Philip Lake

Islet allograft survival in nonhuman primates immunosuppressed with basiliximab, RAD, and FTY720

Objective. In a preclinical, nonhuman primate islet allotransplant model, the authors evaluated a novel immunosuppressive combination of basiliximab for induction and of RAD and FTY720 for maintenance. Methods. Five ABO-compatible and mixed lymphocyte reactivity-mismatched streptozotocin-induced diabetic juvenile cynomolgus monkeys underwent transplantation intraportally with 48-hr cultured 10,000 islet equivalents per kilogram. Induction immunosuppression was with intravenous basiliximab (10 mg on postoperative days 0 and 4). Maintenance immunosuppression was with RAD (everolimus) (0.075 mg/kg per day administered subcutaneously) and FTY720 (0.3 mg/kg per day administered orally), both administered on day −2 through day 180 posttransplant. Results. All five recipients tolerated their transplants and immunosuppressive therapy well, without adverse events or infectious complications. Insulin requirements pretransplant were 2.6 to 4.0 U/kg per day. All recipients became normoglycemic and insulin-independent posttransplant. Posttransplant serum C-peptide levels averaged 2.7 ng/mL (range, 0.6–6.2 ng/mL). Morning blood glucose levels ranged from less than 100 mg/dL to 150 mg/dL. Posttransplant acute C-peptide response to intravenous arginine averaged 1.3 ng/mL (range, 0.23–2.72 ng/mL). In one recipient with subtherapeutic RAD blood levels on day 7 posttransplant, exogenous insulin was resumed 100 days posttransplant; basal C-peptide levels remained positive in this recipient and averaged 2.6 ng/mL. The other four recipients remained insulin-independent for more than 6 months. Conclusions. This study provides preliminary evidence of the safety and efficacy of corticosteroid- and calcineurin inhibitor-free immunosuppression in a relevant preclinical transplant model. These findings provide a strong rationale for evaluating this nondiabetogenic regimen in a clinical trial of islet transplants in type 1 diabetic recipients.

Long-term islet graft survival in streptozotocin- and autoimmune-induced diabetes models by immunosuppressive and potential insulinotropic agent FTY720.

Background. Toxicity of current immunosuppressive agents to islet grafts is one of the major obstacles to clinical islet transplantation (Tx). This study was designed to assess the efficacy of FTY720, a novel immunomodulator with a unique mechanism of action, on islet graft survival and function in streptozotocin (STZ)- and autoimmune-induced diabetic recipients. Methods. Islet allograft from BALB/C mice or islet isografts were transplanted into STZ-induced diabetic CBA mice and autoimmune nonobese diabetic (NOD) mice. FTY720 was administered orally at 0.5 mg/kg per day in STZ diabetic recipients or 3 mg/kg per day in NOD recipients after Tx. Functional status of the islet graft was monitored by measuring blood glucose daily. Insulin secretion from mouse islets was measured with an insulin scintillation proximity assay. Results. Under the treatment of FTY720, long-term normoglycemia (>100 days) was achieved in 100% of STZ diabetic recipients and 50% of diabetic NOD recipients compared with a respective 11 and 7 days in untreated animals after allogeneic islet Tx. Normoglycemia persisted only temporarily (<4 weeks) in untreated NOD recipients of NOD islets, but was maintained for >100days with FTY720 treatment. Histologically, leukocyte infiltration observed in untreated animals was largely inhibited in FTY720-treated ones. Additionally, FTY720 stimulated insulin secretion from isolated islets by approximately twofold under both normoglycemic and hyperglycemic conditions. Conclusions. FTY720 is highly effective in protecting allo- and autoimmune response–mediated islet graft destruction without direct toxicity to the islets. The effect is likely attributable to its action in preventing effector lymphocyte infiltration into the grafted tissue.

Combinations of anti-LFA-1, everolimus, anti-CD40 ligand, and allogeneic bone marrow induce central transplantation tolerance through hemopoietic chimerism, including protection from chronic heart allograft rejection.

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vβ11+ T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening (“chronic rejection”).

