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Keywords:

  • Co-stimulation blockade;
  • monoclonal antibodies;
  • nonhuman primates;
  • pancreatic islet transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

A strategy for inhibiting CD40 has been considered as an alternative approach for immunosuppression because of undesirable effects of anti-CD154 monoclonal antibodies (mAbs). Previously, we demonstrated that ASKP1240, which is a fully human anti-CD40 mAb, significantly prolonged kidney and liver allograft survival in cynomolgus monkeys without causing thromboembolic complications. Herein, we evaluated the effect of ASKP1240 on pancreatic islet transplantation (PITx) in cynomolgus monkeys. Diabetes was induced by total pancreatectomy, and islet allografts were transplanted into the liver. Following PITx (8201–12 438 IEQ/kg), blood glucose levels normalized promptly in all animals. Control islet allografts were rejected within 9 days (n = 3), whereas ASKP1240 (10 mg/kg) given on postoperative days 0, 4, 7, 11 and 14 (induction treatment, n = 5) significantly prolonged graft survival time (GST) to >15, >23, 210, 250 and >608 days, respectively. When ASKP1240 (5 mg/kg) was administered weekly thereafter up to post-PITx 6 months (maintenance treatment, n = 4), GST was markedly prolonged to >96, >115, 523 and >607 days. During the ASKP1240 treatment period, both anti-donor cellular responses and development of anti-donor antibodies were abolished, and no serious adverse events were noted. ASKP1240 appears to be a promising candidate for immunosuppression in clinical PITx.


Abbreviations
ADA

anti-drug antibody

BGLs

blood glucose levels

DM

diabetes mellitus

DSA

donor-specific antibody

FBG

fasting blood glucose

GST

graft survival time

IEQ

islet equivalents

IFN

interferon

IVGTT

intravenous glucose tolerance test

mAbs

monoclonal antibodies

MHC

major histocompatibility complex

MLR

mixed lymphocyte reaction

NHPs

nonhuman primates

PITx

pancreatic islet transplantation

PODs

postoperative days

S.I.

stimulation index

UW

University of Wisconsin.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Clinical pancreatic islet transplantation (PITx) became a promising and realistic treatment option for type 1 diabetes mellitus (DM) after the introduction of the Edmonton protocol [1]. The primary outcomes and safety of PITx have improved steadily in recipients of PITx recently, with fewer islet infusions and adverse events [2, 3]. However, the toxicity associated with immunosuppression is still a major obstacle that prevents long-term allogeneic islet acceptance [4]. Although steroid avoidance has been shown to be feasible, calcineurin inhibitors, which most steroid-free regimens use as the primary immunosuppressant, have significant concomitant side effects, including nephrotoxicity and diabetogenicity [5, 6]. Furthermore, in addition to calcineurin inhibitors, sirolimus also negatively affects β cell viability and regeneration [7, 8] as well as the engraftment and function of transplanted islet grafts [9]. A new immunosuppressive strategy that avoids the use of these conventional immunosuppressants and introduces a suitable, less toxic agent is essential for improvement of clinical PITx.

The CD40–CD154 pathway is representative of various co-stimulatory signaling pathways that play a critical role in T cell activation and alloimmune responses, including humoral immunity. The blockade of this pathway is a key therapeutic strategy to induce donor-specific immunosuppression and/or tolerance in experimental organ transplantation [10, 11]. Indeed, previous studies have demonstrated that co-stimulatory blockade with anti-CD154 monoclonal antibodies (mAbs) allows islet engraftment, insulin independence and long-term function in nonhuman primate recipients of allogeneic islets [12-14]. However, the clinical application of anti-CD154 mAbs is not feasible, mainly because of thromboembolic complications caused by aggregation of platelets induced by the mAb itself [15] and/or the instability of thrombi regulated by soluble CD154 [16]. Alternatively, inhibition of the counter receptor CD40 has recently received attention for the blockade of CD40–CD154 costimulation. A previous study showed that a chimeric anti-CD40 mAb (chi220) markedly prolonged islet allograft survival time in rhesus monkeys when combined with CTLA4Ig [17]. The same investigators have also reported the efficacy of other anti-CD40 mAbs, such as 3A8 [18, 19] and 2C10 [20], on PITx in nonhuman primates (NHPs); long-term islet allograft acceptance was achieved when these mAbs were given in combination with basiliximab, sirolimus and/or CTLA4Ig. In parallel with other researchers, we have been attempting to develop a new anti-CD40 mAb. Previously, we demonstrated that a fully human anti-CD40 mAb, ASKP1240, strongly inhibited both cellular and humoral alloimmune responses, and markedly prolonged renal and hepatic allograft survival in cynomolgus monkeys without causing apparent adverse effects including thromboembolism [21-23]. In this study, we evaluated the effect of ASKP1240 on allogeneic PITx in cynomolgus monkeys.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Animals

