Allograft Survival In Nonhuman Primates Research Articles

P9.10: Mono-Maintenance Therapy With Anti-icam-1 Monoclonal Antibody Induced Long-term Liver Allograft Survival in Nonhuman Primates

P9.10: Mono-Maintenance Therapy With Anti-icam-1 Monoclonal Antibody Induced Long-term Liver Allograft Survival in Nonhuman Primates

Short-term therapy with anti-ICAM-1 monoclonal antibody induced long-term liver allograft survival in nonhuman primates

Tolerance induction remains challenging following liver transplantation and the long-term use of immunosuppressants, especially calcineurin inhibitors, leads to serious complications. We aimed to test an alternative immunosuppressant, a chimeric anti-ICAM-1 monoclonal antibody, MD-3, for improving the outcomes of liver transplantation. We used a rhesus macaque liver transplantation model and monkeys were divided into three groups: no immunosuppression (n=2), conventional immunosuppression (n=4), and MD-3 (n=5). Without immunosuppression, liver allografts failed within a week by acute rejection. Sixteen-week-long conventional immunosuppression that consisted of prednisolone, tacrolimus, and an mTOR inhibitor prolonged liver allograft survival; however, recipients died of acute T cell-mediated rejection (day 52), chronic rejection (days 62 and 66), or adverse effects of mTOR inhibitor (day 32). In contrast, 12-week-long MD-3 therapy with transient conventional immunosuppression in the MD-3 group significantly prolonged the survival of liver allograft recipients (5, 96, 216, 412, 730days; p=.0483). MD-3 effectively suppressed intragraft inflammatory cell infiltration, anti-donor T cell responses, and donor-specific antibody with intact anti-cytomegalovirus antibody responses. However, this regimen ended in chronic rejection. In conclusion, short-term therapy with MD-3 markedly improved liver allograft survival to 2years without maintenance of immunosuppressant. MD-3 is therefore a promising immune-modulating agent for liver transplantation.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n=56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3mg/kg), or subcutaneous iscalimab (3mg/kg), or placebo. Rheumatoid arthritis patients (n=20) received single doses of intravenous iscalimab (10 or 30mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4µg/mL. In subjects receiving 3mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Donor-Derived Regulatory Dendritic Cell Infusion Maintains Donor-Reactive CD4+CTLA4hi T Cells in Non-Human Primate Renal Allograft Recipients Treated with CD28 Co-Stimulation Blockade.

Donor-derived regulatory dendritic cell (DCreg) infusion before transplantation, significantly prolongs renal allograft survival in non-human primates. This is associated with enhanced expression of the immunoregulatory molecules cytotoxic T-lymphocyte-associated antigen (Ag) 4 (CTLA4) and programmed cell death protein 1 (PD1) by host donor-reactive T cells. In rodents and humans, CD28 co-stimulatory pathway blockade with the fusion protein CTLA4:Ig (CTLA4Ig) is associated with reduced differentiation and development of regulatory T cells (Treg). We hypothesized that upregulation of CTLA4 by donor-reactive CD4+ T cells in DCreg-infused recipients treated with CTLA4Ig, might be associated with higher incidences of donor-reactive CD4+ T cells with a Treg phenotype. In normal rhesus monkeys, allo-stimulated CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells exhibited a regulatory phenotype, irrespective of PD1 expression. CTLA4Ig significantly reduced the incidence of CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells following allo-stimulation, associated with a significant reduction in the CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio. In CTLA4Ig-treated renal allograft recipient monkeys, there was a marked reduction in circulating donor-reactive CD4+CTLA4hi T cells. In contrast, in CTLA4Ig-treated monkeys with DCreg infusion, no such reduction was observed. In parallel, the donor-reactive CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio was reduced significantly in graft recipients without DCreg infusion, but increased in those given DCreg. These observations suggest that pre-transplant DCreg infusion promotes and maintains donor-reactive CD4+CTLA4hi T cells with a regulatory phenotype after transplantation, even in the presence of CD28 co-stimulation blockade.

Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells.

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.

ASP0028 in combination with suboptimal-dose of tacrolimus in Cynomolgus monkey renal transplantation model

FTY720, a S1P-receptor modulator, has shown to be effective in several transplant and autoimmune disease models, via modulating lymphocyte homing into secondary lymphoid organs (SLOs), and thereby reducing these cells in peripheral blood. ASP0028, a newly developed S1P1/S1P5-selective agonist, presented comparable efficacy to FTY720 and wider safety margins than FTY720. In this study, we assessed the efficacy and safety of ASP0028 co-administered with suboptimal-dose of tacrolimus in the Cynomolgus monkey renal transplantation model. Seven animals in group-1 or group-2 received mono-tacrolimus 1.0mg/kg once a day (QD), or ASP0028 0.6mg/kg plus tacrolimus 1.0mg/kg QD, respectively. Eight animals in group-3 received ASP0028 1.2mg/kg plus tacrolimus 1.0mg/kg QD. The allograft median survival time (MST) in group-2 and group-3 were significantly extended to 41 and 61.5days, versus that of 28days in group-1 (p=0.036 and 0.001, respectively). ASP0028 administration remarkably reduced absolute numbers of peripheral lymphocytes, particularly subsets of CD4+/ or CD8+/naive and central memory cells, CD4+/Treg cells, and to a lesser extent on B cells, but not CD4+/ or CD8+/effector memory cells and NK cells. These data show ASP0028 combined with suboptimal-dose of tacrolimus effectively prolongs renal allograft survival in nonhuman primates (NHPs) with well tolerated safety, supporting its further investigation to optimize CNI-sparing regimens.

Targeted deletion of Traf2 allows immunosuppression-free islet allograft survival in mice.

Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses. T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4+ T cell effector responses that mediate islet allograft rejection in vivo. In vitro allograft responses were tested using mixed lymphocyte reactions and analysis of IFN-γ and granzyme B effector molecule expression. T cell function was assessed using anti-CD3/CD28-mediated proliferation and T cell polarisation studies. Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4+ T cells exhibited reduced proliferation, activation and acquisition of effector function following T cell receptor stimulation; however, both Traf2TKO CD4+ and CD8+ T cells exhibited impaired alloantigen-mediated proliferation and acquisition of effector function. In polarisation studies, Traf2TKO CD4+ T cells preferentially converted to a T helper (Th)2 phenotype, but exhibited impaired Th17 differentiation. Without TRAF2, thymocytes exhibited dysregulated TNF-mediated induction of c-Jun N-terminal kinase (JNK) and canonical NFκB pathways. Critically, targeting TRAF2 in T cells did not impair the acute phase of CD8-dependent viral immunity. These data highlight a specific requirement for a TRAF2-NFκB and TRAF2-JNK signalling cascade in T cell activation and effector function in rejecting islet allografts. Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.

A Novel, Blocking, Fc-Silent Anti-CD40 Monoclonal Antibody Prolongs Nonhuman Primate Renal Allograft Survival in the Absence of B Cell Depletion

CD40-CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti-CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti-CD40 antibody can still promote graft survival. The parent anti-CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC-mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc-silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98-100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well-tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc-silent anti-CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.

B Cell Depletion Is Not Required for Prolongation of Renal Allograft Survival in Non-Human Primates Using a Novel Anti-CD40 Blocking Antibody.

