A Human Anti-CD40 Monoclonal Antibody, 4D11, for Kidney Transplantation in Cynomolgus Monkeys: Induction and Maintenance Therapy

Authors


* Corresponding author: Satoru Todo, stodo@med.hokudai.ac.jp

Abstract

Blockade of CD40–CD154 signaling pathway is an attractive strategy to induce potent immunosuppression and tolerance in organ transplantation. Due to its strong immunosuppressive effect shown in nonhuman primate experiments, anti-CD154 monoclonal antibodies (mAbs) have been tried in clinical settings, but it was interrupted by unexpected thromboembolic complications. Thus, inhibition of the counter molecule, CD40, has remained an alternative approach. In the previous preliminary study, we have shown that 4D11, a novel fully human anti-CD40 mAb, has a fairly potent immunosuppressive effect on kidney allograft in nonhuman primates. In this study, we aimed to confirm the efficacy and untoward events of the 2-week induction and 180-day maintenance 4D11 treatments. In both, 4D11 significantly suppressed T-cell-mediated alloimmune responses and prolonged allograft survival. Addition of weekly 4D11 administration after the induction treatment further enhanced graft survival. Complete inhibition of both donor-specific Ab and anti-4D11 Ab productions was obtained only with higher-dose maintenance therapy. No serious side effect including thromboembolic complications was noted except for a transient reduction of hematocrit in one animal, and decrease of peripheral B-cell counts in all. These results indicate that the 4D11 appears to be a promising candidate for immunosuppression in clinical organ transplantation.

Introduction

Among many costimulatory signals, the best characterized and perhaps the most important signal within the TNF–tumor necrosis factor receptor (TNFR) family is the CD40–CD154 costimulation. It has been shown that interaction between CD40 and CD154 plays a critical role in activation of various cell types including T cells, B cells, dendritic cells and macrophages, which, in concert, elicit alloimmune responses (1–6). Blockade of the signaling by various approaches has been shown to induce potent immunosuppression and tolerance in experimental organ transplantations in rodents (6–9). Using nonhuman primates, the humanized anti-CD154 monoclonal antibodies (mAbs), such as hu5C8 (10–12), IDEC-131 (13) and ABI793 (14), have been shown to markedly prolong renal allograft survival. Unfortunately, however, clinical application of these mAbs has been hampered by thromboembolic complications (15), presumably due to the nature of the antibody itself (16,17) and/or by activating platelets (18). On the other hand, blocking the counter receptor, CD40, by ch5D12 (19) or chi220 (20,21), have been reported as immunosuppressive in nonhuman primate kidney and islet transplantations, but they are all chimeric Abs and were less potent than the anti-CD154 mAbs.

The 4D11 is a newly developed fully human anti-CD40 mAb. In the previous preliminary study, we have demonstrated that 4D11, when given at doses from 10 mg/kg to 40 mg/kg mainly for 10 weeks, exhibited a fairly potent immunosuppressive effect on preventing renal allograft rejection in cynomolgus monkeys, with using a limited number of animals (22). All of the treated animals showed no evident abnormality, except for one who required euthanasia due to a severe hydronephrosis, having a small, mostly liquidized old cerebral infarction of unknown cause. In the scope of clinical application, this study was conducted to elucidate the immunosuppressive effects of 4D11 under practical and clinically relevant treatment regimens, that is, the 2-week induction therapy and 6-month maintenance therapy. In both of these treatment protocols, we examined the effects of 4D11 from a very low dose of 1 mg/kg up to 20 mg/kg, in order to find out an optimal therapeutic dose and blood concentration of the agent. In addition, we paid attention to investigate the in vivo effects of 4D11 on both cellular and antibody-mediated immune responses, and checked for 4D11-related adverse events, if any.

Materials and Methods

Animals

Forty-one purpose-bred male cynomolgus monkeys, Mocaca fascicularis, were used. The animals were 3.2 to 6.7 (4.7 ± 1.0) years old and weighed 2.5 to 5.7 (4.0 ± 0.9) kg, and were maintained and subjected to the study at the Shin Nippon Biomedical Laboratories (SNBL, Kagoshima, Japan). They were all seronegative for Hepatitis B virus, but seropositive for cytomegalovirus. The experimental protocol and all procedures were approved by the institutional animal care and use committee, and conducted in accordance with the standards described in the Guide for the Care and Use of Laboratory Animals, the National Institutes of Health Office of Animal Care and Use.