In total, 25 purpose-bred male cynomolgus monkeys (Macaca fascicularis) aged 5.01 ± 0.74 years and with a body weight of 5.26 ± 0.97 kg were used in the study at Shin Nippon Biomedical Laboratories (SNBL, Kagoshima, Japan). The experimental protocol was approved by the Animal Care and Use Committee of the SNBL, and all procedures were performed in accordance with the standards described in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (http://www.nap.edu).

Selection of donor–recipient pair

Recipient and donor pairs were selected according to ABO blood type compatibility and the results of a one-way mixed lymphocyte reaction (MLR) assay that revealed a stimulation index (S.I.) of >4.6. In addition, the genetic nonidentity of all monkey pairs was confirmed based on disparities of major histocompatibility complex (MHC) class II (DRβ) loci established by direct sequencing of the second exon of DRB as previously described [21, 22].

Diabetes induction

Diabetes was induced by total pancreatectomy without duodenectomy or splenectomy as previously described [24, 25]. Blood glucose levels (BGLs) were monitored using Accu-Check active II® (Roche, Indianapolis, IN) and controlled in the range of 100–200 mg/dL with regular and intermediate human insulin [Humulin R (HR) and Humulin N (HN); Eli Lilly, Hyogo, Japan].

Pancreas procurement

Donor total pancreatectomy was performed on the same day as PITx. Animals were anesthetized by intramuscular administration of ketamine hydrochloride (Kamud Drugs Pvt. Ltd, Navi Mumbai, India) for induction and inhalation of isoflurane (Forane®: Abbott Japan Co., Ltd., Tokyo, Japan) and nitrous oxide for maintenance. The abdominal organs were perfused through the aorta using cold University of Wisconsin (UW) solution (Astellas Pharma Co., Ltd, Tokyo, Japan) and the pancreas was removed en-bloc with the spleen and duodenum. The spleen and mesenteric lymph nodes were processed and cryopreserved for use as a cell source for later immunological assays.

Pancreatic islet isolation

Pancreatic islets were isolated from the pancreas as described previously [24]. Briefly, the recovered donor pancreas was distended with Liberase MTF-s® (Roche Biochemicals, Indianapolis, IN) and incubated at 37°C under static digestion. Digested pancreas tissues were applied to a three-layer discontinuous gradient (layer densities of 1.112, 1.096 and 1.060) that consisted of the UW solution and Ficoll (Sigma–Aldrich, St. Louis, MO). Purified islets were assessed by counting the number of islets [26], and the data were mathematically converted to determine the number of islets with an average diameter of 150 µm and were expressed as islet equivalents (IEQ). The purity of the isolated islets was estimated based on the percentage of dithizone (Sigma–Aldrich)-positive particles present in the preparation. The viability of the islets was estimated on the basis of fluorescein diacetate (Sigma–Aldrich) and propidium iodide (Sigma–Aldrich) staining.

Islet transplantation

PITx was performed at 14 days after total pancreatectomy as described previously [24]. Exogenous insulin was not administered starting the day before PITx. Purified islets suspended in normal saline with heparin (100 U/kg) were slowly infused into the liver via the mesenteric vein.

