Induction

Historically, polyclonal antibody preparations were used to bolster the effect of steroids and azathioprine in an attempt to reduce the unacceptably high rejection rates typical of the 1960s and 1970s. Generally, a 2- to 3-week course of a polyclonal antibody delayed the onset of acute rejection and reduced the requirement for high-dose steroids in the early postoperative period without significantly altering long-term survival. After the introduction of cyclosporine, the use of polyclonal antibody induction fell from favor with the realization that this potent combination was associated with increased infectious and malignant morbidity. With improved viral prophylaxis, a better understanding of the infectious etiology of PTLD, and more standardized commercial polyclonal products, there has been a marked resurgence of interest in polyclonal antibody induction.

Most modern trials have evaluated polyclonal antibodies added to an otherwise rigorous maintenance regimen (typically triple immunosuppressive therapy). This intense regimen has statistically reduced acute rejection rates, but has reciprocated with increased infectious morbidity without changing the long-term outcome. The increased infectious risk may be acceptable in selected higher-risk patient populations, such as recipients of donation after cardiac death donors, recipients of organs with kidney donor profile index >85%, formerly extended criteria donors, and patients with a high risk of rejection, such as African American recipients, retransplant recipients, and recipients with delayed graft function, particularly when avoidance of prolonged CNIs is desired. A seminal randomized trial between ATG-R and CD25-specific (basiliximab) induction showed that ATG-R reduced the incidence and severity of acute rejection but not the incidence of delayed graft function. Recently, another randomized study with ATG-R and daclizumab induction revealed sustained superiority of ATG-R to prevent acute rejection in highly sensitized patients. In contrast, a randomized trial for rapid steroid withdrawal in nonsensitized patients showed no superiority of ATG-R over basiliximab in preventing acute rejection at 1 year after renal transplantation. Although early patient and graft survival were not influenced by the choice of induction regimen, long-term studies have suggested both a patient and graft survival benefit with ATG-R.

Other trials have attempted to address the increased infectious risk by pairing aggressive polyclonal induction with substantially reduced maintenance therapy. Two pilot studies have shown that ATG-R induction facilitates reduced maintenance immunosuppression in highly selected, closely followed patients, leading to graft and patient survivals comparable to the current standard. These studies have emphasized administration before reperfusion, theoretically to take maximal advantage of antiadhesion molecule effects, and relatively high-dose therapy, to limit the proinflammatory effects of reperfusion and to achieve rapid and lasting T cell depletion. Although these studies indicate that such an approach is possible, it remains to be seen if it can be generalized to noninvestigational settings.

Rescue

Although induction therapy remains an off-label indication for polyclonal antibodies, their use for the treatment of steroid-refractory rejection is an established indication. Many polyclonal preparations have shown their utility in this setting, spanning several decades of associated maintenance regimens. The first randomized trial showing that antilymphocyte serum was superior to high-dose steroids for the treatment of established rejection was reported in 1979. In the context of azathioprine and prednisone maintenance immunosuppression, antilymphocyte serum reversed rejection faster than bolus glucocorticosteroids, reduced the rate of recurrent rejection, and led to improved survival at 1 year. Most rejection episodes in the cyclosporine era and beyond respond to bolus steroids. Polyclonal agents have been indicated as a second-line therapy for steroid-resistant acute cellular rejection. Recurrent rejection can be treated with repeated courses of polyclonal antibodies in situations where antirabbit (or antihorse) antibodies have not formed.

Of the currently available polyclonal preparations, ATG-R is used most commonly for rescue. It has been shown to be superior to ATGAM in terms of reversal of steroid-resistant rejection and persistence of a rejection-free state. This difference has not been shown, however, to influence patient or graft survival.

Non-T cell-specific polyclonal antibody preparations also reverse established cellular acute rejection. Although not typically considered alongside T cell depleting polyclonal antibody preparations, high-dose human IgG fractions (intravenous immunoglobulin) are polyclonal antibodies of random specificity pooled from human donors. Because they are not derived from animals, are not the products of heterologous immunization, and do not target a specific cell type, most of the adverse effects associated with polyclonal antibodies are not applicable. Nevertheless, high-dose human IgG fractions have been shown to reverse rejection despite the absence of any T cell depleting abilities. Although a course of polyclonal anti-T cell antibody typically consists of 5 to 20 mg/kg given over several days, intravenous immunoglobulin is infused at much higher doses, 500 to 1000 mg/kg over 1 to 3 days, and at this dose has been shown to reverse established rejection with the same overall reversal rate as OKT3. At least at high dose, nonspecific antibody infusion can modulate immune responses, perhaps through complement sequestration and Fc receptor binding with resultant downregulatory effects of Fc receptor-expressing antigen-presenting cells (APCs).

Administration and Adverse Effects

The polyclonal preparations used in modern clinical practice are generally given through a large-caliber central vein to avoid thrombophlebitis. In experienced hands, a dialysis fistula can be accessed for this purpose. Some reports have suggested that polyclonal antibodies can be administered peripherally when diluted and formulated with heparin, hydrocortisone, or bicarbonate solutions. An in-line filter is recommended to prevent infusion of precipitates that may develop during storage. The protein content should not exceed 4 mg/mL, and dextrose-containing solutions should be avoided because they induce protein precipitation.

Given the weeks-long half-lives of polyclonal antibodies, divided doses are not required for steady-state levels. The tolerability of these compounds is markedly improved, however, by spaced dosing. The rate of infusion is associated with the severity of side effects, and the course of therapy is generally over several days, with individual doses given over 4 to 6 hours. This time course depends on the dose used and is most applicable to the standard doses of ATG-R and ATG-F (1.5 mg/kg/dose for a total of 7.5–10 mg/kg) or ATGAM (15 mg/kg/dose for a total of 75–100 mg/kg). More recent investigational induction studies have employed substantially higher doses given over 12 to 24 hours or, alternatively, while the patient is anesthetized with comparable safety profiles. With a growing emphasis being placed on reduced length of stay after transplantation, larger infusions over fewer days are being employed.