Kidney transplantation

One donor animal was used for two recipients. Donor and recipient monkey pairs were selected by ABO blood-type compatibility. Major histocompatibility complex (MHC) class II disparity, was determined by denaturing and direct sequencing of DR-beta allele as previously described (23). One-way mixed lymphocyte reaction (MLR) was used to select a pair with a stimulation index greater than 3. Intraabdominal kidney transplantation was performed as described previously (22). In brief, after skeletonization, donor animals were heparinized (100 unit/kg) and the kidneys were perfused in situ with HTK solution (Odyssey Pharmaceuticals Inc., Florham Park, NJ). Renal allograft was implanted by end-to-side anastomoses between the donor renal artery and the recipient distal aorta, and between the donor renal vein and the recipient vena cava. Ureteral reconstruction was performed in an end-to-end fashion using the 4.7 Fr Dretler ureteroscopy stent (Cook-urological Inc., Spencer, IN), but frequently required reanastomoses in some animals due to anastomotic stricture and obstruction. Immediately after grafting, bilateral native nephrectomies were performed. The animals were given cefazolin (100 mg/kg) and intravenous (i.v.) fluid for 3 days. Prophylactic drug for thrombosis was not used. Vital signs, urine output, appetite and attitude were monitored daily. Animals were euthanized when exhibiting any of the following criteria: serum creatinine (Cr) >10 mg/dL, severe weakness, weight loss or abnormal behavior, determined by veterinary staff and investigators.

Experimental groups and treatment protocols

The animals were randomly divided into nontreatment control (n = 3), induction (Group A) and maintenance (Group B) treatment groups. Groups A and B was further divided into four subgroups (n = 3, each) according to the dose of 4D11: Group A/B-I (1 mg/kg), Group A/B-II (5 mg/kg), Group A/B-III (10 mg/kg) and Group A/B-IV (20 mg/kg). The 4D11 was given intravenously on the day of transplantation (immediately before and after operation) and postoperative days (PODs) 4, 7, 11 and 14. Drug administration was ceased after POD 14 in Group A, while half of the initial dose was continued weekly thereafter until POD 180 in Group B.

Biochemical and immunological determinations

Hematology and blood chemistry:  In addition to the examination of serum Cr and blood urea nitrogen (BUN) levels twice per week, hematological and biochemical parameters were analyzed weekly by using the ADIVA120 (Bayer Diagnostics Ltd., Sudbury, UK) and JCA-BM8 (Nippon Denshi Ltd., Tokyo, Japan) analyzers.

Leukocyte phenotype:  Peripheral blood mononuclear cells (PBMC) were labeled with a combination of the following mAbs: CD3 (SP34), CD4 (L200), CD25 (M-A251), CD20 (2H7) (all from BD Biosciences Pharmingen, Mountain View, CA) and CD40 (mAb89: Immunotech, Prague, Czech Republic), and were assessed weekly by a flow cytometry.

Serum 4D11 concentration:  For the pharmacokinetic monitoring of 4D11, peripheral blood samples were obtained immediately before drug administration. Serum 4D11 level was measured by an enzyme-linked immunosorbent assay (ELISA) as described previously (22).

Mixed lymphocyte reaction (MLR):  One-way MLR was performed before transplantation and periodically after grafting. Gamma-irradiated (100 Gy) PBMCs of either donor animals were used as stimulator cells. Responder PBMCs (1 × 105/well) obtained from recipient animals were cocultured with stimulators (3 × 104/well) at 37°C for 6 days using RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). The cells were pulsed with H3-thymidine (1 μCi/well) before termination of culture, and proliferation activity was assessed by H3-thymidine incorporation using a beta-counter.