CD40-CD154 pathway blockade significantly prolongs allograft survival in non-human primates (NHPs). However, antibodies targeting CD154, while efficacious, were associated with an increased incidence of thromboembolic complications precluding clinical development. Current antibodies targeting CD40 effectively prolong renal allograft survival in NHPs without associated thromboembolic events but with accompanying B cell depletion, raising the question of whether CD40 blockade or B cell depletion was the effective mechanism. We developed a novel, Fc-silent (i.e. incapable of antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity) anti-human CD40 monoclonal antibody (OM11-62MF). OM11 blocked recombinant CD154-mediated activation of leukocytes in vitro and inhibited T cell-dependent antibody responses in vivo in the absence of significant peripheral B cell depletion. We tested OM11 and an Fc-competent anti-CD40 antibody in MHC-mismatched cynomolgus monkey renal allograft transplantation as monotherapies. Allograft survival was significantly prolonged in animals dosed with the Fc-competent anti-CD40 antibody (52, 22, 24 days) in comparison to untreated monkeys (survival ˜ 7 days; n = 9). Well-functioning renal allografts survived up to 100 days in OM11-treated animals (100, 100, 100, 98, 78 days) at which point the experiment was terminated and graft morphology examined by histology. In contrast to good graft morphology in OM11-dosed group, acute cellular rejection was observed in animals treated with Fc-competent anti-CD40. Rapid and complete peripheral B cell depletion was only observed with the Fc-competent antibody, despite full peripheral CD40 receptor occupancy by both antibodies throughout the duration of the experiment. OM11 was well-tolerated and we observed no evidence of thromboembolic events. Collectively, our results indicated that CD40 pathway blockade in the absence of B cell depletion inhibited T cell-dependent immune responses and prevented allograft rejection. Thus, use of the Fc-silent anti-CD40 antibody OM11 appears to be an attractive approach for preventing solid organ transplant rejection and treating autoimmune diseases involving T cell-dependent humoral immune mechanisms. DISCLOSURES:Rush, J.: Employee, Novartis Pharmaceuticals Corporation. Cordoba, F.: Employee, Novartis Pharmaceuticals Corporation. Wieczorek, G.: Employee, Novartis Pharmaceuticals Corporation. Audet, M.: Employee, Novartis Pharmaceuticals Corporation. Schneider, M.: Employee, Novartis Pharmaceuticals Corporation. Patel, D.: Employee, Novartis Pharmaceuticals Corporation. Heusser, C.: Other, Novartis Pharmaceuticals Corporation, Contractor. Bruns, C.: Employee, Novartis Pharmaceuticals Corporation. Ulrich, P.: Employee, Novartis Pharmaceuticals Corporation. Slade, A.: Employee, Novartis Pharmaceuticals Corporation. Klupp, J.: Employee, Novartis Pharmaceuticals Corporation.

OM11-62MF: Safety, Tolerability, Pharmacokinetics (PK) and Pharmacodynamics (PD) of a Novel Anti-CD40 Antibody in Healthy Volunteers.

Blocking the CD40-CD154 co-stimulation pathway has been shown to effectively prolong renal allograft survival in non-human primates. OM11-62MF (OM11) is a novel, fully human anti-CD40 monoclonal antibody devoid of antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity activities (i.e., Fc-silent) being developed for use in transplantation. To assess the human safety, tolerability, PK and PD activity of OM11, single doses were administered to healthy male and female volunteers in a Phase 1 trial. METHODS: A double-blind, placebo controlled, time-lagged, ascending, single-dose study using intravenous infusions of 0.03, 0.1, 0.3, 1.0 and 3.0 mg/kg OM11 or 3.0 mg/kg OM11 subcutaneously was conducted. PK and CD40 receptor occupancy (RO) samples were collected during all treatment periods up to 180 days post dose. RESULTS: A total of 48 subjects were enrolled. All doses were well tolerated. There were no serious adverse events (SAEs) or discontinuations due to AEs. A total of 50 adverse events were reported by 64% (23/36) of OM11 treated subjects. The most frequent adverse events on OM11 were headache (12%) and injection site pain (8%). There were no clinically significant changes in laboratory results including coagulation parameters, thromboelastography or multiplex cytokine analysis. There was no effect on leukocyte subsets via FACS or B-cell depletion. OM11 PK concentrations were quantifiable at all dose levels tested. OM11 demonstrates classical target mediated elimination with accelerated clearance below 100% CD40 RO. The concentration-PD response results shown below, demonstrate sustained CD40 target suppression on peripheral B-cells through 28 days with the highest dose tested.Figure: No Caption available.CONCLUSION: The favorable safety and tolerability profile of both intravenous and subcutaneous OM11-62MF coupled with a predictable concentration-CD40 receptor occupancy relationship support future clinical trials of OM11 in transplantation. DISCLOSURE:Slade, A.: Employee, Novartis Pharmaceuticals Corp. Koo, P.: Employee, Novartis Pharmaceuticals Corp. Espie, P.: Employee, Novartis Pharma AG. Rush, J.: Employee, Novartis Pharma AG. Klupp, J.: Employee, Novartis Pharma AG. Lee, D.: Employee, Novartis Pharma AG.

