The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Costimulation blockade with the B7-CD28 pathway-specific agent belatacept is now used in clinical kidney transplantation, but its efficacy remains imperfect. Numerous alternate costimulatory pathways have been proposed as targets to synergize with belatacept, one of which being the inducible costimulator (ICOS)-ICOS ligand (ICOS-L) pathway. Combined ICOS-ICOS-L and CD28-B7 blockade has been shown to prevent rejection in mice, but has not been studied in primates. We therefore tested a novel ICOS-Ig human Fc-fusion protein in a nonhuman primate (NHP) kidney transplant model alone and in combination with belatacept. ICOS-Ig did not prolong rejection-free survival as a monotherapy or in combination with belatacept. In ICOS-Ig alone treated animals, most graft-infiltrating CD4(+) and CD8(+) T cells expressed ICOS, and ICOS(+) T cells were present in peripheral blood to a lesser degree. Adding belatacept reduced the proportion of graft-infiltrating ICOS(+) T cells and virtually eliminated their presence in peripheral blood. Graft-infiltrating T cells in belatacept-resistant rejection were primarily CD8(+) CD28(-) , but importantly, very few CD8(+) CD28(-) T cells expressed ICOS. We conclude that ICOS-Ig, alone or combined with belatacept, does not prolong renal allograft survival in NHPs. This may relate to selective loss of ICOS with CD28 loss.

[Background] Sensitization before transplantation and de novo alloantibodies affect a significant number of patients awaiting kidney transplantation, implicating poorer prognosis both in short- and long-term graft survival. Current approaches to desensitization offer incomplete and temporary removal of antibody-producing cells. In this study, we investigated the kinetics of B cell and alloantibody responses after kidney and skin transplantation in rhesus macaques in order to develop a sustainable preclinical model for evaluating desensitization therapies. [Methods] Nine rhesus macaques underwent unilateral nephrectomy and MHC class I mismatched renal transplantation, with one native kidney preserving normal renal function. Four rhesus macaques underwent skin grafting. B cell phenotypes and donor-specific antibody (DSA) production were assessed with polychromatic flow cytometry. [Results] All renal allografts were rejected on day 7-protocol biopsy, and skin grafts at a mean 10.7 ± 3.86 days. DSA kinetics were assessed by flow crossmatch, revealing a peak DSA titer at 44.3 days for kidney and 39.5 days for skin transplant and stabilization of DSA by approximately 140 days and 70 days, respectively. Baseline pre-transplant DSA levels were similar for both kidney and skin recipients (p=0.743), but peak levels were significantly higher for kidney-transplanted animals (MFI= 49157.1 vs. 6451.3, p=0.005). Memory responses in sensitized skin recipients were tested with a secondary skin transplant. Consistent with sensitization, secondary response resulted in increased magnitude and faster kinetics of DSA production. [Conclusion] This study reveals a unique model of investigating humoral immunity in transplantation for evaluation of novel desensitization techniques.