Antidonor antibody:  Donor-specific antibody (DSA) was assessed by incubating donor splenocytes with serum obtained from transplant recipients. After incubation (30 min, 4°C), cells were washed and stained with either mouse antihuman CD3 or CD20 together with rabbit antihuman IgG- or IgM-F (ab’) 2 mAbs (dilution 1:20; DAKO, Glostrup, Denmark). Cells were analyzed on a FACS Caliber (Becton-Dickinson, Franklin Lakes, NJ) by gating on CD3+ or CD20+ cells. A positive response was defined when the number of IgG- or IgM-bound cells exceeded 20% of the total gated cells.

Antidrug antibody:  The anti-4D11 Ab was examined by applying the surface plasmon resonance technology as described previously (22).

Histopathological determinations

Histological study:  Routine renal biopsies were performed on PODs 14, 28, 56 and monthly thereafter, and whenever graft rejection was suspected, by using the 20-gauge Biopty-cut needle (C.R. Bard Inc., Covington, GA). Necropsy was performed immediately after euthanasia to evaluate abnormality of the graft and extrarenal organs. Tissues were obtained from the brain, lung, heart, gastrointestinal tract, liver, spleen, lymph nodes, thymus and thyroid. They were fixed in 10% neutral buffered formalin, embedded in paraffin, and were stained with hematoxylin and eosin (H&E), periodic acid Schiff, periodic acid–methenamine–silver, elastica van Gieson and Masson trichrome. Graft histopathology was evaluated based on the Banff classification (24,25) by a single pathologist without knowledge of the treatments or clinical findings.

Immunohistochemistry:  Four-micron thick deparaffinized sections were stained with rabbit antihuman CD3 (Zymed Laboratories Inc., South San Francisco, CA), CD4 (1F6), CD8 (1A5), CD20 (L26) (all from Ventana Medical Systems) and CD83 (HB15e: Oxford Biotechnology Ltd., Kidlington, UK), after confirming the reactivity with monkey tissues. Slides were set on NexES® Immunohistochemistry Staining System with iVEW(tm) DAB Detection Kit (Ventana Medical Systems Inc., Tucson, AZ). Also, graft frozen sections were stained for C4d by the indirect immunofluorescent technique, using the antihuman C4d mAb (1:50; Quidel, San Diego, CA) and FITC-conjugated rabbit antimouse IgG polyclonal Ab (1:50; Zymed Laboratories Inc.).

Statistical analysis

All values were described as a mean ± standard deviation (SD). Intergroup statistical analysis was performed by a Mann–Whitney U-test. Kaplan–Meier analysis with a log-rank test was used to compare graft survival. Differences were considered statistically significant when a p-value was less than 0.05.

Results

Animal survival

Graft survival, histological diagnosis of biopsies, development of DSA and final cause of graft failure are summarized in Figure 1. Untreated control animals rejected renal allograft within a week (mean survival time [MST]= 6 days). In contrast, the induction treatment with 4D11 at doses of 1 mg/kg (Group A-I), 5 mg/kg (Group A-II), 10 mg/kg (Group A-III) and 20 mg/kg (Group A-IV) significantly prolonged graft MST to 54.7 (p < 0.025), 137.0 (p < 0.025), 167.7 (p < 0.025) and 121.3 days (p < 0.025), respectively, as compared to the control group (Figure 1A). After stopping the treatment at POD 14, most animals revealed a gradual elevation of serum Cr level between PODs 35 and 70 (Figure 2), and all grafts finally failed due to rejection that was confirmed by histopathology. When the 4D11 treatment was maintained by a weekly administration at a half of the initial dose following the induction treatment, higher doses of 4D11 further prolonged allograft survival: graft MST at 1 mg/kg (Group B-I), 5 mg/kg (Group B-II), 10 mg/kg (Group B-III) and 20 mg/kg (Group B-IV) was 56.3, 120.3, 218.0 and 179.3 days, respectively (Figure 1B). Although these survivals were significantly prolonged when compared with that of the control group (all p < 0.025), there was no significant difference among the 4D11 treatment groups. In Groups B-I and B-II, serum Cr level elevated in between PODs 40 and 90 (Figure 2), and grafts were rejected despite the continued treatment. On the other hand, with most of the animals given 4D11 at higher doses (Group B-III and B-IV), serum Cr level was kept under 2.5 mg/dL throughout the treatment course (Figure 2), but gradually increased after the drug cessation. One animal in Group B-III was euthanized on POD 27 due to pyelonephritis. Gram-negative rod bacteria were detected in the renal pelvis and urine. All other animals were euthanized due to histology-confirmed rejection (Figure 1B).

