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Previously, we reported the fundamental pharmacological characteristics of a novel Igβ and Fc gamma receptor IIB cross-linking antibody, ASP2713, as a new treatment option for systemic lupus erythematosus. The aims of the present study were to investigate ASP2713's characteristics with regard to pharmacological effect, pharmacokinetics (PK), and receptor occupancy, and to predict its human PK and clinically effective dose. The relationship between the concentration and receptor occupancy of ASP2713 for Igβ of B cell receptors was examined using whole blood B cells. Calculated EC

Systemic lupus erythematosus (SLE) is an autoimmune disease involving multi-organ systems with a widely heterogeneous clinical presentation. Renal involvement, observed mainly in lupus nephritis (LN), is the most common organ lesion associated with SLE and a determinant of prognosis. However, treatment of LN remains controversial and challenging, prompting the need for novel therapeutic approaches. In particular, development of a clinically relevant LN animal model would greatly facilitate the development of new treatments. Here, we report a novel murine model for LN established by administering polyinosinic-polycytidylic acid (Poly (I:C)) to NZB/W F1 mice. We investigated the effectiveness of administering Poly (I:C) to NZB/W F1 mice for accelerating nephritis onset and explored the optimal conditions under which to enroll mice with nephritis with similar pathology for studying treatment candidates. Gene-expression analysis revealed that activation of macrophages, which are reported to be involved in the progression of LN in patients, was a unique characteristic in this accelerated nephritis model. Evaluation of the therapeutic effect of mycophenolate mofetil (MMF), a recommended first-choice agent for LN, in this novel LN model showed that MMF significantly reduced proteinuria. The cathepsin S (CatS) inhibitor ASP1617, which has been reported to prevent development of lupus-like glomerulonephritis in the spontaneous NZB/W F1 mouse model, also showed marked therapeutic effect in this model. Our novel Poly (I:C) accelerated LN model would thus be very useful for screening clinical candidates for LN, and CatS may be an attractive therapeutic target for the treatment of LN.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the dysregulation of various cell types and immunological pathways. Autoantibodies play an important role in its pathogenesis. The presence of autoantibodies suggests that self-antigen presentation through major histocompatibility complex (MHC) class II on antigen presenting cells is involved in the pathogenesis of autoimmune diseases, including SLE. Cathepsin S (CatS) is a key protease for antigen peptide loading onto lysosomal/endosomal MHC class II molecules through invariant chain degradation to promote antigen presentation. Inhibition of CatS is therefore expected to suppress antigen presentation via MHC class II, T and B cell activation, and antibody production from B cells. Here, we report the pharmacological profile of ASP1617, a novel CatS inhibitor. ASP1617 induced invariant chain accumulation and decreased the expression level of MHC class ΙΙ on the cell surface in both mouse and human B cells. Further, ASP1617 prevented DO11.10 mice T cell proliferation to ovalbumin antigen. We investigated the effects of ASP1617 and mycophenolate mofetil (MMF) on the development of lupus-like nephritis in NZB/W F1 mice, a widely used SLE mouse model. Oral administration of ASP1617 suppressed anti-dsDNA IgG, prevented progression of lupus-like glomerulonephritis, and significantly prevented proteinuria excretion. In contrast, MMF did not suppress anti-dsDNA IgG. Further, we found that plasma and/or urine CatS levels were increased in specimens from NZB/W F1 mice and several SLE patients. These results indicate that CatS may be an attractive therapeutic target for the treatment of SLE.

The T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) is a validated immune checkpoint protein expressed on memory CD4+T-cellls, Tregs, CD8+T-cell and natural killer (NK) cells. ASP8374 is a fully human monoclonal immunoglobulin (Ig) G4 antibody designed to block the interaction of TIGIT with its ligands and inhibit TIGIT signaling. ASP8374 exhibited high affinity binding to TIGIT and increased interferon (IFN)-γ production of cultured peripheral blood mononuclear cells (PBMCs) in a titratable manner. When used in combination with pembrolizumab, an anti-programmed death-1 (PD-1) antibody, ASP8374 induced higher T-cell activation in vitro than either treatment alone. An anti-mouse TIGIT antibody surrogate, mSEC1, displayed anti-tumor efficacy in an MC38 syngeneic mouse tumor model alone and in combination with an anti-programmed death-ligand 1 (PD-L1) antibody. In an additional syngeneic mouse tumor model (CT26), while mSEC1 alone did not demonstrate anti-tumor efficacy, mSEC1 combined with an anti-PD-1 antibody enhanced anti-tumor efficacy above that of the anti-PD-1 antibody alone. These data provide evidence that ASP8374 has therapeutic potential for advanced malignancies.