Generally, rabbit-derived polyclonal preparations seem to be significantly better tolerated and more efficacious than ATGAM when used in a quadruple regimen for renal transplantation. The most common acute symptoms associated with polyclonal antibody use are the result of transient cytokine release. Chills and fever occur in at least 20% of patients and are generally treatable by premedication with methylprednisolone, antipyretics, and antihistamines. The use of polyclonal antibodies, particularly in the treatment of rejection, has been associated with an increase in the reactivation and development of primary viral disease caused by CMV, herpes simplex virus, EBV, and varicella. It is likely, however, that this is not a class-specific association, but rather an indication of more intensive immunosuppression in general.

Dosage adjustment is warranted to counter leukopenia and thrombocytopenia. Peripheral cell counts drawn immediately after infusion tend to exaggerate cytopenic effects, and most side effects are promptly remedied by time. T cell counts or, more easily, absolute lymphocyte counts can be monitored to ensure that the preparation is achieving its desired effect. Absolute lymphocyte counts less than 100 cells/μL are typical. Attempts to tailor therapy to a specific peripheral cell count have been made to limit the use of these costly preparations. Rejection can occur and persist with very low T cell counts, however, and there is little evidence that dose variation by cell count alters efficacy.

As discussed earlier, polyclonal antibody preparations evoke a humoral immune response to themselves. This response can be detected by enzyme-linked immunosorbent assay for antirabbit or antihorse antibody, but these tests are typically unavailable in clinical settings. Failure to achieve significant T cell depletion suggests the presence of these antibodies. Serum sickness and anaphylaxis also can occur. Preemptive skin testing is not often practiced because these tests have not correlated well with clinical outcome. Rather, slow infusion rates should be employed during initial exposure. Xenospecific antibodies are most likely to occur in individuals with prior exposure to the preparation involved, but can also exist in individuals with significant prior exposure to the animals themselves.

The most common adverse symptoms related to polyclonal antibodies are fever, urticaria, rash, and headache. These are most likely related to the release of pyrogenic cytokines, such as tumor necrosis factor (TNF)-α, IL-1, and IL-6, which result from activating antibody binding to targeted cell surface receptors and subsequent cell lysis. Infrequently, pulmonary edema and severe hypertension or hypotension can result in death. As the number of target cells decreases with repeated dosing, this response typically abates. The most concerning response is within the first 24 hours of the first dose, and patients should be monitored closely during this period. The response is limited considerably by methylprednisolone premedication. The rash associated with polyclonal antibody administration, conversely, tends to occur late in treatment or, at times, after the last dose. It is generally self-limiting and requires only symptomatic treatment for urticaria. Antiendothelial antibodies in polyclonal antibodies have been suggested to bind to donor endothelia and activate complement, inducing humoral rejection in some patients.

Muromonab (OKT3; Murine Anti-CD3)

OKT3 (muromonab) is no longer available for clinical use. However, it is considered here given its unique place in the historical continuum of MAb therapy. OKT3 is CD3-specific; CD3 is a transmembrane complex of proteins that links to the TCR and conveys its activating signal to the nucleus via a calcineurin-dependent pathway, thus serving as the fundamental signal in antigen-specific T cell activation. CD3 is present on essentially all T cells, defining the cell type. The TCR signal is generally known as signal 1 because it is primarily required for T cell activation and defines the antigen specificity of the T cell. Given that T cells are a crucial mediator of acute cellular rejection, CD3 was one of the first molecules to be targeted with MAbs, and OKT3 (muromonab) was the first MAb to gain clinical approval for therapeutic use in humans.

Although the molecular target of OKT3 is singular and precise, its effects are many. The mechanism by which OKT3 mediates its immunosuppressive effect remains ill defined. OKT3 is an IgG2a mouse antibody that binds to the ε component of human CD3. On binding, the antibody mediates complement-dependent cell lysis and ADCC and, in doing so, rapidly clears T cells from the peripheral circulation. This binding event also leads to pan-T cell activation before their elimination, resulting in systemic cytokine release. The result is a marked cytokine release syndrome responsible for most of the adverse effects associated with the drug (see later).

When antigen binds to the TCR, TCR-CD3 internalization occurs; physiologically, this ensures that antigen binding is reflective of antigen burden and avoids activation mediated by continuous binding of a low-prevalence antigen. Similarly, OKT3 binding to CD3 leads to TCR-CD3 internalization. Uncleared T cells are often rendered void of surface TCR, are incapable of receiving a primary antigen signal, and are immunologically inert.

Bulk T cell clearance likely is not the primary mechanism of action of OKT3. Clinical rejection can occur with exceptionally low T cell counts achieved by other means, and stable graft function can occur with large T cell infiltrates within the graft itself. Although the peripheral circulation is rapidly cleared by OKT3, many T cells can be found in the periphery and in the allograft itself. A substantial amount of the rapid T cell clearance from the circulation is likely related to lymphocyte marginalization, perhaps induced by the cytokines released and by the methylprednisolone that is given with OKT3. The overall effect of OKT3 is likely an aggregate effect of interrupted TCR binding, TCR internalization, cytokine-mediated regulatory changes, disrupted trafficking, and cell depletion. OKT3 has proven efficacy as an induction and rescue agent. Its immunogenicity has prevented its use as a maintenance agent, and the drug is effective only in combination with other immunosuppressive compounds.

Interleukin-2 Receptor (CD25)-Specific Monoclonal Antibodies

The receptor for IL-2 is composed of three chains (α, β, and γ), of which the α and γ chains are constitutively expressed, and the β chain is induced with activation. The presence of the β chain, now designated as CD25, indicates prior T cell activation and identifies cells that have undergone some degree of effector maturation. CD25 has been targeted to suppress activated cells, while sparing resting cells.