Alefacept Promotes Immunosuppression-Free Renal Allograft Survival in Nonhuman Primates via Depletion of Recipient Memory T Cells

Renal allograft tolerance has been achieved in MHC-mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow transplantation (DBMT). To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel-conditioning regimen, the “delayed protocol” in which donor bone marrow (DBM) is transplanted several months after kidney transplantation. However, activation/expansion of donor-reactive CD8+ memory T cells (TMEM) occurring during the interval between kidney and DBM transplantation impaired tolerance induction using this strategy. In the current study, we tested whether, Alefacept, a fusion protein which targets LFA-3/CD2 interactions and selectively depletes CD2highCD8+ effector memory T cells (TEM) could similarly induce long-term immunosuppression-free renal allograft survival but avoid the deleterious effects of anti-CD8 mAb treatment. We found that Alefacept significantly delayed the expansion of CD2high cells including CD8+ TEM while sparing naïve CD8+ T and NK cells and achieved mixed chimerism and long-term immunosuppression-free renal allograft survival. In conclusion, elimination of CD2high T cells represents a promising approach to prevent electively the expansion/activation of donor-reactive TEM and promotes tolerance induction via the delayed protocol mixed chimerism approach.

Regulatory Dendritic Cell Infusion Prolongs Kidney Allograft Survival in Nonhuman Primates

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5–10 × 106/kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day −2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95+ T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

ASKP1240, a Fully Human Anti-CD40 Monoclonal Antibody, Prolongs Pancreatic Islet Allograft Survival in Nonhuman Primates

A strategy for inhibiting CD40 has been considered as an alternative approach for immunosuppression because of undesirable effects of anti-CD154 monoclonal antibodies (mAbs). Previously, we demonstrated that ASKP1240, which is a fully human anti-CD40 mAb, significantly prolonged kidney and liver allograft survival in cynomolgus monkeys without causing thromboembolic complications. Herein, we evaluated the effect of ASKP1240 on pancreatic islet transplantation (PITx) in cynomolgus monkeys. Diabetes was induced by total pancreatectomy, and islet allografts were transplanted into the liver. Following PITx (8201–12 438 IEQ/kg), blood glucose levels normalized promptly in all animals. Control islet allografts were rejected within 9 days (n = 3), whereas ASKP1240 (10 mg/kg) given on postoperative days 0, 4, 7, 11 and 14 (induction treatment, n = 5) significantly prolonged graft survival time (GST) to >15, >23, 210, 250 and >608 days, respectively. When ASKP1240 (5 mg/kg) was administered weekly thereafter up to post-PITx 6 months (maintenance treatment, n = 4), GST was markedly prolonged to >96, >115, 523 and >607 days. During the ASKP1240 treatment period, both anti-donor cellular responses and development of anti-donor antibodies were abolished, and no serious adverse events were noted. ASKP1240 appears to be a promising candidate for immunosuppression in clinical PITx.