T-CELL DEPLETION AND GRAFT SURVIVAL INDUCED BY ANTI-HUMAN CD3 IMMUNOTOXINS IN HUMAN CD3 TRANSGENIC MICE

BACKGROUND Anti-CD3 immunotoxins are broad-spectrum immunosuppressive agents in a wide range of organ transplantation animal models with potential use in eliciting antigen-specific tolerance. However, the anti-CD3 immunotoxins used in animal studies do not cross-react with human T cells, limiting extrapolation to humans and hindering clinical development. METHODS Three anti-human CD3-directed immunotoxins, DT389-scFv(UCHT1), scFv(UCHT1)-PE38, and UCHT1-CRM9, were compared in vitro and in transgenic mice, tg(epsilon)600+/-, that have T cells expressing both human and murine CD3epsilon antigens. RESULTS These immunotoxins were extraordinarily potent in vitro against human or transgenic mouse T cells, with IC50 values in cellular assays ranging from pM to fM. Systemic administration of these immunotoxins dose-dependently depleted >99% of tg(epsilon)600+/- lymph node and spleen T cells in vivo. Depletion was specific for T cells. The loss of the concanavalin A-induced, but not the lipopolysaccharide-induced, splenic proliferative response from immunotoxin-treated animals further demonstrated specific loss of T-cell function. Immunotoxin treatment prolonged fully allogeneic skin graft survival in tg(epsilon)600+/- recipients to 25 days from 10 days in untreated animals. T-cells recovered to approximately 50% of normal levels after approximately 22 days in animals with or without skin grafts; T-cell recovery correlated with skin graft rejection. All three immunotoxins elicited >100 day median survival of fully allogeneic heterotopic heart grafts. By 100 days, T cells recovered to normal numbers in these animals, but the grafts showed chronic rejection. CONCLUSION These immunotoxins profoundly deplete T cells in vivo and effectively prolong allogeneic graft survival.

Design and synthesis of potent and selective inhibitors of integrin VLA-4

The synthesis and identification of a novel series of inhibitors of integrin VLA-4 are described. Their in vitro activity and selectivity against closely related integrins are also presented.

Pharmacodynamic evaluation of the neutralization of endotoxin by PMX622 in mice.

ABSTRACT Polymyxin B (PMB) binds to and neutralizes endotoxin, but its systemic clinical utility is limited by neuro- and nephrotoxicity. PMX622 is a covalent conjugate of PMB and Dextran-70 designed to retain the ability of PMB to neutralize endotoxin and to retain the favorable colloidal, pharmacokinetic, and metabolic properties of Dextran-70. PMX622 has demonstrated efficacy in a number of animal models and effectively neutralized endotoxin in phase I clinical trials. Here, we systematically evaluated the pharmacodynamic properties of PMX622 in a murine model of endotoxin-induced lethality in galactosamine-sensitized mice. PMX622 completely and dose dependently inhibited lethality in this model. A stoichiometric relationship was found between the endotoxin challenge dose and the dose of PMX622 needed for protection. PMX622 neutralized endotoxin from four different genera of gram-negative bacteria but not Neisseria meningitidis. PMX622 was significantly less toxic than PMB in the mouse, suggesting that PMX622 has a better margin of safety than PMB. The timing of PMX622 administration relative to endotoxin was crucial. PMX622 was active for several hours prior to the endotoxin challenge; however, PMX622 did not protect mice if administered ≥15 min after endotoxin challenge. This suggests that PMX622 would best be clinically used prophylactically rather than therapeutically. These studies will be crucial in designing and interpreting human clinical trials assessing PMX622 efficacy.

Development of a Combined Heart and Carotid Artery Transplant Model to Investigate the Impact of Acute Rejection on Cardiac Allograft Vasculopathy

BACKGROUND Cardiac allograft vasculopathy (CAV) is the leading cause of late allograft loss after heart transplantation. Although clinical studies are suggestive of an association between episodes of acute rejection and subsequent emergence of CAV, direct experimental evidence in support of a causal relationship is lacking. METHODS We developed a new murine model of CAV in which a carotid artery and a heart graft are simultaneously transplanted into a single recipient. Transplants were performed across full or partial major histocompatibility complex (MHC) mismatched strain combinations. The heart grafts were either syngeneic with the carotid graft or from a third-party strain. Carotid arteries were harvested after 30 days and evaluated by morphometry and immunohistochemistry. RESULTS In the fully mismatched combination, all heart grafts were rejected within 7 days, as determined by loss of pulsation. At 30 days, carotid allografts in the combined transplant group had significantly more intimal hyperplasia compared with isolated carotid allografts. The neointima consisted of abundant smooth muscle cells and leukocytes. Intimal hyperplasia was also significantly enhanced by acute rejection of the third-party donor heart. In the partial MHC mismatched combination, the heart graft survived indefinitely, and this was associated with diminished intimal hyperplasia in the cotransplanted carotid artery compared with the isolated carotid allograft. CONCLUSION We present direct experimental evidence that CAV is promoted by acute parenchymal rejection of the heart. This interaction between acute rejection and CAV is mediated by both allospecific and non-allospecific processes. Effective therapeutic strategy against CAV should therefore target non-allospecific mediators as well as prevent episodes of acute rejection.

Combinatorial approaches to inhibitors of VLA-4: Piperazine-peptoid-bisarylureas

A combinatorial approach towards identifying inhibitors of VALA-4 was investigated. A library of piperazine-peptoid-bisarylureas was assembled in solid phase and screening in the novel v-well assay enabled the identificationof active compounds