Two commercially available anti-CD25 antibodies have been developed, both of which have been engineered to avoid antimurine antibody responses. Daclizumab is a humanized anti-CD25 IgG1, and basiliximab is a chimeric mouse–human anti-CD25 IgG1. Both agents avoid immune clearance and can be used for prolonged periods without inducing a neutralizing antibody. CD25 was the first molecule to be targeted successfully with a humanized MAb in transplantation. These agents also avoid the serum sickness associated with mouse-, rabbit-, or horse-derived proteins. Daclizumab was voluntarily withdrawn in 2009, largely based on market rather than biologic considerations. A subcutaneous form of daclizumab that may have future implications in transplantation was reintroduced and US Food and Drug Administration (FDA)-approved in 2016 for relapsing multiple sclerosis. Currently, basiliximab remains the only CD25-specific MAb available for use in transplantation. Studies for both of these agents are considered as the efficacy and mechanisms of action of these agents appear to be practically interchangeable.

Anti-CD25 antibodies are thought to work primarily through steric hindrance of IL-2 binding to CD25 and deprive T cells of this cytokine during early activation. There is little evidence for a depletional effect, or if there is one, it is limited to a few cells. More recently, it has become clear that CD25 induction is involved not only in the activation of cytotoxic T cells but also in the activation of cells with potentially salutary effects on the allograft, such as T regulatory cells. Previously activated T cells that are responding in an anamnestic response are less dependent on IL-2 for proliferation. Heterologous responses (cross-reactive responses between a previously encountered pathogen and an alloantigen) or memory alloimmune responses seem not to be affected significantly by CD25 interruption. Given this biology, primarily focused on naïve T cell early activation, CD25-directed antibodies have found a role in induction, but have no role in the treatment of established rejection. Although there has been anecdotal experience using these antibodies for maintenance immunosuppression in the setting of CNI toxicity with recurrent rejection, no study has formally evaluated this approach.

Alemtuzumab (Humanized Anti-CD52)

Given the reduction in rejection achieved with prolonged polyclonal antibody-mediated T cell depletion, the ease of administration and consistency of MAbs, and the benefits of humanization, clinicians have sought agents with a combination of these traits. The CD52-specific humanized MAb alemtuzumab has emerged as a promising depletional MAb.

Alemtuzumab (Campath-1H) is a humanized IgG1 derivative of a rat antihuman CD52. CD52 is a nonmodulating, glycosylphosphatidylinositol-anchored membrane protein of unknown function found in high density on most T cells, B cells, and monocytes. CD52 is not found on hematopoietic precursor cells and does not seem to be an adhesion molecule; it is not necessary for T cell activation. Several versions of the nonhumanized anti-CD52 predecessors of alemtuzumab have been shown to be effective in mediating rapid T cell depletion and reversing steroid-resistant rejection. The humanized form has been studied in several indications and is currently approved for the treatment of multiple sclerosis and marketed as Lemtrada. Initially approved in 2001 to treat lymphogenous malignancies, it was withdrawn from the market in 2014. It remains available for this indication through the worldwide Campath Distribution Program. Although not approved for use in solid-organ transplantation, alemtuzumab has been used off-label as an induction agent. Its mechanism of action seems to be predominantly related to bulk T cell depletion, with lesser depletion of B cells and monocytes. It rapidly depletes CD52-expressing lymphocytes centrally and peripherally in renal transplant recipients. The use of alemtuzumab as a rescue drug is burgeoning, and there has been anecdotal investigation in this drug as a maintenance therapy. Alemtuzumab is currently unavailable for commercial use in transplantation, but is being supplied for transplant use by its maker (Sanofi).

Rituximab (Humanized Anti-CD20)

Rituximab is a chimeric MAb-specific for CD20. CD20 is a cell surface glycoprotein involved in B cell activation and maturation whose natural ligand is unknown. Similar to alemtuzumab, it has been developed and approved for use in lymphogenous malignancies, particularly CD20 ⁺ B cell lymphomas and PTLD. Given its specificity for B cells (and despite its lack of specificity for antibody-producing plasma cells), rituximab has been suggested to be a therapy for antibody-mediated rejection and rejections involving vasculitis. Rituximab also has been used in regimens designed to facilitate transplantation in sensitized individuals, such as ABO-incompatible donor–recipient pairs or transplants across a positive crossmatch after antibody removal. Lastly, rituximab has been used with varying efficacy to treat recurrent diseases posttransplantation, including membranous nephropathy, focal segmental glomerulosclerosis (FSGS), antineutrophil cytoplasmic autoantibody mediated vasculitis, and IgA nephropathy. At present, the role of rituximab in transplantation is largely investigational; however, similar to alemtuzumab, its off-label use is increasing considerably, despite a randomized trial adding rituximab to standard immunosuppression that showed a marked increase in rejection in the rituximab arm.

The mechanism of action of rituximab is presumed to be depletional, primarily through induced apoptosis. Treatment with this antibody rapidly and specifically clears CD20 ⁺ cells from the circulation. The role of CD20 ⁺ cells in alloimmune responses is currently incompletely defined. Although these cells are precursors to antibody-producing plasma cells, they do not produce antibody without further maturation. Their role in acute antibody production is not well established, and it is unlikely that they have a direct effector cell role in rejection. Several authors have documented CD20 ⁺ infiltrates as a marker for particularly recalcitrant acute rejection. These cells are known to have APC function and it has been postulated that they serve to facilitate intragraft antigen presentation. Although the mechanism of action for rituximab and recurrent disease remains unclear, rituximab has been shown to stabilize the renal podocyte by binding to SMPDL-3b protein, thereby decreasing proteinuria in FSGS. Currently, rituximab is being used in induction and rescue indications.

Belatacept

Maintenance

Belatacept is the first fusion protein to achieve FDA approval for use in kidney transplantation. It is a fusion of a recombinant, high-affinity form of the T cell coinhibitory molecule CTLA4 (CD152) fused to a human IgG Fc portion. Composed of two human proteins, it evokes little heterologous antibody response and can be given indefinitely. As such, belatacept is the first antibody or fusion protein in transplantation given as a maintenance agent rather than for induction or rescue. Indeed, belatacept has no efficacy as an induction or rescue agent per se, and will thus be discussed here only for its role as a maintenance immunosuppressant.

