F. Leopardi

Alefacept promotes co-stimulation blockade based allograft survival in nonhuman primates

Memory T cells promote allograft rejection particularly in co-stimulation blockade–based immunosuppressive regimens. Here we show that the CD2-specific fusion protein alefacept (lymphocyte function–associated antigen-3–Ig; LFA -3–Ig) selectively eliminates memory T cells and, when combined with a co-stimulation blockade–based regimen using cytotoxic T lymphocyte antigen-4 (CTLA-4)-Ig, a CD80- and CD86-specific fusion protein, prevents renal allograft rejection and alloantibody formation in nonhuman primates. These results support the immediate translation of a regimen for the prevention of allograft rejection without the use of calcineurin inhibitors, steroids or pan–T cell depletion.

Islet xenotransplantation using gal-deficient neonatal donors improves engraftment and function.

Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose‐α1,3‐galactose, the gal epitope, has emerged as the principal target of rejection in pig‐to‐primate models of solid organ transplant, the importance of gal‐specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal‐expressing wild‐type (WT) or gal‐deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin‐independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies.

LFA-1–specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.

IDEC‐131 (Anti‐CD154), Sirolimus and Donor‐Specific Transfusion Facilitate Operational Tolerance in Non‐Human Primates

CD154‐specific antibody therapy prevents allograft rejection in many experimental transplant models. However, initial clinical transplant trials with anti‐CD154 have been disappointing suggesting the need for as of yet undetermined adjuvant therapy. In rodents, donor antigen (e.g., a donor blood transfusion), or mTOR inhibition (e.g., sirolimus), enhances anti‐CD154s efficacy. We performed renal transplants in major histocompatibility complex‐(MHC) mismatched rhesus monkeys and treated recipients with combinations of the CD154‐specific antibody IDEC‐131, and/or sirolimus, and/or a pre‐transplant donor‐specific transfusion (DST). Therapy was withdrawn after 3 months. Triple therapy prevented rejection during therapy in all animals and led to operational tolerance in three of five animals including donor‐specific skin graft acceptance in the two animals tested. IDEC‐131, sirolimus and DST are highly effective in preventing renal allograft rejection in primates. This apparently clinically applicable regimen is promising for human renal transplant trials.

Nondepleting Anti‐CD40‐Based Therapy Prolongs Allograft Survival in Nonhuman Primates

Costimulation blockade of the CD40/CD154 pathway has been effective at preventing allograft rejection in numerous transplantation models. This strategy has largely depended on mAbs directed against CD154, limiting the potential for translation due to its association with thromboembolic events. Though targeting CD40 as an alternative to CD154 has been successful at preventing allograft rejection in preclinical models, there have been no reports on the effects of CD40‐specific agents in human transplant recipients. This delay in clinical translation may in part be explained by the presence of cellular depletion with many CD40‐specific mAbs. As such, the optimal biologic properties of CD40‐directed immunotherapy remain to be determined. In this report, we have characterized 3A8, a human CD40‐specific mAb and evaluated its efficacy in a rhesus macaque model of islet cell transplantation. Despite partially agonistic properties and the inability to block CD40 binding of soluble CD154 (sCD154) in vitro, 3A8‐based therapy markedly prolonged islet allograft survival without depleting B cells. Our results indicate that the allograft‐protective effects of CD40‐directed costimulation blockade do not require sCD154 blockade, complete antagonism or cellular depletion, and serve to support and guide the continued development of CD40‐specific agents for clinical translation.

Alternative immunomodulatory strategies for xenotransplantation: CD40/154 pathway-sparing regimens promote xenograft survival.

Immunosuppressive therapies that block the CD40/CD154 costimulatory pathway have proven to be uniquely effective in preclinical xenotransplant models. Given the challenges facing clinical translation of CD40/CD154 pathway blockade, we examined the efficacy and tolerability of CD40/CD154 pathway‐sparing immunomodulatory strategies in a pig‐to‐nonhuman primate islet xenotransplant model. Rhesus macaques were rendered diabetic with streptozocin and given an intraportal infusion of ∼50 000 islet equivalents/kg wild‐type neonatal porcine islets. Base immunosuppression for all recipients included maintenance therapy with belatacept and mycophenolate mofetil plus induction with basiliximab and LFA‐1 blockade. Cohort 1 recipients (n = 3) were treated with the base regimen alone; cohort 2 recipients (n = 5) were additionally treated with tacrolimus induction and cohort 3 recipients (n = 5) were treated with alefacept in place of basiliximab, and more intense LFA‐1 blockade. Three of five recipients in both cohorts 2 and 3 achieved sustained insulin‐independent normoglycemia (median rejection‐free survivals 60 and 111 days, respectively), compared to zero of three recipients in cohort 1. These data show that CD40/CD154 pathway‐sparing regimens can promote xenoislet survival. Further optimization of these strategies is warranted to aid the clinical translation of islet xenotransplantation.