Figure 1.

Figure 1.

Clinical course and graft histopathological changes of individual monkeys receiving the induction (A) or maintenance (B) 4D11 treatments. The horizontal bars represent the time length of animal survival after renal transplantation. The graft survival time and the final histolopathogical diagnosis are shown at the end of the survival bar. Periodical biopsy results are depicted on each horizontal bar (N = normal; BC = borderline change; AR = acute rejection; CAN = chronic allograft nephropathy). The presence, including period of serum donor-specific antibody (DSA: oblique line) and antidrug antibody (ADA: gray) are indicated within the bars. The black bars represent presence of both DSA and ADA. One animal in Group B-III (animal no. a14) was euthanized on POD 27 due to pyelonephritis. All the other animals were euthanized because of histology-confirmed rejection.

Figure 1.

Figure 1.

Clinical course and graft histopathological changes of individual monkeys receiving the induction (A) or maintenance (B) 4D11 treatments. The horizontal bars represent the time length of animal survival after renal transplantation. The graft survival time and the final histolopathogical diagnosis are shown at the end of the survival bar. Periodical biopsy results are depicted on each horizontal bar (N = normal; BC = borderline change; AR = acute rejection; CAN = chronic allograft nephropathy). The presence, including period of serum donor-specific antibody (DSA: oblique line) and antidrug antibody (ADA: gray) are indicated within the bars. The black bars represent presence of both DSA and ADA. One animal in Group B-III (animal no. a14) was euthanized on POD 27 due to pyelonephritis. All the other animals were euthanized because of histology-confirmed rejection.

Figure 2.

Serum creatinine levels in renal allograft recipients. In the nontreated control group (dashed line), serum creatinine level raised rapidly at POD 3, and all animals rejected allograft within a week. In most of the animals given induction 4D11 treatment, serum creatinine level increased in between PODs 35 and 70 (solid lines). The serum creatinine level began to elevate at approximately 40 to 90 PODs in most of the animals treated with maintenance 4D11 at 0.5 or 2.5 mg/kg (Group B-I and B-II). On the other hand, when 4D11 was maintained in weekly administration at 5 or 10 mg/kg (Groups B-III and B-IV), serum creatinine level was kept under 2.5 mg/dL during the treatment course.

Serum 4D11 concentration

The serum drug level on POD 14 was 8.2 ± 2.3, 128.0 ± 6.8, 299.0 ± 61.2 and 611.7 ± 38.0 μg/mL in Group A, and 6.5 ± 1.6, 56.4 ± 13.1, 318.5 ± 22.7 and 612.7 ± 38.0 μg/mL in Group B at a dose of 1 mg/kg, 5 mg/kg, 10 mg/kg and 20 mg/kg, respectively (Figure 3). After drug cessation, blood 4D11 became undetectable at POD 40 in Group A-I, POD 77 in Groups A-II and A-IV and at POD 103 in Group A-III (Figure 3). In the lower-dose maintenance treatment groups, the trough level was less than 1 μg/mL after POD 28 in Group B-I (maintained at 0.5 mg/kg) and after POD 56 in Group B-II (2.5 mg/kg), although the weekly 4D11 administration continued. In contrast, the trough levels were maintained during the treatment period at 50 μg/mL and 100 μg/mL in Groups B-III and B-IV, respectively.

Figure 3.

Changes of serum 4D11 trough levels. Dashed lines and solid lines indicate the mean value of serum 4D11 trough levels of each animal in the induction (A) and maintenance (B) treatment groups, respectively. In all treated groups, the serum trough level peaked at POD 14, and decreased thereafter. In the group B-III and B-IV animals, the trough levels were maintained during the treatment period at 50 μg/mL and 100 μg/mL, respectively.