Purpose: Effector memory T cells (TEM) and NK cells, identified as important mediators of costimulation blockade-resistant rejection (CoBRR) in solid organ transplantation and in the early immune response to bone marrow transplantation (BMT), are known to have high surface expression of LFA-1. Leukotoxin (Leukothera®, LtxA) is a product of the bacterium A. actinomycetemcomitans capable of killing leukocytes via a LFA-1 specific mechanism. We sought to study the effect of this compound on TEM and NK cells and the potential novel use of LtxA in transplantation. Methods: Fresh peripheral blood mononuclear cells (PBMCs) were isolated from rhesus macaques. To examine the effect of LtxA by cell phenotype, PBMCs were incubated for 90 minutes with LtxA over a 4-log range of concentrations. Cells were stained for viability and apoptotic markers, and defined for memory T cell and NK cell phenotypes by flow cytometry. To examine the effect of cell activation on LtxA-mediated cell death, PBMCs were studied with or without exposure to PMA/I. Finally, to examine the effect of LtxA in vivo, 2 rhesus macaques received a single dose of LtxA IV. Blood was drawn at multiple time points post-infusion and analyzed by flow cytometry. Results: LtxA induced cell death in PBMCs in a dose-dependent fashion. TEM were particularly susceptible to LtxA-based killing with cell viability of 22±3% (Mean±SEM, n=8 pooled from 3 experiments) at [LtxA]=10μg/mL. Central memory (TCM) and naïve (TN) T cells had viability of 75±3% (p < 0.0001) and 83±2% (p<0.0001), respectively. NK cells were readily killed (16±11% viable). Co-incubation with PMA/I increased the susceptibility of all cell types to LtxA. In vivo, LtxA induced a transient lymphopenia (Mean lymphocyte count 841 cell/μL at 5 hours). Circulating CD3+ cells were decreased with relative reduction in TEM versus TCM and TN. NK cells were rapidly depleted. These effects lasted less than 24 hours. Conclusion:In vitro, LtxA potently kills TEM and NK cells in a dose-dependent manner, exhibiting greater potency against activated cells. In vivo, LtxA is capable of transient depletion of NK cells and TEM from the circulation. These data demonstrate the potential of this bacterial protein to selectively target TEM and NK cells, warranting further investigation of LtxA for the prevention/treatment of CoBRR in solid organ transplantation and for use in BMT. DISCLOSURES:Kachlany, S.: Stockholder, Actinobac Biomed, Inc.

Interactions between CD40 & CD154 represent a critical pathway for effective T cell activation. Over two decades ago there were tremendous expectations surrounding clinical trials of anti-CD154 antibodies. Unfortunately, outcomes were disappointing as unanticipated thromboembolic events led to cessation of trials. One prevailing hypothesis suggested that anti-CD154 therapy contributed to the development of immune complexes composed of anti-CD154, platelet Fc receptors & soluble CD154. These complexes purportedly led to platelet aggregation & thromboembolism. We have developed a novel CD154 domain antibody (dAb), which lacks Fc binding but retains the ability to block CD40-CD154. Initial screens identified monomeric dAbs against hCD154, which were then formatted as bivalent Fc-silent proteins. Platelet activation studies were performed with soluble CD154 & dAbs targeting CD154 with or without Fc binding capacity. Only reagents with Fc binding activity promoted platelet activation in the presence of soluble CD154. This activation was abolished with the addition of anti-FcγRIIa. Additional in-vitro studies evaluating CD154-dependent B & T cell activation were used to select an optimal clone (BMS-986004). Further evaluation of the novel anti-CD154 dAb was assessed in non-human primates. A single dose of anti-CD154 dAb effectively eliminated the T-dependent Ab response after KLH immunization. Next efficacy was evaluated in a non-human primate renal transplant model. Rhesus macaques received anti-CD154 dAb (20mg/kg iv, qwk to day 70) and underwent a MHC-mismatched renal transplant. BMS 986004 effectively prolonged survival in the majority of animals (>77, >77, >77, 68, 67, >42, 8, & 8 days). 3 of 8 animals were sacrificed after finishing therapy & underwent a full pathologic evaluation, which failed to identify any gross or histologic evidence of thromboembolism. Protective immunity, as assessed by CMV viral reactivation and general clinical condition, was maintained. This report identifies a novel dAb targeting CD154 which effectively prolongs transplant survival yet lacks thromboembolic potential & thus may provide an attractive candidate molecule for clinical development. DISCLOSURES:Price, K.: Employee, Bristol-Myers Squibb. Nadler, S.: Employee, Bristol-Myers Squibb. Adams, A.: Grant/Research Support, Bristol-Myers Squibb.