Belatacept has higher binding affinity to CD86 and CD80 than its parent compound abatacept, which is mostly used for rheumatologic diseases. Significant interest in belatacept was driven by a pivotal phase II study conducted to compare the efficacy of belatacept as a maintenance immunosuppressant to the standard CNI cyclosporine in combination with steroids, MMF, and a basiliximab induction. The study demonstrated that belatacept-based immunotherapy did not increase rates of acute rejection at 6 months and improved renal function at 1 year compared with cyclosporine. This laid the basis for a large, international phase III trial, the Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial (BENEFIT), designed to evaluate less intensive and more intensive belatacept- versus cyclosporine-based regimens in approximately 650 adult patients receiving kidney transplants from living or standard criteria deceased donors. Overall, the trial was a success. Belatacept immunotherapy resulted in significantly higher patient and graft survival, and mean estimated glomerular filtration rate over the 7-year period ( Figs. 19.5 and 19.6 ). Patients who received expanded criteria donor kidneys (i.e., older age and more comorbid conditions than standard criteria donors, BENEFIT-EXT) and were treated with belatacept experienced similar long-term benefits for renal allograft function, although there was no difference in patient or allograft survival compared with cyclosporine at 7 years. Additionally, patients treated with belatacept had lower rates for developing de novo donor-specific antibodies, likely due to suppression of follicular helper T cells and B cell clonal expansion in germinal centers of the lymph nodes and spleen.

Long-term patient and graft survival for belatacept- versus cyclosporine-based regimens from the BENEFIT trial.

From Vincenti F, Larsen C, Durrbach A, et al. Costimulation blockade with belatacept in renal transplantation. N Engl J Med 2005;353:770–81. Used with permission.

Patients on belatacept-based regimens had a significant increase in mean estimated glomerular filtration rate compared with cyclosporine over an 84-month study period in the BENEFIT trial.

From Vincenti F, Rostaing L, Grinyo J, et al. Belatacept and long-term outcomes in kidney transplantation. N Engl J Med 2016;374:333–43. Used with permission.

Belatacept was generally well tolerated by patients. The long-term safety profile of belatacept was comparable to cyclosporine with similar rates of adverse events, including serious infections and cancers. As there was an increased risk of posttransplant lymphoproliferative disorder in EBV-seronegative patients, belatacept is only approved for EBV-seropositive recipients. Surprisingly, the rates and grades of acute allograft rejection were higher in the belatacept-based more intensive (MI) and less intensive (LI) regimens (22% and 17%, respectively) versus the cyclosporine group (7%), and CD57 ⁺ CD4 T cells have been implicated ( Fig. 19.7 ). The majority of acute rejection episodes occurred early (within the first 3 months), showed no sign of recurrence, and resolved with treatment. More importantly, the use of depletional therapy in conjunction with belatacept has eliminated the risk of acute rejection in some exploratory studies.

Increased rate and grade of rejection in patients treated with belatacept from the BENEFIT trial. Shown are rejection rates and grades for belatacept LI (less intensive)-treated patients compared with cyclosporine-treated patients. Belatacept is associated with more rejection and with higher grades of rejection.

Other exploratory open-label phase II trials have examined whether the use of belatacept-based immunosuppression can entirely preclude CNIs and corticosteroids. One study randomized recipients of living and standard criteria deceased donors to receive belatacept-MMF, belatacept-sirolimus, or tacrolimus (TAC)-MMF, a standard steroid avoidance regimen. The belatacept-MMF group had a higher rate of acute rejection at 6 months (12%) versus the belatacept-sirolimus (4%) and TAC-MMF (3%) groups, although 12% is much lower than reported in other CNI- or steroid-avoiding regimens. Belatacept-based therapy also appeared to confer superior renal function, compared with TAC. Another study investigated a combined therapy with alemtuzumab, sirolimus, and belatacept, indicating that this base regimen facilitates a low rate of steroid-sensitive acute rejection and preserves excellent 3-year allograft function. It also facilitates a long-term therapy of belatacept monotherapy in selected recipients of living donor kidneys. Belatacept monotherapy is particularly attractive for nonadherent EBV-seropositive adolescents. Furthermore, anecdotal reports have shown that adolescents who are nonadherent with their oral immunosuppression will comply with a monthly belatacept infusion. A pharmacokinetic, safety, and tolerability study of belatacept in adolescents has recently been completed and a conversion study is imminent.

CD2-Specific Approaches

CD2, also known as LFA-2, is an adhesion molecule expressed on T cells and natural killer cells that binds to CD58 (LFA-3) on APCs and facilitates TCR binding and signal transduction. It has been targeted by the rat IgG2b anti-CD2 MAb BTI-322, and more recently by siplizumab (also known as MEDI-507), a humanized IgG1 version of BTI-322. BTI-322 was investigated initially as an induction and rescue agent for deceased donor renal and hepatic allografts, and for graft-versus-host disease, and was shown to have biologic activity and to give results consistent with standard therapies available at the time.

Clinical trials in psoriasis using siplizumab began in 1999 and were met with an unexpected propensity toward agent immunogenicity. This agent has been used in nonhuman primate transplant tolerance trials with success in mixed chimerism-directed approaches and human trials are ongoing. It has been used clinically as part of a nonmyeloablative conditioning regimen to achieve mixed hematopoietic chimerism. Siplizumab has also been investigated in phase I and II trials for T lymphocytic malignancies, graft-versus-host disease, and psoriasis.