Leukocyte proliferation against donor or third-party antigens

MLR stimulation index (SI) against donor and third-party antigens was determined at 1, 3, 6 and 12 months following transplantation. All 4D11-treated animals exhibited a substantial reduction of lymphocyte proliferation against both donor (Figure 4) and third-party antigens (data not shown). Besides, the degree of SI suppression did not correlate with the dose, type of 4D11 treatment, degree of rejection or duration of graft survival time.

Figure 4.

MLR stimulation indexes at pre- and posttransplantation. The mean SI values against the donor antigens in the induction groups (dashed lines) and maintenance groups (solid lines) are depicted. All treated animals exhibited a substantial reduction irrespective of the treatment protocol and the dose of 4D11.

Antidonor antibody production

All animals were seronegative for DSA before transplantation. In the induction treatment groups, DSA was not detected during 4D11 administration. However, after drug cessation, transplant recipients started to develop DSA after 3 weeks in Groups A-I, A-II and A-III, and in between 6 and 8 weeks in Group A-IV (Figure 1A). On the other hand, in the maintenance treatment groups, two of three animals in Group B-I and all animals in Group B-II developed DSA during the treatment course, and rejected their grafts within 2 months after DSA formation except for one animal in Group B-II (Figure 1B). In the higher-dose 4D11-treated groups, Groups B-III and B-IV, DSA did not develop during the drug treatment (Figure 1B).

Anti-4D11 antibody production

Eight of 24 animals developed anti-4D11 Ab. In the induction treatment groups, six animals in total formed anti-4D11 Ab after drug cessation irrespective of the 4D11 doses (Figure 1A). In the maintenance treatment groups, one monkey each in Groups B-I and B-II developed anti-4D11 Ab on PODs 28 and 140, whereas that was not detected in the animals treated with higher 4D11 doses (Figure 1B).

Hematology

No significant changes in red blood cells (RBC), hematocrit (Ht), white blood cells (WBC) and platelet counts were noted in all animals except for one in Group B-III that experienced a transient anemia from PODs 42 to 84 with having a normal renal function without a sign of clinical bleeding. This anemia, however, was spontaneously normalized without any alteration of treatment schedule. The number of peripheral CD20+ B cells reduced to one-third to two-third of the preoperative value within 4 weeks after transplantation (Figure 5). In all animals given the induction treatment and low-dose maintenance treatment, CD20+ cell counts recovered to a normal level before POD 90. However, in Groups B-III and B-IV, the B-cell counts remained reduced during the treatment course or until their deaths. The population of CD3+, CD4+, CD8+, CD40+ and CD4+CD25+ cells in PBMC showed no changes in all groups (data not shown).

Figure 5.

Changes of peripheral CD20+ cell counts following the induction (A) and maintenance (B) 4D11 treatments. The number of peripheral CD20+ B cells reduced to one-third to two-third of the preoperative value within 4 weeks after transplantation. In all of the induction and low-dose maintenance (Groups B-I and B-II) 4D11 treatment groups, peripheral CD20+ B-cell counts recovered to a normal level within POD 90. However, in the high-dose 4D11 maintenance treatment groups (Groups B-III and B-IV), CD20+ B-cell counts did not recover during the treatment period. The gray area indicates the range of the preoperative peripheral CD20+ cell numbers (mean ± SD) of all the 27 transplant recipients.

Biochemistry

Serum liver enzymes, lipid, glucose and all other biochemical parameters determined were unaffected in all of 4D11-treated animals (data not shown).