Infused Bone Marrow Fails to Prolong Vascularized Composite Allograft Survival in Nonhuman Primates

Introduction: Clinical protocols for hand and face vascularized composite allografts (VCA) have included varying amounts donor bone marrow cell (BMC) infusion after lymphocyte depletion as an immunomodulatory strategy. We previously demonstrated that vascularized bone marrow (VBM) protects facial VCA from rejection in unconditioned nonhuman primates. To investigate the potential immunologic benefit of infused BMC, we substituted infused BMC for VBM in the transplanted facial segment. Methods: Three cynomolgus macaques underwent heterotopic facial segment transplantation with mandibular segment removed, followed by central venous infusion of BMC from the same MHC-mismatched donor on postoperative day 1. Bone marrow cells were collected from the donor lumbar vertebrae following euthanasia. Cells counts and viability were confirmed using trypan blue and automated complete blood count. Donor BMC were infused at 200 million cells per kg of recipient weight, a standard for bone marrow transplantation. CD34+ hematopoeitic stem and progenitor cells were identified using a modified ISHAGE gating strategy. Immunosuppression was maintained with tacrolimus and mycophenolate mofetil. Rejection episodes were not treated. Chimerism was measured in blood samples using flow cytometry. Results: Mean cell content of the mandibular segments set aside before transplantation was 48.2 million cells/kg of donor weight. Hematopoeitic stem and progenitor cells comprised 5.63% of vertebral bone marrow BMC and 7.60% of mandibular BMC. Animals in the previous VCA+VBM treatment group had no graft loss while on immunosuppression. Additionally, graft survival was longer vs. VCA alone (205-430 vs. 9-42 days), as was rejection-free survival (44-358 vs. 7-15 days). In the VCA + BMC group all animals developed acute rejection (Banff grade I or higher) by day 14. One progressed to grade IV rejection and graft loss by day 28, while another resolved but progressed to chronic rejection by day 50 and endpoint at day 115 for post-transplant lymphoproliferative disease with chronic rejection. Only one animal receiving BMC demonstrated transient macrochimerism days 85-98. Chimerism was similarly observed transiently in only one animal receiving VCA without either VBM or BMC. In contrast, 3 of 4 animals with VBM demonstrated macrochimerism.Figure: [Graft Survival]Conclusion: Despite larger cell counts, BMC infusion failed to prolong facial VCA survival compared to VBM. These data support that VBM is a more effective immunomodulatory strategy than infusion of donor BMC without depletional pre-conditioning. The addition of irradiation and T cell depletion therapies to VBM may further enhance the amount and durability of chimerism, and potentially allow for the development tolerance.

Long-Term Hepatic Allograft Acceptance Based on CD40 Blockade by ASKP1240 in Nonhuman Primates

Blockade of the CD40-CD154 costimulatory signal is an attractive strategy for immunosuppression and tolerance induction in organ transplantation. Treatment with anti-CD154 monoclonal antibodies (mAbs) results in potent immunosuppression in nonhuman primates (NHPs). Despite plans for future clinical use, further development of these treatments was halted by complications. As an alternative approach, we have been focusing on the inhibition of the counter receptor, CD40 and have shown that a novel human anti-CD40 mAb, ASKP1240, markedly prolongs renal allograft survival in NHPs, although allografts eventually underwent chronic allograft nephropathy. On the basis of our previous findings that a CD40-CD154 costimulation blockade induces tolerance to hepatic, but not cardiac, allografts in rodents, we tested here our hypothesis that a blockade of CD40 by ASKP1240 allows acceptance of hepatic allografts in NHPs. A 2-week ASKP1240 induction treatment prolonged liver allograft survival in NHPs; however, the graft function deteriorated due to chronic rejection. In contrast, a 6-month ASKP1240 maintenance monotherapy efficiently suppressed both cellular and humoral alloimmune responses and prevented rejection on the hepatic allograft. No serious side effects, including thromboembolic complications, were noted in the ASKP1240-treated monkeys. We conclude that CD40 blockade by ASKP1240 would be a desirable immunosuppressant for clinical liver transplantation.