Alefacept is a human fusion protein of the CD2 ligand (CD58, LFA-3) with IgG1 shown to inhibit T cell proliferation. Its administration has also been shown to have a relative selective depleting effect on effector memory T cells, the same cells that have been relatively spared by other depleting MAbs and polyclonal preparations. It gained increased attention more recently in experimental transplantation. Alefacept is currently approved for the treatment of plaque-like psoriasis. Preclinical trials in nonhuman primate transplantation have shown that alefacept has minimal effect on graft survival when used alone, but that it does extend graft survival when used with adjuvant therapies. In particular, its pairing with belatacept was shown to eliminate belatacept-resistant memory T cells and facilitate belatacept-based maintenance therapy in nonhuman primates and in vitro in humans. Moreover, it has been effective in promoting immunosuppression-free renal allograft survival in nonhuman primates.

Alefacept was studied in a phase II trial pairing it with TAC-based maintenance immunosuppression. As the addition of alefacept was associated with an increased risk of malignancy and no clear efficacy benefit, its development in transplantation was halted.

CD3-Specific Antibodies

Targeting CD3 is a proven strategy, as shown by the success of OKT3. Significant effort has been directed toward modernizing the anti-CD3 approach to avoid the many side effects associated with CD3 activation. Several CD3-specific antibodies, including huOKT3γ1 (MGA031, teplizumab), aglycosyl CD3 (otelixizumab), visilizumab (HuM291), and foralumab (28F11-AE, NI-0401), have been humanized and otherwise engineered to eliminate their undesirable activating properties and immunogenicity. Phase I studies have indicated that modified versions of a CD3-specific antibody can achieve T cell depletion without the confounding problems of cytokine release or an antibody neutralization. Phase II trials using visilizumab in marrow transplantation have shown initial efficacy against graft-versus-host disease, and teplizumab has shown promise as a prophylactic agent in new-onset diabetes mellitus. These studies have shown that the side effects related to OKT3 use are not inherent in CD3-directed therapies, opening the door for more refined targeting of this receptor complex. Currently, teplizumab has completed a single clinical study in islet transplantation, the results of which have yet to be reported. Visilizumab exhibited initial potential in phase III clinic trials for ulcerative colitis. Unfortunately, it was associated with cytokine release syndrome, infections, and vascular/cardiac symptoms, and further development was terminated. Foralumab, a fully humanized, oral anti-CD3 antibody, has been shown to increase regulatory T cells and decrease disease activity in patients with resistant chronic hepatitis C infection and nonalcoholic steatohepatitis (NASH). A phase III clinical trial will soon begin enrollment for NASH and type 2 diabetes mellitus. Its use to treat inflammatory and autoimmune disease shows promise and may have applications in transplantation.

CD4-Specific Antibodies

CD4 is a cell surface glycoprotein that binds to a monomorphic region of MHC class II molecules and in doing so stabilizes the interaction between the TCR and MHC class II. It is expressed on approximately two-thirds of peripheral T cells and has partially defined several functional T cell subsets, including helper T cells and T regulatory cells. CD4 is also expressed by peripheral monocytes and other APCs, where its function is poorly characterized. It likely plays a crucial role in facilitating cell-to-cell communication among lymphoid cells, and it has lesser effects on physiologic effector functions. Given its central role in cellular immune responses, CD4 has long been a target for immune manipulation, and several antibodies have been tested in transplantation. Generally, the efficacy has been exceptional in defined rodent models and modest in more clinically relevant settings; this may relate to the growing recognition that CD4 ⁺ T cells have a potential role in tempering immune responses.

Many studies have shown that anti-CD4 antibody induction dramatically inhibits the development of acute rejection in rodents, particularly when combined with a supplementary donor antigen, such as donor-specific transfusion. Given that the distribution of MHC class II molecules differs substantially between rodents and humans, however, these studies have not been predictive of the anticipated effect in humans. Depleting and nondepleting antibodies have shown an effect in experimental models, suggesting that cell elimination, disruption of cell–cell communication, or signal transduction through CD4 may be mechanistically relevant. Two humanized anti-CD4 preparations have shown significant prolongation of nonhuman primate renal allograft survival.

Initial clinical transplantation trials using anti-CD4 MAbs employed murine-derived antibodies, including OKT4A, BL4, MT151, and B-F5. Predictably, these agents were subject to immune clearance, but nevertheless were shown to lead to CD4 ⁺ T cell clearance. Regardless, patients experienced rejection rates of 50%, and the agents were not sufficiently efficacious to warrant further development. Subsequent trials investigating the humanized OKT4A and the chimeric cM-T412 have been evaluated in conjunction with cyclosporine-based maintenance therapy in kidney and heart transplant recipients. In both cases, the antibody was well tolerated, and treated patients had low rates of rejection, suggesting that this approach is promising. Antibody responses toward the remnant murine portions of the MAb, however, were surprisingly frequent. CD4 ⁺ T cell depletion was not achieved using OKT4A, but was common with cM-T412.

The mouse antihuman CD4 MAb, Max.16H5, has been tested in pilot fashion as a clinical rescue agent. Max.16H5 depletes CD4 ⁺ T cells and was associated with reversal of rejection in most treated patients. Neutralizing antibodies were not detected. No trials investigating humanized anti-CD4 MAbs have been reported for rescue therapy.

Many human or humanized CD4-specific MAbs, including HuMax-CD4 (zanolimumab), TNX355, MT412, and 4162 W94, have been evaluated in phase I, II, and III trials for nontransplant indications, such as psoriasis and rheumatoid arthritis, as well as oncologic indications. These studies have shown that CD4-specific antibodies can influence immune responses and that their use is relatively safe in humans. Currently, there are no active anti-CD4 MAb trials registered in transplantation.

Costimulation-Based Therapies

Interest in the costimulation pathways as targets for immune manipulation has exploded in recent years. Generally, these agents interfere with pathways that influence the outcome of antigen binding to the TCR. Costimulatory molecules can exert positive or negative influences on the efficiency of antigen presentation and recognition and alter the threshold for activation of naive T lymphocytes without having a primary activating or inhibitory function. Costimulatory molecule manipulation influences only cells with ongoing TCR activation and should affect only cells actively undergoing antigen recognition; this may allow for antigen-specific immune manipulation.