Histopathological study

Renal allograft:  The failed grafts in the control group animals exhibited characteristic histological features of acute cellular rejection with a dense mononuclear cell infiltration, tubular epithelial cell necrosis and interstitial hemorrhage. At 1-month biopsies, one animal in Group A-II and one animal each in Groups A-III and B-II showed grade IA or IB rejection (Figure 1). At the same point in time, however, all of the other treated animals showed only borderline changes with a mild-to-moderate interstitial cell infiltration with or without mild tubulitis, regardless of 4D11 dosages and treatment protocol (Figure 1, Figure 6A, B). In the induction treatment groups, biopsies taken at 2 and 3 months exhibited a moderate-to-severe interstitial cell infiltration and tubulitis (Figure 6C), and progressed to grade IIA or IIB within 5 months postoperation (Figure 6D). In the maintenance treatment groups, most grafts of the monkeys treated with a lower-dose 4D11 exhibited the same histopathological changes as that of the induction-treated animals. On the other hand, the grafts of animals given 4D11 at high doses showed less cellular infiltrate at 2 and 3 months after operation (Figure 6E), but almost all of the long-term surviving grafts exhibited a moderate-to-severe interstitial fibrosis and tubular atrophy without allograft glomerulopathy or arterial intimal fibrosis (Figure 6F).

Figure 6.

Microscopic appearance of renal allografts. (A)–(F) photographs show histology of graft biopsy specimens (H&E staining, magnification ×200). Graft biopsies in Group B-I (animal no. a23) (A) and Group B-IV (animal no. a11) (B) show borderline changes with mild-to-moderate interstitial cell infiltration and mild tubulitis at POD 28. (C) Graft biopsy in Group A-III (animal no. a2) at POD 84 shows a severe interstitial cell infiltration and tubulitis. (D) Graft biopsy in Group A-IV (animal no. a7) at POD 150 shows a severe interstitial cell infiltration and interstitial fibrosis. The animal rejected allograft soon after this biopsy with a grade IIA rejection. (E) Graft biopsy in Group B-IV (animal no. a11) at POD 84 shows mild interstitial cell infiltration and tubulitis. (F) Graft biopsy in Group B-IV (animal no. a11) shows a severe interstitial fibrosis and tubular atrophy at POD 150. All photographs are taken with an ×200 magnification. (G)∼(J) Graft immunohistochemistry for CD3, CD4, CD8 and CD20 (magnification ×200). The graft specimen obtained from the recipient in Group A-IV (animal no. a9) at POD 28 shows that a majority of interstitial infiltrating cells were CD3+ (G) and CD8+ (I) cells, but CD4+ (H) or CD20+ (J) cells were barely detected. (K) Autopsy-obtained graft specimen in Group A-III (animal no. a30) at POD 320 shows capillary staining for C4d (magnification ×400). (L) Autopsy-obtained graft specimen in Group B-III (animal no. a5) at POD 374 (magnification ×400). In this graft, C4d was negative in the peritubular capillaries.

Extra renal tissues:  No histopathological remarks of thromboembolism were noted in all animals during the 4D11 treatment, after the drug cessation or at the time of autopsy. Besides, no abnormal findings were seen with tissue specimens, for example, the brain, heart or lung on autopsy. Depletion of germinal center was noted in the spleen in 10 (one animal each in Groups A-III, B-II and B-III, two each in Groups A-I and B-I, and all in Group B-IV) of 22 animals treated with 4D11. In these 10 spleens, the ratio of primary against secondary follicles was increased to 86.5 ± 8.9%, which was in contrast to that of the other animals (27.9 ± 16.4%). In addition, germinal center was depleted in the peripheral and intraabdominal lymph nodes in these 10 animals (data not shown).

Immunohistological study:  Irrespective of the dose and duration of 4D11 treatment, a majority of interstitial infiltrating cells in the grafts at early postoperative days were CD3+ and CD8+ cells. The CD4+, CD20+ and CD83+ cells were barely detected (Figure 6G–J). Within the later biopsies taken from the grafts, CD8+ cells still persisted, while the number of CD20+ cells increased gradually. Most grafts obtained from the induction treatment groups (one animal each in Groups A-I, A-IV and B-II, two in Group A-III and all three in Group A-II) were C4d positive at the time of autopsy (Figure 6K), while that was barely detectable in the maintenance treatment groups (Figure 6L).