Belatacept is the first costimulatory blockade agent approved for maintenance immunosuppression in kidney transplant recipients. Clinical results have indicated that belatacept is associated with superior renal function and improved metabolic profile; however, higher incidence of acute rejection and posttransplant lymphoproliferative disorder are the shortcomings of this agent. In this study, ASP2409, a new cytotoxic T-lymphocyte associated protein 4-immunoglobulin possessing 14-fold higher in vitro CD86 binding affinity than belatacept, was tested for renal allograft survival in cynomolgus monkeys. ASP2409 monotherapy dose-dependently prolonged renal allograft survival. Low-dose ASP2409 in combination with a subtherapeutic dose of tacrolimus showed much longer median survival time than monotherapy. Similar allograft survival results were observed in regimens based on high-dose ASP2409, belatacept, and therapeutic-dose tacrolimus. The results of renal allograft histopathology with high-dose ASP2409-based regimens were not inferior to the belatacept-based regimen. Moreover, higher frequencies of FoxP3-positive regulatory T cells in renal allografts were observed in ASP2409- and belatacept-based regimens compared with tacrolimus-based regimens. No serious side effects related to ASP2409 administration were found during the study. These data suggest that ASP2409 is a promising candidate for calcineurin inhibitor-sparing or -avoidance regimens.

The histone deacetylase (HDAC) inhibitor FR276457, a hydroxamic derivative, was identified during chemical library screening and was found to exhibit potent inhibitory effects on the activity of mammalian HDACs. It has been shown that FR276457 exhibited marked immunosuppressive effects in a rat heterotopic cardiac transplant model. To predict clinical efficacy of FR276457, we investigated the inhibitory effect of the proliferation of Jurkat cells in vitro and immunosuppressive effect of orally administered FR276457 on allograft rejection as a monotherapy or in combination with tacrolimus (0.04 mg/kg) injected intramuscularly (i.m.) in a canine renal transplant model. Animal survival, the plasma creatinine level, and histopathology were evaluated. FR276457 inhibited the proliferation of Jurkat cells probably by targeting activity of NF-kappaB. FR276457 prolonged the median survival time (MST) of transplanted grafts from 11.5 days (untreated group) to 29.0 days (FR276457-treated group). FR276457 administered 1 mg/kg twice a day in combination with tacrolimus prevented allograft rejection. In addition, a dose of 1.5 mg/kg twice a day or 5.0 mg/kg once a day prolonged the MST from 18 days (control group) to >73 or >90 days, respectively. Histopathological analysis showed that FR276457 suppressed the score for mononuclear cell infiltration and vasculitis. In conclusion, the HDAC inhibitor FR276457 inhibited the proliferation of T cell line established from human in vitro. And more, FR276457 clinically prolonged allograft survival when administered as a monotherapy, and had additive or synergistic effects when combined with tacrolimus with the canine renal transplant model. These results showed HDAC inhibitor is a promising biological target for treatment in transplant field.

Many studies have examined the efficacy of tacrolimus in rats and dogs, but few have reported its evaluation in cynomolgus monkeys. The aim of this study was to clarify the efficacy of tacrolimus in a cynomolgus monkey renal transplant model based on the efficacy of various doses. Monkeys that had undergone renal transplant were treated with a vehicle or 0.5, 1.0, or 2.0 mg/kg of tacrolimus by oral administration. Tacrolimus administration prolonged animal survival in a dose-dependent manner, and the median survival time (MST) was 11, 21, and >90 days for the 0.5, 1.0, and 2.0 mg/kg tacrolimus groups, respectively. The MST of the vehicle group was 6 days. Histopathological analyses of all transplanted kidneys were also performed. Typical pathological findings of acute rejection were observed in both the vehicle and tacrolimus (0.5 and 1.0 mg/kg)-treated groups. Only limited mononuclear cell infiltration and hemorrhage were present in the tacrolimus (2.0 mg/kg)-treated group. In conclusion, 2.0 mg/kg was considered to be a therapeutic dose in this model, and 0.5 or 1.0 mg/kg could be used for a study when efficacy of a new compound is evaluated in a combination therapy with tacrolimus. Keywords:: tacrolimus, cynomolgus monkey, monkey renal transplant model, therapeutic dose, subtherapeutic dose