Post-PITx care and monitoring of blood glucose

Animals were given prophylactic antibiotics, i.e., cefazolin sodium (Astellas Pharma Co., Ltd.), at a dose of 100 mg/body (i.v.) on postoperative days (PODs) 0, 1 and 2. Buprenorphine hydrochloride (Otsuka Co., Ltd., Tokyo, Japan) was administered on the day of transplantation and PODs 1 and 2. The animals were fed morning, afternoon and night meals of Hi-Fiber Primate (Purina Mills, LLC, Gray Summit, MO); the amount of food provided at each meal was 108 g (426.6 kcal, including 62.1% carbohydrates, 13.6% fat and 24.3% protein) [24]. Pancreatic exocrine insufficiency was compensated for by using pancreatin (Pancreatin®, Maruishi, Osaka, Japan). Fasting blood glucose (FBG) level was defined as the BGL at prebreakfast (15 h of fasting). Animals were not sedated with ketamine to obtain these measurements.

Experimental groups and treatment protocols

The islet recipients were randomly divided into three groups. Three animals receiving no treatment served as the control (n = 3). For the induction treatment group (n = 5), ASKP1240 (10 mg/kg) was given intravenously on days 0, 4, 7, 11 and 14. For the maintenance treatment group (n = 4), weekly ASKP1240 (5 mg/kg) administration was subsequently continued for up to 6 months after PITx. No additional therapy was given to prevent rejection.

Definition of rejection and BG control after rejection

Rejection was considered to occur when the FBG level exceeded 250 mg/dL for three consecutive days. The date of graft rejection was defined as the first of the three consecutive days. Subsequent to rejection, recipient animals were treated with exogenous insulin (Humulin R or N) to maintain BG levels in the range of 100–200 mg/dL.

Measurement of plasma C-peptide and insulin

Serum C-peptide and insulin levels were measured by a chemiluminescent enzyme immunoassay method (Fujirebio Inc., Tokyo, Japan) using human antibodies. The amino acid sequence identities of C-peptide and insulin between human and cynomolgus monkeys are 97% and 100%, respectively [27]. The lower limit of detection for C-peptide was 0.03 ng/mL and that for insulin was 0.30 µIU/mL.

Intravenous glucose tolerance test (IVGTT)

Food was withheld for 15 h before testing. The test animals were given 0.5 g/kg glucose in a 25% glucose solution intravenously over 1 min. Blood samples were collected before glucose injection and at 1, 3, 5, 10, 15, 20, 25, 30, 45 and 60 min after glucose injection for assessment of BG, serum C-peptide and insulin levels. IVGTTs were performed before and after total pancreatectomy, and sequentially after PITx.

Serum ASKP1240 trough level

For pharmacokinetic monitoring of ASKP1240, peripheral blood samples were obtained immediately before drug administration. Serum ASKP1240 trough levels were measured using an enzyme-linked immunosorbent assay as described previously [21, 22].

Donor-specific antibody (DSA)

DSAs were assessed by incubating donor splenocytes with serum obtained from transplanted recipients as described previously [21, 22].

Anti-ASKP1240 antibody

Anti-drug antibodies (ADAs) were examined by applying surface plasmon resonance technology as described previously [21, 22].

ELIspot assay

The frequency of interferon (IFN)-γ-secreting T cells reactive to donor antigens or third-party antigens (leukocytes from a third-party monkey) was determined using the ELIspotPLUS Monkey IFN-γ kit (Mabtech, Cleveland, OH), as described previously [23].

Phenotype analyses of peripheral leukocytes

Peripheral blood mononuclear cells (PBMCs) were labeled with the following mAbs: CD4 (L200), CD8 (RPA-T8), CD20 (2H7) and CD25 (M-A251), all of which were from BD Biosciences (Pharmingen, Mountain View, CA), and FoxP3 (PCH101), which was from eBioscience (San Diego, CA). Subsequently, they were assessed by flow cytometry as described previously [21, 22].

Statistical analysis

All values are presented as the mean (SD). An intergroup statistical analysis was performed using an analysis of variance with a post hoc test. Differences were defined as statistically significant when p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Characteristics of transplanted islets and recipient animals

The isolated islets were suitable for transplantation in terms of purity (>90%) and viability (>90%). An adequate number of viable islets to achieve PITx was obtained from a single donor, except for one animal who was in the control group (C-#3) and required two donors. The MLR S.I. of the recipient and donor pairs was 4.6–9.9, and the number of transplanted islets was 8201–12 438 IEQ/kg. These parameters did not statistically differ among the experimental groups (Table 1).