Discussion

Ligation of the CD40-CD154 axis plays pivotal roles in the initiation and progression of alloimmune responses. The signal activates antigen-presenting cells (APC) to upregulate CD80, CD86, MHC class II, adhesion molecules and cytokines (1,2,26). It promotes T-cell activation to attack the graft (6), and induces B-cell proliferation, immunoglobulin switching, antibody production and germinal center formation (3–5). It also has a close association with the activation of endothelial cells, fibroblasts and platelets (1,27). All of these cellular and humoral responses are intimately involved in the establishment of acute and chronic rejection (2). Thus, blockade of the CD40–CD154 signal using mAbs, targeting either one with or without interrupting the other costimulatory molecules, has been attempted in many experimental transplantation models (7–10,20) and some clinical cases (28). The first mAb developed against CD154, hu5C8, showed encouraging immunosuppressive effect on kidney allografts in nonhuman primate (10–12), but the clinical application was held due to unexpected thromboembolic complication. Subsequently developed anti-CD154 mAbs, IDEC-131 (13) and ABI793 (14) were the same. Therefore, blockade of the counter molecule, CD40, by anti-CD40 mAb has been an attractive alternative to achieve the goal.

The 4D11 is a novel fully human anti-CD-40 mAb, which interrupts the CD40–CD154 axis by masking but not causing cytotoxic activities. In preliminary studies, 4D11 inhibited soluble human CD154-induced PBMC proliferation at IC50 of 5.8 ng/mL in human and at 25.1 ng/mL in monkey (unpublished data). The binding rates of CD40 were comparable between human and monkey, but the dissociation rate was slightly slower in human (unpublished data). The delayed-type hypersensitivity reaction and antibody production against tetanus toxoid were completely abolished in monkey with 4D11 at 10 mg/kg (29). In addition, weekly administration of 4D11 at 100 mg/kg for 4 weeks did not induce drug-related adverse events such as platelet activation or thromboembolism (29). Finally, in the previous kidney transplant experiment (22), we have reported that 4D11 exhibited a fairly potent immunosuppressive effect in cynomolgus monkeys without notable side effects, except for one who had an old small cerebral infarction at the time of autopsy. This animal experienced a severe hydronephrosis during the course.

Current regimens of standard immunosuppression include calcineurin inhibitors (CIs), cyclosporine or tacrolimus. They allowed successful early graft survival, but caused untoward various complications later. Augmentation of antirejection by induction treatment with anti-IL-2R mAb, anti-thymocyte globulin, anti-CD52 mAb (Campath-1H) and/or the use of multidrug cocktail, aiming at inhibition of different molecules associated with the initiation and progress of rejection, are the means to reduce CIs, and to even eliminate these drugs during maintenance immunosuppression (30). In addition to its use as an induction therapy, the blockade of costimulatory molecules appears to have a unique role for the maintenance of immunosuppression as shown recently. Larsen et al. (31) reported that, although three animals died during the treatment, administration of LEA29Y (belatacept) at 20 mg/kg, biweekly for upto 70 days, yielded survivals of 99 days and 134 days in the other two animals after kidney transplantation in rhesus monkeys. Based on these observations, Vincenti et al. (32) carried out a clinical trial and demonstrated that, when compared to cyclosporine-based immunosuppression, combination of belatacept with basiliximab, mycophenolate mofetil and tapering of methylprednisolone showed an equivalent efficacy in preventing acute rejection at 6 months, and reduced the rate of adverse events at 12 months. Belatacept is a modified version of CTLA-4Ig that competes with surface CD80 and CD86 molecules on APCs upon engaging to CD28. This study has shown that 4D11, which interrupts costimulatory molecule CD40 on APCs, is also an effective drug as a maintenance treatment when half of the initial dose is administered weekly for 180 days: graft MST was extended to 56.3, 120.3, 218.0 and 179.3 days when given at doses of 0.5, 2.5, 5 and 10 mg/kg, respectively. Moreover, most of the animals given higher 4D11 doses (5 and 10 mg/kg in Groups B-III and B-IV) showed no histological evidence rejection in the graft during the drug administration. Appropriate maintenance dose appears to be 5 to 10 mg/kg with blood trough levels of 50 to 100 μg/mL in our experimental model. We tested a weekly maintenance treatment in this study, because 4D11 has a short half-life (96.3 h with 30 mg/kg, i.v.) in cynomolgus monkeys (29), and a monthly 4D11 administration at 40 mg/kg was unable to prevent rejection in our earlier study (22).

