematopoietic cells. Four patients developed persistent mixed chimerism, and eight developed transient chimerism [136]. All those with persistent mixed chimerism, and several of those with transient chimerism, were weaned from their maintenance immunosuppression. With these proofs of concept studies, the next challenge has been to attempt tolerance in HLA-mismatched transplant pairs. Kawai et al. followed up their initial study of myeloma patients with ten patients who received HLA-mismatched kidney transplants with an induction regiment consisting of thymic radiation, anti-CD2 mAb, and cyclophosphamide +/− rituximab followed by ~9 months of calcineurin inhibitors. Seventy percent of the patients were weaned from all maintenance immunosuppression up to 5 years posttransplant, even though donor hematopoietic chimerism was transient [137].

Another approach to allograft tolerance has been the induction of full donor chimerism, whereby the recipients’ immune constitution is replaced by that of the donor. Studies in the bone marrow transplant literature where full donor chimerism was induced have been plagued by high rates of GVHD and engraftment syndrome. Recently, attempts have been made with some success, to induce full donor chimerism in renal allograft recipients. In one of the largest groups to date, Leventhal et al. reported on 15 patients who underwent HLA-mismatched kidney transplantation after an induction regiment of pretreatment with fludarabine, 200-cGy TBI, and cyclophosphamide followed by infusion of tolerance-promoting CD8+, TCR-facilitating cell (FC)-based hematopoietic stem cell (HSC) graft infusion, and posttransplant immunosuppression with tacrolimus and mycophenolate mofetil. Postoperative monitoring for donor chimerism was used to establish decision for immunosuppressive drug weaning. Ten of these 15 patients have achieved durable full or mixed hematopoietic chimerism without GVHD or engraftment syndrome. Eight of these ten achieved durable, high-level (>90 %) hematopoietic chimerism. Six of the eight have successfully completed immunosuppression withdrawn without allograft rejection or graft loss (range of between 10 and 22 months off IS). The two remaining patients with high-level chimerism are currently undergoing immunosuppression withdrawal. Two subjects achieved sustained, mixed chimerism, while three participants achieved transient chimerism [138]. The key to this experience relies on the coadministration of the proprietary “facilitating cell” first identified by Ildstad and coworkers [139].

Todo and coworkers recently reported on their prospective liver tolerance study – they utilized a protocol of T-regulatory cell (Treg) expansion ex vivo to determine whether Treg-based cell therapy affords COT in living donor LT (LDLT). The group from Hokkaido University treated ten consecutive LDLT adult patients with Tregs created from peripheral blood mononuclear cells collected from both donors and recipients by leukapheresis and expanded ex vivo with a 2-week culture of recipient PBMNs with irradiated donor PBMNs under the presence of anti-CD80/anti-CD86 mAbs. These cells were infused into the recipient on postoperative day (POD) 13 along with cyclophosphamide given on POD 5. Steroids and MMF were stopped within 1 month, while the patients were left on tacrolimus monotherapy. At 6 months after LDLT, when graft function and histology were normal, immunosuppression was gradually tapered by spaced doses until it was discontinued 12 months later. Thus far, of the ten recipients, seven are free from immunosuppression [140] (update provided by Todo S. personal communication, April 2014). Protocols for deceased donor liver transplantation are planned.

The potential downside of facilitating increasing levels of chimerism is the prospect of developing GVHD, which is a rare but serious complication after liver transplantation. It occurs when immunocompetent donor lymphocytes transferred through the liver allograft become activated and are able to carry out an immune response against recipient tissues. Acute GVHD is more commonly seen after hematopoietic stem cell transplantation and uncommonly after solid organ transplantation. The true incidence of GVHD after LT is not clear, but according to the more recent reports, it is estimated to be around 1–2 % [141]. GVHD usually presents with fever, skin rash, diarrhea, and pancytopenia 2–10 weeks after liver transplantation. The diagnosis is confirmed by demonstration of substantial number of donor chimerism in the patient’s peripheral blood. Studies have shown that these donor chimeric cells are usually CD8+ T cells [142]; however, multilineage donor hematopoietic chimerism has also been described [143–145]. Despite a variety of protocols for treatment of GVHD after LT, with different strategies to decrease or increase immunosuppression [141, 142, 145–147], response rate remains poor with 85 % mortality rate in affected patients. Mortality is usually as a result of multiorgan failure and especially bone marrow failure and infection. To date, only two forms of therapy have been successful, reprogramming the recipient’s immune system with infusion of pre-transplant recipient bone marrow [148] and the promising use of alefacept, a fusion protein comprising the extracellular CD2-binding portion of the human leukocyte function antigen-3 (LFA-3) linked to the Fc portion of human-IgG1, and selectively targets memory T cells [149].