Histone deacetylase (HDAC) is a known modulator of gene transcription, and the immunosuppressive activity of HDAC inhibitors has been demonstrated in recent several reports. In this study, the HDAC inhibitor FR276457, a hydroxamic derivative, was found to have a similar inhibitory effect on all mammalian HDACs tested, but no isozyme selectivity. Both FR276457 and tacrolimus exerted an immunosuppressive effect on in vitro rat splenocyte proliferation stimulated with Concanavalin A. Next, the effect of FR276457 on allograft rejection when administered either as a monotherapy or in combination with tacrolimus was investigated in a rat heterotopic cardiac transplant model. Orally administered FR276457 prolonged the median survival times (MST) of the transplanted grafts in the vehicle group from 6 d to 17 or 21 d at doses of 20 or 40 mg/kg, respectively. Histopathological analysis showed the structures of the myocardium were not affected, but interstitial cellular infiltration could not be suppressed completely. Tacrolimus (0.032 mg/kg) prolonged allograft MST to 16 d. FR276457, when combined with tacrolimus, prevented allograft rejection at a dose lower than that of the monotherapy. The combination dose prolonged the MST in the groups treated with 10 and 20 mg/kg to >28 d, and cellular infiltration was suppressed completely. In conclusion, this study demonstrated that the oral administration of HDAC inhibitor FR276457 can prevent allograft rejection as a monotherapy, and has additive or synergistic effects when combined with tacrolimus.

The Th1 and Th2 preference induced by cells from the Peyer's patch (PP) and spleen (SPL) with various doses of an antigen was examined. The same splenic T cell receptor-transgenic CD4+ T cells were first incubated with PP or SPL cells in the presence of various doses of an antigen, and the cytokine response was observed after secondary stimulation. A Th2-type pattern was only obtained for primary stimulation at 10 microM of the antigen with PP cells, whereas a Th1 pattern was induced at both higher and lower concentrations. SPL cells in the presence of 0.1 to 1 microM of the antigen induced the secretion of Th2-type cytokines. Ten and 100 microM of the antigen plus SPL cells did not induce the release of a large quantity of cytokines. PP cells induced a different cytokine pattern at the antigen concentration that induced a similar level of T cell proliferation with SPL cells. Our findings suggest that the antigen-dose dependent development of Th1/Th2 cells is differentially modulated by the antigen-presentation function of cells in PP and SPL.

T cells play a central role in mediating transplant rejection by orchestrating the inflammatory events that ultimately lead to graft destruction. Immunosuppressants have been the primary treatment for preventing transplant rejection for the past 50 years. The aim of immunosuppressive therapies is to block T cell activation, thereby attenuating these processes. However, long-term use of immunosuppressants carries an increased risk of infection and cancer (1–3). As a new immunotherapy, monoclonal antibody (mAb) therapy has been used in transplantation, cancer, and autoimmune diseases (4–6). Activation of naive T cells requires costimulatory signals provided by the B7 and tumor necrosis factor (TNF) families, in which the CD40-CD154 pathway is preeminent in the T cell response after alloantigen presentation (7–10). Blocking the CD40-CD154 costimulatory pathway for T-cell activation has been shown to be effective in several models of transplantation and autoimmune diseases (11–13). Recent experimental studies (14–19) and early clinical trials (20) have reported that a humanized anti-CD154 antibody can induce long-term allograft survival, but the clinical trial was interrupted by unexpected thromboembolic complications. Thus, inhibition of the counter molecule for CD154, CD40, has become an alternative approach in transplantation. ASKP1240 has been demonstrated to inhibit human CD154-induced proliferation of peripheral blood mononuclear cells, suppress the delayed-type hypersensitivity reaction and tetanus toxoid-specific antibody formation in nonhuman primates, and exhibit potent immunosuppressive effects to prolong renal allograft survival in Cynomolgus monkeys (21–23). This study aimed to investigate the serum concentration of ASKP1240 in a pharmacokinetic (PK) study, evaluate the binding ability of ASKP1240 in CD20+ B cells in a pharmacodynamics (PD) study, and clarify the PK/PD relationship in normal and renal transplanted monkeys. These results will be helpful to select the optimal dose and timing for ASKP1240 therapy in clinical trials.