Table 1. Characteristics of recipient animals and transplanted islets
TreatmentNAge (months)BW (kg)Transplanted islets (IEQ/kg)MLR S.I.
  1. IEQ/kg, islet equivalents/kilogram.

Control355.0 ± 3.614.2 ± 0.79361 ± 2286.7 ± 0.6
Induction559.2 ± 10.64.7 ± 0.88853 ± 3737.0 ± 0.9
Maintenance470.5 ± 10.85.0 ± 0.810 565 ± 20238.0 ± 2.0

Allogeneic islet graft survival and outcome

Islet graft survival time (GST) and the corresponding recipient outcome are summarized in Figure 1. Without immunosuppression, control allogeneic islet grafts were promptly rejected within 9 days (C-#1, C-#2 and C-#3). In contrast, the ASKP1240 induction treatment prolonged the survival time of pancreatic allogeneic islet grafts. In this treatment group, one animal (I-#1) died at 15 days after PITx because of an internal hernia as determined by autopsy, and another animal (I-#2) was lost on day 23 because of peritonitis arising from stomach perforation at the site of pyloroplasty. At the time of sacrifice, the islet allografts in these two islet recipients were functioning well. Another two animals, I-#3 and I-#4, rejected the islet allografts at 210 and 250 days after PITx, respectively. The other animal (I-#5) maintained normoglycemia during the study without any signs of rejection and was euthanized at 608 days after PITx. The maintenance ASKP1240 treatment also markedly prolonged the survival time of islet allografts to 523 (M-#3) and >607 days (M-#4). Despite the pyloroplasty and compensation with pancreatin, two of the four animals in the maintenance treatment group experienced gastric dysmobility and malnutrition that caused progressive loss of body weight, and they were sacrificed at 96 (M-#1) and 115 (M-#2) days after PITx, although both animals harbored functional islet allografts. Another animal in this treatment group demonstrated rejection of the islet allograft at 523 (M-#3) days after PITx, whereas the other islet recipient (M-#4) maintained normoglycemia up to 607 days after PITx until the day of euthanasia. No serious side effects, including thromboemboli, were observed in animals receiving the ASKP1240 treatment.

image

Figure 1. Clinical course of islet-transplanted animals. Islet allograft survival time and corresponding recipient outcomes are summarized. Horizontal bars represent the duration of graft survival in each animal after pancreatic islet transplantation (PITx). GST and final diagnosis are shown at the end of the survival bar. The superscript symbol at the end of the GST represents animals that died because of surgical complications but that had functional islet allografts. The course of death was determined by clinical findings and necropsy. In the figure, C-#1, C-#2 and C-#3 represent the nontreated control animals; I-#1, I-#2, I-#3, I-#4 and I-#5 represent the ASKP1240 induction-treatment animals; and M-#1, M-#2, M-#3 and M-#4 represent the ASPK140 maintenance-treatment animals. In the control animals, transplanted islet allografts were promptly rejected within 9 days. The ASKP1240 induction treatment prolonged allograft survival to 210 (I-#3), 250 (I-#4) and more than 608 (I-#5) days; two animals with functional islet grafts died on days 15 and 23 because of internal hernia (I-#1) and peritonitis (I-#2), respectively. The ASKP1240 maintenance treatment also markedly prolonged islet allograft survival time to 523 (M-#3) and more than 96 (M-#1), 115 (M-#2) and 607 days (M-#4). Two animals in the ASKP1240 maintenance treatment group developed gastric dysmobility and were sacrificed at 96 (M-#1) and 115 (M-#2) days after PITx without apparent rejection.