The most studied costimulatory receptor on T cells is CD28. It has two known ligands, CD80 (B7-1) and CD86 (B7-2), both of which are expressed on APCs. CD28 is constitutively expressed on most T cells and on ligation reduces the threshold for TCR activation. CD152 (cytotoxic T lymphocyte associated antigen 4 [CTLA-4]) is an induced molecule expressed on T cell activation that is structurally similar to CD28 and competitively binds CD80 and CD86, transmitting an inhibitory signal that acts to terminate the immune response. CD28 and CD152 serve reciprocal roles, both stimulated by the B7 molecules and facilitating (CD28) or quelling (CD152) a T cell response.

An additional receptor–ligand pair that has gained considerable attention involves CD40 and CD154. CD154, also known as CD40 ligand, is expressed on activated T cells and other cells, including platelets. CD40 is expressed on APCs. Although the specific effect of CD154 on T cells is incompletely defined, CD40 has a major influence on APC activation. CD40 ligation leads to marked APC activation, including increased expression of B7 molecules and MHC, and stimulatory cytokine production, greatly facilitating antigen presentation. CD154 is released from the alpha granules of activated platelets and acts locally to greatly augment alloimmune responses. It can serve as the sole source of CD154 responsible for rejection. CD154 exists as a large inducible reservoir that can be triggered by platelet activation and augment antigen presentation at the time of a traumatic injury, including a transplant procedure. Its release is local and governed by alpha degranulation.

Seminal studies using small animals and nonhuman primates revealed that simultaneous blockade of the CD28 and CD40 pathways with CTLA-4 Ig and anti-CD154 inhibited T cell responses, provided durable protection from allograft rejection and held promise for both induction and maintenance therapy. Furthermore, costimulation blockade with belatacept, a high-affinity CTLA4 fusion protein, combined with mammalian target of rapamycin (mTOR) inhibition promoted a sustained CNI-free, steroid-sparing immunomodulatory regimen for kidney transplantation in nonhuman primates. However, long-term host allograft tolerance with costimulation blockade has remained a challenge. The combination of anti-B7-1 and B7-2 antibodies with either cyclosporine A or sirolimus was not sufficient to induce long-term tolerance. Moreover, a regimen consisting of donor antigen-pulsed autologous regulatory dendritic cell infusions, CTLA 4 IgG, and tapered sirolimus did not show a beneficial effect on graft survival in nonhuman primates. Although most experimental use of MAbs directed against costimulatory molecules in transplantation has focused on tolerance induction (elimination of a need for any maintenance therapy), the clinical focus has been on pairing costimulation-directed biologics with maintenance minimization strategies, particularly calcineurin-sparing approaches. Agents interfering with the CD28/B7 and the CD40/CD154 pathways have reached clinical trials.

Two humanized MAbs specific for CD154, hu5c8, and IDEC-131, have been shown in nonhuman primates to prevent acute rejection for months to years without additional immunosuppression and have been paired with sirolimus monotherapy and donor-specific transfusion to lead to operational tolerance in some cases. Early human trials with hu5c8 were hindered by unimpressive efficacy and concerns for thromboembolic risk.

CD154-specific therapies have not been studied clinically in recent years, and most preclinical attention has turned toward intervention with CD40 as opposed to CD154. Pursuant to CD40 blockade, ASKP1240 (also known as 4d11) and CFZ533 have reached the clinical arena. ASKP1240 has completed phase I and II trials, whereas CFZ533 is undergoing phase II trials. ASKP1240 and CFZ533 are fully human CD40-specific MAbs that have been shown in numerous nonhuman primate studies to prevent kidney and liver allograft rejection, particularly when used in combination with tacrolimus. Investigational interest in CD154 manipulation also remains intense.

A cocktail of two humanized MAbs specific for the B7 molecules CD80 and CD86 has been shown to facilitate prolonged renal allograft survival in nonhuman primates. These antibodies reached clinical trials in organ transplantation and were shown to have initial safety in humans. Their development has not been pursued.

Many other costimulatory molecules have been investigated successfully in animal models, but none has yet been exploited as a target in the clinic. Costimulatory molecules can be targeted with blocking MAbs to inhibit their stimulatory effects. Because it is difficult to determine prospectively whether a MAb is stimulatory or inhibitory in vivo, and because costimulatory molecules have stimulatory and inhibitory effects, it has been challenging to find therapeutically reliable agents. For example, CD152 and CD154 are upregulated on activated T cells and may serve as targets for selective elimination of activated effector cells. Selective targeting of CD152 with the MAb ipilimumab has been used to augment immunity in the setting of metastatic melanoma, with salutary oncologic results balanced by considerable induction of autoimmune side effects including enteritis and vasculitis. This situation likely relates to the fundamental role that costimulation molecules have in general immunity and immune homeostasis. Similarly, severe septic-like responses have been reported after the administration of TGN1412, a CD28-specific MAb tested in phase I trials. A fundamental balance between the two B7-specific T cell molecules CD28 and CD152 seems to be required to avoid dysregulated autoimmunity. However, the conceptual promise of CD28-specific blockade has led to the development of numerous agents, including single-chain domain-specific antibodies that have proven themselves efficacious in nonhuman primate transplantation. Their clinical translation is anticipated.

Cytokine-Based Approaches

Sequestration of cytokines using MAbs has long been contemplated as a therapeutic strategy in many inflammatory diseases. Although many cytokine-specific agents have been developed, only TNF-α-specific agents have gained widespread clinical use. TNF-α is a cytokine produced by many immune cells that is ubiquitously present in most inflammatory responses and has numerous general proinflammatory effects, including increased chemotaxis, vascular permeability, and fever. It has been considered as an attractive target for many inflammatory aspects of transplantation, including depletion-associated cytokine release syndrome, ischemia-reperfusion injury, and rejection. Three TNF-α-specific agents are currently approved for nontransplant conditions, and their use in transplantation is emerging, particularly in islet transplantation.