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.

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

Memory T-cells promote allograft rejection particularly in costimulation blockade (CoB)-based immunosuppressive regimens. Here we show that the CD2-specific fusion protein alefacept (LFA3-Ig) selectively eliminates memory T-cells and when combined with a CoB-based regimen utilizing CTLA4-Ig, prevents renal allograft rejection and alloantibody formation in primates. These results support the development of an immediately translatable regimen for the prevention of allograft rejection without the use of calcineurin inhibitors, steroids, or pan-T-cell depletion.

Portal Venous Donor-Specific Transfusion in Conjunction with Sirolimus Prolongs Renal Allograft Survival in Nonhuman Primates

Pretransplant exposure to donor antigen is known to modulate recipient alloimmunity, and frequently results in sensitization. However, donor-specific transfusion (DST) can have a protolerant effect that is dependent on route, dose and coadministered immunosuppression. Rodent studies have shown in some strain combinations that portal venous (PV) DST alone can induce tolerance, and uncontrolled clinical use of PVDST has been reported. In order to determine if pretransplant PVDST has a clinically relevant salutary effect, we studied it and the influence of concomitant immunosuppression in rhesus monkeys undergoing renal allotransplantation. Animals received PVDST with unfractionated bone marrow and/or tacrolimus or sirolimus 1 week prior to transplantation. Graft survival was assessed without any posttransplant immunosuppression. PVDST alone or in combination with tacrolimus was ineffective. However, PVDST in combination with sirolimus significantly prolonged renal allograft survival to a mean of 24 days. Preoperative sirolimus alone had no effect, and peripheral DST with sirolimus prolonged graft survival in 2/4 animals, but resulted in accelerated rejection in 2/4 animals. These data demonstrate that PVDST in combination with sirolimus delays rejection in a modest but measurable way in a rigorous model. It may thus be a preferable method for donor antigen administration.

B lymphocyte–directed immunotherapy promotes long-term islet allograft survival in nonhuman primates

We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.

A Novel Fully Human Anti-CD40 Monoclonal Antibody, 4D11, for Kidney Transplantation in Cynomolgus Monkeys

CD40-CD154 pathway blockade by anti-CD154 monoclonal antibodies (mAbs) significantly prolongs allograft survival in nonhuman primates. However, thromboembolic complications have prevented clinical application. Thus, blockade of the counter molecule by a novel fully human anti-CD40 mAb, 4D11, is an attractive alternative. Kidney transplantations were performed between outbred cynomolgus monkeys (stimulation index >3 in a mixed lymphocyte reaction). The animals were divided into five groups: nontreatment control (Group 1, n=3), 10-week treatment with either 10 mg/kg (Group 2, n=3), 20 mg/kg (Group 3, n=3), or 40 mg/kg (Group 4, n=1), and 4-week treatment (Group 5, n=1 each) with 10 mg/kg, 20 mg/kg, or 40 mg/kg followed by monthly administration. Graft survival, biochemistry, complete blood counts, lymphocyte phenotypes, blood drug levels, antidonor and antidrug antibodies, and renal histology were examined. Survival (days) was as follows: Group 1 (5, 6, 7), Group 2 (150, 108, 108), Group 3 (84, 108, 379), Group 4 (147), and Group 5 (147, 102, 112). Two animals in Group 3 with normal graft function were killed upon development of hydronephrosis and cerebral infarction. B lymphocytes fell to one-third of the preoperative value at 4 weeks after transplantation in all animals. Antidonor antibodies developed in most of the animals after stopping drug treatment or at the time of death. No animals except for one formed anti-4D11 antibody. 4D11 appears to be a promising agent for antirejection treatment in clinical organ transplantation.