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FBG and serum C-peptide levels after PITx

After total pancreatectomy, complete induction of insulin-dependent diabetes was confirmed in all animals based on the lack of response of serum C-peptide and insulin levels on IVGTT. Following allogeneic PITx, FBG levels were normalized promptly in all animals. However, in the control animals, FBG levels exceeded 250 mg/dL and rejection occurred within 9 days. Fasting serum C-peptide levels were reduced to an undetectable level on the next day of rejection (animals C-#1, C-#2 and C-#3). In contrast, treatment with ASPK1240 maintained normoglycemia in the long term, and serum C-peptide was detected during the period of normoglycemia. In three of the ASKP1240-treated animals, FBG levels increased and allograft rejection occurred at 210 (I-#3), 250 (I-#4) and 523 (M-#3) days after PITx. Inversely, the fasting serum C-peptide level decreased to an undetectable level within 2 weeks of the day of graft rejection in animals I-#3 and I-#4, whereas C-peptide was persistent in the sera, albeit at a low level, in animal M-#3 at the time of sacrifice on postoperative day 598 (Figure 2).

image

Figure 2. Fasting blood glucose and serum C-peptide levels after PITx. Fasting blood glucose (FBG) levels (left panels) and fasting C-peptide levels (right panels) after PITx are presented. The shaded areas represent the periods of ASKP1240 administration. After allogeneic islet grafting, FBG levels normalized promptly in all animals without exogenous insulin injection. In the control animals, FBG levels exceeded 250 mg/dL shortly after blood glucose normalization and rejection occurred within 9 days (GST: 7 days in C-#1, 8 days in C-#2 and 9 days in C-#3). Fasting serum C-peptide levels were reduced to an undetectable level on the day after rejection (upper panels). In contrast, treatment with ASPK1240 facilitated allograft acceptance in all animals. In the ASKP1240 induction treatment group (middle panels), FBG levels gradually increased after ASKP1240 cessation in 2 animals (I-#3 and I-#4) and rejection occurred thereafter at 210 and 250 days after PITx, respectively. Fasting serum C-peptide levels were also reduced to undetectable levels after rejection (I-#3; white circle and I-#4; white triangle in the middle right panel). The other animal (I-#5) demonstrated survival of the islet allograft for more than 600 days, and C-peptide was detected throughout the duration of the study (black square in the middle right panel). Maintenance treatment with ASKP124 also facilitated long-term allograft acceptance (bottom panels). The maintenance treatment resulted in long-term islet allograft acceptance for more than 600 days in one animal (M-#4). C-peptide levels in this animal were virtually constant throughout the study (black diamond in the bottom right panel). In another animal (M-#3), FBG levels gradually increased, and the islet allograft was rejected at 523 days after PITx.

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Function of islet allografts in ASKP1240-treated cynomolgus monkeys

To evaluate the function of the transplanted islet allografts, IVGTT was performed sequentially. As representative data, graft-rejected (M-#3) and graft-accepted (M-#4) animals are shown in Figure 3. At 7 days after total pancreatectomy, changes in C-peptide and insulin production were not observed following IVGTT. When examined on day 180 after PITx, the BGL, C-peptide level, and insulin production in response to IVGTT showed a normal pattern similar to that before pancreatectomy. In graft-rejected animals, e.g. animal M-#3, BGLs in response to IVGTT that was performed at 7 days after rejection showed a diabetic pattern, and C-peptide and insulin levels did not respond to glucose injection. In contrast, blood glucose, C-peptide and insulin levels remained normal even at the end of the study in animals that accepted islet allografts, e.g. animal M-#4.

image

Figure 3. Islet allograft function in ASKP1240-treated cynomolgus monkeys. IVGTT was performed, and representative data for islet-rejected (M-#3) and islet-accepted (M-#4) animals are shown. IVGTT was performed before (pre-T.P.; white square) and after total pancreatectomy (post-T.P.; black square), at 180 days after PITx (bar), after rejection (x-marks) or at 606 days after PITx in the animal demonstrating long-term acceptance (x-marks). In animals demonstrating rejection and acceptance, C-peptide and insulin production were unaffected after pancreatectomy. At 180 days after PITx, the BGL, C-peptide and insulin production in response to IVGTT were normal, i.e. identical to that observed for IVGTT performed before total pancreatectomy. In the animal showing rejection (M-#3; GST; 523 days), BGLs in response to IVGTT performed at 7 days after rejection showed a diabetic pattern, and C-peptide and insulin levels did not respond to glucose injection (A). In contrast, in the animal showing graft acceptance (M-#4), blood glucose, C-peptide and insulin levels remained normal even at the end of the study (B).