Infliximab is a chimeric IgG1 MAb that binds to cell-bound and circulating TNF-α, sequestering it from the TNF receptor and inhibiting TNF-dependent proinflammatory effects. It has been developed for the treatment of numerous autoimmune disorders, including rheumatoid arthritis (its primary approved clinical use), psoriasis, Crohn’s disease, and ulcerative colitis. It has been used in pilot studies of many transplant indications, including renal, bone marrow, intestinal, and islet transplantation, with suggestive success. Its predominant therapeutic effect in transplantation seems to be to limit paracrine cytokine-mediated activation within the graft and to mute the clinical sequelae of rejection without altering the overall infiltrate of inciting allosensitization. Similarly, adalimumab is a TNF-α-specific MAb approved for the treatment of psoriatic arthritis.

Etanercept is a soluble recombinant TNF receptor–IgG fusion protein that acts to absorb soluble TNF-α and limit its availability in the circulation. It is approved for the treatment of rheumatoid arthritis and has been increasingly evaluated for a role in the treatment of graft-versus-host disease. The use of etanercept in islet transplantation has been reported.

Golimumab is a fully human TNF-α-specific MAb that is in phase II trials for rheumatoid arthritis. No reports have been made of this agent in transplantation, although there are numerous trials in autoimmune indications.

Several other cytokine-directed MAbs are now approved for immune indications outside of transplantation and are likely to have relevance to transplantation. Ustekinumab is a humanized MAb specific for the p40 subunit common to the receptors for both IL-12 and IL-23. It is approved for clinical use in the treatment of psoriasis, particularly in patients who have failed therapy with etanercept. The IL-12/23 axis is increasingly recognized as relevant in transplantation, particularly in the setting of costimulation blockade resistant rejection, and it is likely that ustekinumab will be evaluated as an adjuvant therapy in combination with costimulation blockade based strategies in the foreseeable future. Similarly, tocilizumab is an IL-6 receptor MAb approved for use in rheumatoid arthritis. Given the role of IL-6 in alloantibody production, tocilizumab has been used to treat chronic antibody-mediated rejection and as part of a desensitization regimen in patients awaiting kidney transplantation with promising results. Additionally, belimumab, a MAb that inhibits BAFF, a cytokine critical to B cell activation, proliferation, and differentiation, mitigated autoantibody responses in systemic lupus erythematous. Both intravenous and subcutaneous formulations are currently FDA-approved, and current trials in both sensitized and nonsensitized kidney transplant recipients are either completed with results pending or ongoing.

Targeting Cell Adhesion

Given the fundamental requirement for adhesion molecules in most inflammatory responses, there has been long-standing interest in blocking adhesion interactions to prevent platelet and leukocyte adherence and infiltration. As discussed previously, polyclonal antibodies are thought to bind to and inhibit some adhesion molecules. Several MAbs have been developed to target adhesion pathways.

Among the most prominent is the LFA-1/ICAM-1 pathway. LFA-1 (the heterodimer integrin molecule CD11a/CD18) is expressed on mature T cells and binds to ICAM-1 (CD54) expressed on APCs and endothelial cells. The pathway greatly facilitates initial lymphocyte recruitment at sites of injury and inflammation. Adhesion pathways have been studied in several preclinical settings, including rodents and nonhuman primates, with survival being markedly prolonged in rodents and prolonged 30 days in primates. Data pairing the LFA-1-specific MAb TS1/22 with belatacept has led to exceptionally prolonged survival in nonhuman primate islet transplantation.

Enlimomab, a murine anti-CD54 MAb, was successfully tested in a phase I trial involving high-risk deceased donor kidneys and subsequently evaluated in a placebo-controlled phase II study combined with conventional triple-drug maintenance therapy. No significant difference was detected between the treated and the placebo groups, and further development was not pursued. Similarly, odulimomab, a murine anti-LFA-1 MAb, was studied as an induction agent compared with ATG-R in renal transplantation, with no significant difference being found between the groups. A single rescue trial using the anti-LFA-1 murine MAb 25-3 failed to show efficacy.

The most promising and well-studied LFA-1-specific MAb is efalizumab. Efalizumab (Raptiva) is a recombinant humanized MAb that binds to human CD11a and inhibits the LFA-1/ICAM-1 interaction. Efalizumab has been tested in phase I/II studies for renal transplantation where it was paired with conventional triple immunosuppression. In that setting, the combined regimen was overly immunosuppressive and its development waned. It reemerged recently in two clinical islet trials as the centerpiece maintenance immunosuppressant. In these trials, the efalizumab-based regimen was shown to facilitate initial engraftment and function, and to prevent islet rejection successfully. Trials based on efalizumab as a calcineurin alternative were initiated in kidney transplantation but had to be halted when the maker of efalizumab withdrew the drug from the market. It was used clinically and approved by the FDA for treatment of mild-to-moderate psoriasis. However, its use was associated with a low incidence of progressive multifocal leukoencephalopathy (approximately 1 in 10,000 exposures), which was cited as an undue risk for patients with psoriasis. In general, this risk profile, although perhaps adverse for psoriasis, is acceptable in kidney transplantation, and there is genuine hope that this agent will resurface for use in transplant indications in the future.

Other integrin-based adhesion molecules have been shown to be clearly involved in peripheral immune responses, the most studied of which is VLA4 (α ₄ -integrin). The MAb natalizumab targets this molecule and has gained FDA approval for the treatment of multiple sclerosis. It was subsequently withdrawn from the market due to an elevated risk of progressive multifocal encephalopathy, but returned with a more robust warning relative to this side effect. VLA-4-specific MAbs have been shown to attenuate costimulation blockade resistant rejection in mice, and promising preliminary results in nonhuman primates have been observed. Its availability for off-label use in the clinic may facilitate clinical transplant trials.