[Background] Sensitized patients comprise up to a third of the kidney waiting list. Preformed and de-novo alloantibody post transplantation (TX) significantly deteriorate graft survival. Currently used desensitization strategies are time-consuming, expensive and moderately effective. To better understand the mechanisms involved in allograft rejection in sensitized patients we aim to establish a rigorous model of solid organ TX in sensitized non-human primates (NHP). [Methods] Fully mismatched rhesus macaques were sensitized with kidney or skin TX. Immunologic responses were monitored by T-cell crossmatch by flow cytometry. After stabilization the animals underwent native nephrectomy and kidney TX from their prior kidney/skin donor. Immunosuppression (IS) included Basiliximab (0.3mg/kg IV day 0,4), Tacrolimus (0.05mg/kg IM BID, trough level 10-15ng/ml) and Mycophenolate mofetil (MMF; 30mg/kg PO BID) combined with tapered methylprednisolone. Assessment comprised laboratory testing, urine output and clinical monitoring. After sacrifice kidney grafts were submitted to pathology for H&E, PAS and C4d staining. Survival results were compared to a group of non-sensitized monkeys with comparable mismatch and IS for 140 days post kidney TX. [Results] 5 monkeys were transplanted. T-cell flow crossmatch showed mean fluorescence intensities between 1.7 and 6.5 fold over baseline on the day of the TX. Survival calculated by Kaplan-Meier method was median 6 days compared to 183 days in the non-sensitized group (p<0.001). All sensitized animals had to be sacrificed due to worsening clinical state and concomitant kidney failure. Histology revealed mixed cellular and humoral rejection in 2/5 grafts, BANFF ACRIIa, C4d1 and ACRIII, C4d2; these had the longest survival, 4 and 8 days respectively. Other grafts showed acute tubular injury/necrosis without signs of rejection. [Conclusion] These results show that kidney TX into sensitized recipients using conventional IS leads to aggressive and rapid mixed rejection. These results support the use of more potent induction therapies, such as T cell depletion, in order to overcome the immune barrier established in sensitized patients. Currently we are optimizing such therapies for the use in NHP. We believe that this preclinical model will be an invaluable tool in the development of novel desensitization therapies.

A 25-year-old, female rhesus macaque (Macaca mulatta) presented with a history of weight loss despite a normal appetite and supportive care. The animal was humanely destroyed due to poor prognosis. Post-mortem examination revealed a focally extensive, firm, white annular constriction at the ileocaecal junction and an incidental finding of a pale white nodule approximately 0.8 cm in diameter in the left renal pelvis. Based on the microscopical findings, ileocaecal adenocarcinoma and renal pelvis transitional cell carcinoma (TCC) were diagnosed. The use of cytokeratin (CK)-7 and -20 and uroplakin III as potential renal TCC markers was evaluated. The neoplastic cells were labelled intensely with antibodies to uroplakin III, but not to CK-7 or -20. Spontaneous intestinal adenocarcinoma has been documented in the rhesus macaque, but concurrent renal pelvis TCC is highly unusual.

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.

Introduction: The liver, which is currently the only approved site for clinical islet transplantation, presents numerous immunologic and non-immunologic barriers to islet engraftment and survival. The bone marrow cavity represents an attractive alternative site: It avoids liver-specific first-pass metabolism, is easily accessed, and intraosseous delivery has successfully been used as an alternative to intravenous delivery for human bone marrow transplantation. In light of the successful transplantation of islets into the bone marrow of rodents, we hypothesized that the bone marrow could be a feasible site for islet transplantation in nonhuman primates. Methods: Five rhesus macaques were rendered diabetic with streptozotocin. A mean of 13,991 (±3,114) MHC-mismatched islet equivalents per kilogram were transplanted into the bone marrow cavity at five separate sites, including bilateral humeri, bilateral femurs and the iliac crest. Recipients were treated with an immunosuppression regimen that we have previously shown significantly prolongs islet allograft survival: induction therapy with TS1/22, a monoclonal antibody to LFA-1, and belatacept maintenance therapy. Rejection was defined as fasting blood glucose greater than 130mg/dL on two consecutive days. Results: Four of five recipients achieved immediate insulin-independent normoglycemia. The fifth recipient required insulin therapy to maintain normoglycemia; this rate of engraftment with insulin independence is similar to that seen with intraportal islet transplantation. Rejection-free survival for recipients with functional grafts is currently 80, >79, >51 and >29 days. Conclusion: The bone marrow cavity may be a feasible alternative to intraportal islet transplantation and provide an attractive option for clinical islet transplantation.