The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti-pig antibodies significantly improved survival in a pig-to-rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan-T cell depletion and an anti-CD154-based maintenance regimen. Selective depletion of CD4+ T cells but not CD8+ T cells resulted in long-term survival (median survival time >400 days vs 6 days). These studies suggested that CD4+ T cells may have a more prominent role in xenograft rejection compared with CD8+ T cells. Although animals that received selective depletion of CD8+ T cells showed signs of early cellular rejection (marked CD4+ infiltrates), animals receiving selective CD4+ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4+ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti-pig antibody and CD4 depletion resulted in consistent, long-term xenograft survival.

Belatacept, the CD28-B7 costimulation pathway inhibitor, has been approved as a calcineurin inhibitor (CNI) alternative in kidney transplantation. Although costimulation blockade (CoB) allows for CNI avoidance, it is associated with increased rates of early rejection, prompting a search for agents to pair with belatacept. Methotrexate (MTX) is an antimetabolite that has been found to be complimentary with abatacept, a lower affinity CD28-B7-specific analogue of belatacept, in the treatment of rheumatoid arthritis (RA). We examined whether this synergy would extend to prevention of kidney allograft rejection. Rhesus macaques underwent kidney transplantation treated with abatacept maintenance therapy with either a steroid taper, MTX, or both. The combination of abatacept maintenance with steroids prolonged graft survival compared to untreated historical controls and previous reports of abatacept monotherapy. The addition of MTX did not provide additional benefit. These data demonstrate that abatacept with adjuvant therapy may delay the onset of acute rejection, but fail to show synergy between abatacept and MTX beyond that of steroids. These findings indicate that MTX is unlikely to be a suitable adjuvant to CoB in kidney transplantation, but also suggest that with further modification, a CoB regimen used for advanced RA may suffice for RA patients requiring kidney transplantation.

Human rhinovirus (RV) is the leading cause of human infectious disease. RV contributes to exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Years ago, inactivated RV was established as a protective vaccine, and virus-neutralizing antibodies (nAb) correlated with protection. However, co-circulation of over 150 discouraged vaccine development. We hypothesize that a highly polyvalent RV vaccine is feasible with sufficiently high antigen load per RV type. Polyvalent vaccines consisted of high-titer RVs. Rhinoviruses were inactivated with formalin, mixed with alum adjuvant, and administered by intramuscular injection in a prime and boost regimen. BALB/c mice were vaccinated with 1, 3, 5, 7, 10, or 25-valent RV vaccine. Rhesus macaques were vaccinated with 25-valent or 50-valent RV. Immunogenicity of these vaccines was measured by in vitro virus nAb assays against each RV type. In mice, we observed that antisera neutralized 100% of RV types in the 10-valent vaccine and 96% of RV types in the 25-valent vaccine. Antisera from rhesus macaques neutralized 100% of RV types in the 25-valent and 98% of RV types in the 50-valent vaccine. RV vaccine immunogenicity correlated with the quantity of input antigens, and valency was not a major determinant for potency or breadth of the immunogenicity. This is the first study showing a polyvalent inactivated RV vaccine capable of inducing broad nAb responses to numerous and diverse RV types. The approach demonstrated here provides proof of principle for the feasibility of a polyvalent rhinovirus vaccine approach that could alleviate RV-induced asthma exacerbation.

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.

As the predominant etiological agent of the common cold, human rhinovirus (HRV) is the leading cause of human infectious disease. Early studies showed monovalent formalin-inactivated HRV vaccine can be protective, and virus-neutralizing antibodies (nAb) correlated with protection. However, co-circulation of many HRV types discouraged further vaccine efforts. We approached this problem straightforwardly. We tested the hypothesis that increasing virus input titers in polyvalent inactivated HRV vaccine will result in broad nAb responses. Here, we show that serum nAb against many rhinovirus types can be induced by polyvalent, inactivated HRVs plus alhydrogel (alum) adjuvant. Using formulations up to 25-valent in mice and 50-valent in rhesus macaques, HRV vaccine immunogenicity was related to sufficient quantity of input antigens, and valency was not a major factor for potency or breadth of the response. We for the first time generated a vaccine capable of inducing nAb responses to numerous and diverse HRV types.

Vascularized composite allografts (VCAs) are technically feasible. Similar to other organ transplants, VCAs are hampered by the toxicity and incomplete efficacy associated with conventional immunosuppression. Complications attributable to calcineurin inhibitors remain prevalent in the clinical cases reported to date, and these loom particularly large given the nonlifesaving nature of VCAs. Additionally, acute rejection remains almost ubiquitous, albeit controllable with current agents. Costimulation blockade offers the potential to provide prophylaxis from rejection without the adverse consequences of calcineurin-based regimens. In this study, we used a nonhuman-primate model of VCA in conjunction with immunosuppressive regimens containing combinations of B7-specific costimulation blockade with and without adhesion blockade with LFA3-Ig to determine what adjunctive role these agents could play in VCA transplantation when combined with more conventional agents. Compared to tacrolimus, the addition of belatacept improved rejection free allograft survival. The combination with LFA3-Ig reduced CD2(hi) memory T cells, however did not provide additional protection against allograft rejection and hindered protective immunity. Histology paralleled clinical histopathology and Banff grading. These data provide the basis for the study of costimulation blockade in VCA in a relevant preclinical model.

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.

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.

Human post-transplant lymphoproliferative disorder (PTLD) is an abnormal lymphoid proliferation that arises in 1-12% of transplant recipients as a consequence of prolonged immunosuppression and Epstein-Barr viral infection (EBV). Nonhuman primates, primarily rhesus macaques (Macaca mulatta), have been used extensively in research models of solid organ transplantation, as the nonhuman primate immune system closely resembles that of the human. Lymphocryptovirus of rhesus monkeys has been characterized and shown to be very similar to EBV in humans in regards to its cellular tropism, host immune response, and ability to stimulate B lymphocyte proliferation and lymphomagenesis. Thus, it appears that the NHP may be an appropriate animal model for EBV-associated lymphoma development in humans. The clinical management of post-transplant nonhuman primates that are receiving multiple immunosuppressive agents can be complicated by the risk of PTLD and other opportunistic infections. We report 3 cases of PTLD in rhesus macaques that illustrate this risk potential in the setting of potent immunosuppressive therapies for solid organ transplantation.