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Histological features of transplanted islet allografts

In the livers of ASKP 1240-treated animals who were normoglycemic but were euthanized prematurely because of surgical complications (I-#1, I-#2, M-#1 and M-#2), islet allografts were present in the portal area. In these samples, cellular infiltrates were not observed (data not shown). In the long-term graft-accepted animals (I-#5 and M-#4), durable islet allografts also were confirmed by insulin staining, without evidence of cellular infiltration of CD3+, CD4+ and CD8+ cells into or surrounding the islet allografts as determined by immunohistochemistry (Supplemental Figure S1). In the liver of the animal who became diabetic at 523 days after PITx (M-#3), only a few islets were identified after extensive histological analysis. A focal cellular infiltrate was noted around these islets (Supplemental Figure S1). However, in the livers of other rejected animals (C-#1, C-#2, C-#3, I-#3 and I-#4), islet allografts were not identified by microscopic examination.

ASKP1240 pharmacokinetic study

The serum ASKP1240 trough level peaked at 14 days after PITx and decreased thereafter. In the induction group, the serum ASKP1240 concentration reduced to an undetectable level at 8 weeks after discontinuation of treatment. In the maintenance treatment group, ASKP1240 trough levels were maintained at 30–100 µg/mL during the treatment period and persisted for up to 200 days after PITx (Figure 4).

image

Figure 4. ASKP1240 pharmacokinetic study. Serum ASKP1240 trough levels were periodically assessed before and after PITx. The shaded areas indicate the periods of ASKP1240 administration. The serum ASKP1240 trough level peaked at 14 days after PITx and decreased thereafter. In the ASKP1240 induction group, the serum ASKP1240 level was reduced to an undetectable level at 8 weeks after the discontinuation of treatment. In the ASKP1240 maintenance treatment group, trough levels were maintained at 30–100 µg/mL during the treatment period and for up to 200 days after PITx.

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Hematology

The number of peripheral CD4+ cells increased in some of the ASKP1240-treated animals, especially in recipients that accepted islet allografts in the long-term without rejection (I-#5 and M-#4) (Figure 5A). No significant changes were noted in the lymphocyte subset of CD8+ cells in all the ASKP1240-treated animals (Figure 5B). The number of peripheral CD4+CD25+Foxp3+ T cells did not increase, even in the animals (I-#5 and M-#4) who accepted islet allografts over the long-term without rejection (Figure 5C). The number of peripheral CD20+ cells slightly decreased during the period of ASKP1240 administration, and it increased after the discontinuation of ASKP1240 administration in some islet recipient animals (I-#4, I-#5 and M-#4) (Figure 5D).

image

Figure 5. Changes in peripheral lymphocyte counts following ASKP1240 treatments. Counts of peripheral CD4+ (A), CD8+ (B), CD4+CD25+Foxp3+ T cells (C) and CD20+ (D) lymphocytes following the induction (left panels) and maintenance (right panels) treatment are shown. The shaded areas represent the periods of ASKP1240 administration. The number of peripheral CD4+ cells increased in some of the ASKP1240-treated animals, especially in recipients that accepted islet allografts without rejection (I-#5 and M-#4) (A). No significant change was noted in the counts of peripheral lymphocyte subset of CD8+ cells in all the ASKP1240-treated animals (B). The CD4+CD25+Foxp3+ regulatory T cells did not increase in the periphery, even in animals who accepted islet allografts over the long-term (C). The number of peripheral CD20+ cells slightly decreased during ASKP1240 administration, whereas it increased after the discontinuation of ASKP1240 administration in some animals (I-#4, I-#5 and M-#4) (D).

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Anti-ASKP1240 antibody formation

Antibodies against ASKP1240 were not detected in any animal during the study, irrespective of the type of ASKP1240 treatment (data not shown).