Previous study demonstrated that animals receiving either anti-CD80, anti-CD86 or anti-CD154 mAbs developed antidonor IgG Abs (10), while the treatment with anti-CD40 mAb, ch5D12, did not (33). In our current study, all of the monkeys given induction or lower-dose maintenance treatment finally developed DSA. In contrast, 4D11 maintenance treatment with higher doses inhibited DSA formation, which was also confirmed by graft C4d staining. In addition, the animals receiving higher-dose maintenance treatment did not develop anti-4D11 Ab. This was in contrast to the observation reported by Haanstra et al. (19), in which 2 of the 4 monkeys given ch5D12 developed anti-ch5D12 Ab even with the high-dose maintenance treatment. Because conventional immunosuppressants cannot fully abolish DSA development and antibody-mediated rejection, CD40 blockade by the 4D11 seems to have great advantages for preventing humoral type of rejection and treating presensitized patients.

Besides suppression of DSA and antidrug Ab formation, treatment with 4D11 reduced peripheral CD20+ B cells to one-third to two-third of the preoperative values. This was, in part, comparable to the chi220, which has been shown to reduce circulating B cells in nonhuman primates (20), while this was not the case with the other antihuman CD40 mAb, ch5D12 (19). Along with the peripheral B-cell reduction, suppression of germinal center formation in the spleen and lymph nodes was noted at the time of autopsy in some of the 4D11-treated monkeys, especially when administered at higher doses. This phenomenon was also found in our previous preliminary study (22), which was in line with the effects of ch5D12 on lymphoid organ/tissues shown in cynomolgus monkeys (34). Although rituximab (anti-CD20 mAb) and Campath-1H also reduces circulating B-cell numbers and inhibits formation of germinal centers (34,35), the mechanism of action on B cells seems to be different since the 4D11 do not cause antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or apoptosis (29). Moreover, the number of circulating CD40+ cells was unaffected in our monkeys following 4D11 treatment (data not shown). Thus, it seems that the suppression of DSA, antidrug Ab and germinal center formation by the 4D11 can be attributed to the inhibitory effects of CD40 signaling pathway that has been shown essential for proliferation, Ig production, isotype switching and memory cell development in B cells (1–5), rather than B-cell depletion. In order to elucidate the precise mechanism, however, further in vitro studies are warranted.

Because clinical trials on CD40-CD154 blockade by the anti-CD154 mAbs in autoimmune diseases and transplantation were terminated due to unwarranted thromboembolic complications (15,26), the side effects of 4D11 have always been our major concern. Indeed, in the previous preliminary study, we found a small old cerebral infarction in one of the 10 4D11-treated animals at the time of necropsy (22). In this study, however, none of the animals treated for upto 180 days have demonstrated histopathological abnormalities reflecting the presence of thromboembolism. Andre et al. (36) have shown that CD154 is involved in stabilizing arterial thrombi via the integrin glycoprotein IIb/IIIa-dependent pathway, and that the absence of CD154 delays arterial occlusion. In contrast, they have described that the absence of CD40 had no apparent effect on thrombus development or arterial occlusion in vivo (36). Others have shown that CD154-deficient mice have developed unstable thrombi, whereas this was not the case in CD40 knockout mice (37).

In conclusion, both induction and maintenance treatments with a fully human anti-CD40 mAb, 4D11, induced potent immunosuppressive effects on nonhuman primate renal allografts without causing serious side effects including thromboembolic complications. Our results indicate that the 4D11 is a promising agent for clinical organ transplantation. Further studies, such as combination with conventional immunosuppressants or various immunosuppressive Abs are warranted, and some are already under way.

Acknowledgments

The authors thank Ms. M. Goto for serum 4D11 concentration and Dr. K. Harada for anti-4D11 Ab analyses; Mr. R. Mitsuo and Mr. T. Nakamura for study conduction; Mr. Ikeda and Mr. Mizuyoshi and others in SNBL for the excellent animal care.

This work was supported in part by grants from the Kyowa Hakko Kirin Co., Ltd. and from the Ministry of Public Health and Welfare and the Ministry of Education, Japan.

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