Background: A recent study indicated that FR276457, a pan-histone deacetylase inhibitor, prevented allograft rejection in an ACI-to-Lewis rat heart transplant model, seemingly without affecting cellular infiltration into the transplanted heart. In this report, we investigated alterations in infiltrating cells in allografts by treatment of FR276457. Materials and Methods: Heterotopic cardiac allografts which were removed from August Copenhagen Irish rats were transplanted into the necks of Lewis rats. FR276457 40 mg/kg or vehicle was subsequently administered orally for 5 consecutive days beginning on the operation day after recovery from anesthesia (Day 0). The allograft recipients were sacrificed under anesthesia on Day 5 after transplantation. At first, we identified infiltrating cells into the allografts by the histopathological analysis. In addition, we isolated the infiltrating cells from allografts, sorted CD8 positive (CD8 + ) T cells, and then investigated cytotoxicity of CD8 + T cells against spleen cells from an August Copenhagen Irish rat. Results: Histopathological analysis of allografts on Day 5 after the heart transplant showed that infiltration of T cells was not suppressed in FR276457-treated allograft recipients, compared with those in vehicle-treated allograft recipients, although the infiltration of ED1 positive cells was tended to decrease. Ex vivo analysis revealed that alloantigen-specific cytotoxicity of CD8 + T cells, isolated from allografts in FR276457-treated allograft recipients, against ACI rat splenocytes was much lower than that in vehicle-treated allograft recipients. Conclusion: FR276457 treatment suppressed the alloantigen-specific cytotoxicity of CD8 + T cells infiltrated into

Chronic allograft nephropathy (CAN) is a major cause of late allograft loss. One morphological characteristic of CAN is renal interstitial fibrosis. Mycophenolate mofetil (MMF), the inosine 5′-monophosphate dehydrogenase (IMPDH) inhibitor, has been reported to attenuate the progression of renal interstitial fibrosis. However, the question of whether the newly synthesized IMPDH inhibitors with structures different from MMF have an antifibrotic effect remains unanswered. We evaluated the antifibrotic effects of BMS-566419, a chemically synthesized IMPDH inhibitor, using an experimental rat model, unilateral ureteral obstruction (UUO), in comparison with those of MMF. Expression levels of monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-β1), which play important roles in UUO-induced renal fibrosis, were also investigated to determine the mechanism by which BMS-566419 affects the progression of renal fibrosis. After 14 days of UUO, interstitial fibrosis was frequently observed in the renal cortex of rats administered vehicle control. BMS-566419 by oral administration showed a significant and dose-dependent suppressive effect on UUO-induced renal fibrosis in histopathological experiments. BMS-566419 treatment also decreased collagen content, as indicated by hydroxyproline concentration, and the expression of collagen type 1 mRNA. BMS-566419 also decreased the expression of mRNA for both MCP-1 and TGF-β1. The antifibrotic effects of treatment with BMS-566419 at 60 mg/kg seemed comparable to those with MMF at 40 mg/kg. These results suggest that BMS-566419 and other chemically synthesized IMPDH inhibitors have beneficial pharmacological effects similar to those of MMF, and are potential pharmaceutical candidates in the treatment of fibrotic renal disease, including CAN.

Surgical complications are important causes of graft loss in the nonhuman primate kidney transplantation model. We reviewed the incidence and intervention methods in 182 kidney transplantations performed in our lab recently 2 years in Cynomolgus monkeys. There were six renal artery thromboses (3.3%), eight urine leakages (4.4%), and five ureteral stenoses (2.7%). All renal artery thrombosis cases were found within 3 days after surgery. Urine leakage appeared from the 5th to 12th day after surgery and all cases were caused by ureter rupture. Reexploration was performed in five cases to reanastomose ureter with stent. Four cases reached long-term survival. The rest one died of graft rejection. Ureteral stenoses were found in long-term survival cases. Ureter reanastomoses with stent were performed in two cases. The postoperative renal functions of these two monkeys recovered to normal and they survived until study termination. From this large number of study, our experience indicated that kidney transplantation in the nonhuman primate is a safe procedure with low complications. Reexploration is recommended for salvage of the graft with urine leakage and ureteral stenosis.

Acute rejection following renal transplantation has become manageable with the introduction of calcineurin inhibitors, FK506 and cyclosporine A. However, chronic allograft dysfunction accompanied by renal interstitial fibrosis, which induces graft loss, remains unresolved. Here, we evaluated the effect of FR276457, a pan-histone deacetylase (HDAC) inhibitor, on interstitial fibrosis in the injured kidneys of a rat model of unilateral ureteral obstruction. The injured kidneys, harvested on Day 14 following the operation, showed progression of interstitial fibrosis, increases of hydroxyproline contents, and mRNA expression of collagen type Ialpha1 and monocyte chemotactic protein 1 (MCP-1). However, these changes were found to be prevented with daily oral administration of FR276457. In addition, given that MCP-1 is believed to contribute to progressive fibrosis, we investigated the direct effect of FR276457 on MCP-1 production by activated THP-1 cells in vitro. Results showed that FR276457 administration decreased MCP-1 production in these cells in a concentration-dependent manner. Findings from the present study suggested that a pan-HDAC inhibitor may exert a prophylactic effect against renal interstitial fibrosis by inhibiting MCP-1 production.