A Novel Monoclonal Antibody to CD40 Prolongs Islet Allograft Survival

The importance of CD40/CD154 costimulatory pathway blockade in immunosuppression strategies is well‐documented. Efforts are currently focused on monoclonal antibodies specific for CD40 because of thromboembolic complications associated with monoclonal antibodies directed towards CD154. Here we present the rational development and characterization of a novel antagonistic monoclonal antibody to CD40. Rhesus macaques were treated with the recombinant anti‐CD40 mAb, 2C10, or vehicle before immunization with keyhole limpet hemocyanin (KLH). Treatment with 2C10 successfully inhibited T cell‐dependent antibody responses to KLH without significant peripheral B cell depletion. Subsequently, MHC‐mismatched macaques underwent intraportal allogeneic islet transplantation and received basiliximab and sirolimus with or without 2C10. Islet graft survival was significantly prolonged in recipients receiving 2C10 (graft survival time 304, 296, 265, 163 days) compared to recipients receiving basiliximab and sirolimus alone (graft survival time 8, 8, 10 days). The survival advantage conferred by treatment with 2C10 provides further evidence for the importance of blockade of the CD40/CD154 pathway in preventing alloimmune responses. 2C10 is a particularly attractive candidate for translation given its favorable clinical profile.

CD40 Blockade Combines with CTLA4Ig and Sirolimus To Produce Mixed Chimerism in an MHC-defined Rhesus Macaque Transplant Model

In murine models, T‐cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti‐CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, nondepleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well‐established primate bone marrow chimerism‐induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and nondepletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.

Belatacept and Sirolimus Prolong Nonhuman Primate Renal Allograft Survival Without a Requirement for Memory T Cell Depletion

Belatacept is an inhibitor of CD28/B7 costimulation that is clinically indicated as a calcineurin inhibitor (CNI) alternative in combination with mycophenolate mofetil and steroids after renal transplantation. We sought to develop a clinically translatable, nonlymphocyte depleting, belatacept‐based regimen that could obviate the need for both CNIs and steroids. Thus, based on murine data showing synergy between costimulation blockade and mTOR inhibition, we studied rhesus monkeys undergoing MHC‐mismatched renal allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on costimulation blockade‐resistant rejection, some animals also received CD2 blockade with alefacept (LFA3‐Ig). Belatacept and sirolimus therapy successfully prevented rejection in all animals. Tolerance was not induced, as animals rejected after withdrawal of therapy. The regimen did not deplete T cells. Alefecept did not add a survival benefit to the optimized belatacept and sirolimus regimen, despite causing an intended depletion of memory T cells, and caused a marked reduction in regulatory T cells. Furthermore, alefacept‐treated animals had a significantly increased incidence of CMV reactivation, suggesting that this combination overly compromised protective immunity. These data support belatacept and sirolimus as a clinically translatable, nondepleting, CNI‐free, steroid‐sparing immunomodulatory regimen that promotes sustained rejection‐free allograft survival after renal transplantation.

Belatacept and Sirolimus Prolong Nonhuman Primate Islet Allograft Survival: Adverse Consequences of Concomitant Alefacept Therapy

Calcineurin inhibitors (CNI) and steroids are known to promote insulin resistance, and their avoidance after islet transplantation is preferred from a metabolic standpoint. Belatacept, a B7‐specific mediator of costimulation blockade (CoB), is clinically indicated as a CNI alternative in renal transplantation, and we have endeavored to develop a clinically translatable, belatacept‐based regimen that could obviate the need for both CNIs and steroids. Based on the known synergy between CoB and mTOR inhibition, we studied rhesus monkeys undergoing MHC‐mismatched islet allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on CoB‐resistant rejection, some animals also received CD2 blockade with alefacept (LFA3‐Ig). Nine rhesus macaques were rendered diabetic with streptozotocin and underwent islet allotransplantation. All received belatacept and sirolimus; six also received alefacept. Belatacept and sirolimus significantly prolonged rejection‐free graft survival (median 225 days compared to 8 days in controls receiving basiliximab and sirolimus; p = 0.022). The addition of alefacept provided no additional survival benefit, but was associated with Cytomegalovirus reactivation in four of six animals. No recipients produced donor‐specific alloantibodies. The combination of belatacept and sirolimus successfully prevents islet allograft survival in rhesus monkeys, but induction with alefacept provides no survival benefit and increases the risk of viral reactivation.