PSGL1-Ig (YSPSL) is a fusion protein combining the extracellular domains of P-selectin glycoprotein ligand-1 (CD162) with the Fc portion of IgG1. CD162 is a ligand for P-selectin, E-selectin, and L-selectin, all of which have been shown to facilitate leukocyte and platelet adhesion. Because cell adhesion has been implicated as a primary event in reperfusion injury and in allorecognition, this drug has been contemplated as a therapy to limit the effect of events occurring during initial implantation. Treatment with PSGL1-Ig has been shown to attenuate ischemia-reperfusion injury, most prominently in rodent models of hepatic warm ischemia. Recent testing in a phase I/II evaluation in liver transplantation has shown that this agent attenuates the clinical and biochemical syndrome of hepatic ischemia-reperfusion injury. In kidney transplantation, PSGL-1Ig was well tolerated and safe, though it did not decrease the rate of delayed graft function requiring dialysis.

Targeting the T Cell Receptor

T cells bind their cognate antigen through their heterodimeric glycoprotein TCR. There are two general forms, an α/β form, expressed on 95% of peripheral T cells and responsible for specifying most alloimmune responses, and a γ/δ form, involved in innate immune responses and appearing late in allograft rejection. The TCR is a result of somatic gene rearrangement similar to that seen in antibody formation, and the specificity of each T cell can be defined by its individual TCR. Rejections based on specific TCR/MHC interactions select for specific TCR types, showing that each MHC mismatch is recognized by a few clones, rather than by the entire T cell repertoire. Although this finding fostered initial enthusiasm for targeting antigen-specific T cells through custom MAbs specific for a given TCR, this approach has been deemed impractical given the vast number of TCRs generated during T cell maturation and their variable cross-reactivity with variable MHC polymorphisms. Nevertheless, the success of targeting TCR-associated proteins such as CD3 has generated some interest in targeting monomorphic portions of the TCR directly. The realization that TCR signaling is required for T cell apoptosis and regulation has made preservation of the TCR a competing strategy.

T10B9, also known as Medi-500, is a murine IgM specific for a monomorphic determinant on α/β and γ/δ TCRs. It is effective in mediating T cell depletion in vitro and in vivo and has been studied as a rescue and induction agent in renal and cardiac transplantation. In both trials, the antibody-mediated T cell depletion was well tolerated. Its efficacy as a rescue agent seemed to be similar to that of OKT3, and the cardiac trial suggested efficacy as an induction agent. Nevertheless, the agent has not been developed further in organ transplantation, likely as a result of comparably effective humanized MAbs. T10B9 has been studied as a conditioning agent and ex vivo depletional agent for bone marrow transplantation.

TOL101 is an IgM MAb specific for the α/β TCR. It has been shown to have numerous depletional and nondepletional effects on human T cells. In kidney transplantation, TOL101 used for induction therapy was well-tolerated overall and had an acceptable rate of acute cellular rejections (13.9%) without development of donor-specific antibodies. Moreover, TOL101 was not associated with patient or allograft loss after 6 months. Long-term safety and efficacy studies are required to assess its clinical utility.

Targeting Complement

Proteins of the complement cascade have long been known to be crucial in mediating antibody-associated cytotoxicity. Many approaches have been contemplated to achieve complement elimination in the setting of antibody presensitization, including plasmapheresis and intravenous immunoglobulin administration. More recently, it has been shown that complement, specifically that produced locally within the kidney itself, is a contributing factor facilitating peripheral T cell maturation and rejection. Polymorphisms in complement expression have been shown to influence the incidence of rejection and renal allograft survival in ways not previously recognized.

Three complement-specific agents have been used clinically and have been shown to be biologically active with promise for application in transplantation. Eculizumab is a humanized MAb specific for C5a, a key initiation factor in complement membrane attack complex formation. It has been shown to be effective therapy for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome, and it is FDA-approved for both indications in children and adults. Numerous single-center studies and case reports have previously shown that eculizumab, when used as part of a multimodal treatment strategy, can facilitate the prevention or reversal of numerous complement-mediated maladies in transplantation, including hemolytic uremic syndrome (HUS) and antibody-mediated rejection. Although the results with eculizumab for desensitization and chronic antibody-mediated rejection have been disappointing, its use for selected cases of acute antibody-mediated rejection and long-term recurrent atypical HUS remains promising. Accordingly, it is likely that this agent will become an established part of the transplant armamentarium in the future for these indications.

TP-10 (soluble complement receptor type 3) is a recombinant soluble protein that binds and inactivates the central activating component of the complement cascade, C3. It has been used in numerous preclinical settings and shown to be effective in preventing humoral xenograft rejection in a pig-to-nonhuman-primate model. It was investigated in a clinical trial for its role in preventing cardiopulmonary bypass-related complications, with disappointing results. Despite its clear ability to arrest C3-mediated complement activation, it has not been systematically investigated in clinical transplantation.

Inhibition at the level of C3 and C5 does not prevent the generation of important upstream inflammatory mediators. Therefore therapeutic strategies for antibody-mediated rejection have recently focused on upstream targets of the complement pathway such as the C1 complex, which consists of C1q, C1r, and C1s, and is the first to interact with donor-specific antibody on the endothelium. Preclinical in vitro studies have shown that blockade of C1 prevented antibody-mediated complement activation using sera from sensitized patients. Pilot studies with C1 inhibitor and anti-C1s monoclonal antibody have shown modest results with decreasing delayed graft function, short-term prevention of chronic transplant glomerulopathy, and reduction of C4d deposition in renal allografts. Multicenter studies to confirm efficacy are ongoing. Inhibiting the interaction between complement and donor-specific antibody is an alternative strategy for reducing complement-mediated allograft damage. Recently a novel approach using Ig-G enzyme derived from Streptococcus pyogenes (IdeS) as an adjunctive therapy for desensitization was associated with significant antibody reduction and allowed for HLA-incompatible transplantation in 24 of 25 patients. IdeS is an endopeptidase which cleaves human IgG into F(ab′)2 and Fc fragments, inhibiting complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. These promising findings are being validated in an ongoing phase II trial.