Cellular immune response after PITx

Both direct and indirect anti-donor cellular responses were examined by assessing the frequency of IFN-γ-secreting alloreactive T cells in the periphery (Figure 6A and B). In the induction treatment group, these alloreactive T cells were suppressed when ASKP1240 was present in the sera; however, their number increased after disappearance of serum ASKP1240 in animals I-#3 and I-#4, which demonstrated rejection of islet allografts at 210 and 250 days after PITx, respectively. In contrast, inhibition of cellular responses against donor antigens persisted in animal I-#5, which demonstrated long-term acceptance of islet allografts, even though ASKP1240 treatment was discontinued at 2 weeks after PITx. A similar trend was observed in the maintenance treatment group. The number of alloreactive T cells responding to donor antigens, both directly and indirectly, increased before the rejection of islet allografts at 523 days after PITx in animal M-#3, whereas these T cells were completely abolished in the periphery, and islet allografts were accepted in animal M-#4 despite the cessation of ASKP1240 treatment. In recipient animals that accepted islet allografts without rejection (I-#5 and M-#4), however, cellular responses against third-party antigens tended to recover after the termination of ASKP1240 treatment (Supplemental Figure S2).

image

Figure 6. Cellular alloimmune responses and anti-donor antibody formation. The frequencies of directly (A) and indirectly (B) stimulated donor antigen-reactive IFN-γ-secreting T cells in the induction (left panels) and maintenance (right panels) treatment groups were evaluated by using an ELIspot assay, and the results are presented as the fold change relative to the number before PITx. The formation of anti-donor IgM (C) and IgG (D) antibodies in sera following ASKP1240 induction (left panels) and maintenance (right panels) treatments was also monitored by flow cytometric analysis. The shaded areas indicate periods of ASKP1240 administration. In animals rejecting islet allografts (I-#3: GST, 210 days; dotted square in the left panels, I-#4: GST, 250 days; black square in the left panels, and M-#3: GST, 523 days; black square in the right panels), the frequency of donor-antigen reactive T cells in the periphery increased gradually after PITx. Anti-donor IgG antibodies were evident at 127, 126 and 383 days after PITx, and the islet allograft was rejected thereafter in animals I-#3, I-#4 and M-#3, respectively. In contrast, in the animals accepting allografts over the long-term (I-#5: GST, more than 608 days; white square in the left panels and M-#4: GST, more than 607 days; white square in the right panels), both direct and indirect responses against donor antigens were strongly suppressed. In addition, formation of anti-donor IgM and IgG antibodies was also abolished throughout the study period.

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Humoral immune responses after PITx

Formation of anti-donor IgM and IgG antibodies (Abs) in sera was suppressed during the treatment course, whereas serum levels of anti-donor IgG Abs tended to increase after ASKP1240 cessation in animals that rejected islet allografts (Figure 6C and D). After induction of ASKP1240 treatment, two animals (I-#3 and I-#4) that demonstrated rejection of islet transplants developed anti-donor IgG Abs at 126 days (I-#4) and 127 days (I-#3) after PITx, and the anti-donor IgG Abs remained present thereafter. Additionally, in the maintenance treatment group, one animal (M-#3) became seropositive for anti-donor IgG antibodies at 383 days after PITx and demonstrated rejection of islet allografts at 523 days. In contrast to these allograft-rejected animals, one animal each from the induction and maintenance treatment groups—who demonstrated acceptance of islet transplants in the long-term (I-#5 and M-#4)—developed neither anti-donor IgM nor IgG Abs during the entire course of the study.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

In this study, we adopted induction and maintenance treatment protocols for ASKP1240 at a dose of 10 mg/kg in an allogeneic islet transplantation model in cynomolgus monkeys, based on our previous findings in kidney and liver transplantation studies [21-23]. Herein, we demonstrate that ASKP1240 monotherapy markedly prolonged the survival of pancreatic islet allografts. Notably, allografts survived for more than 600 days in two animals, even though the ASKP1240 treatment was terminated shortly after PITx, and the function of the transplanted islets in these animals was confirmed by C-peptide production and IVGTT evaluated at the time of euthanasia. The result was different from that in our previous ASKP1240 trial in kidney transplantation, in which the grafts became positive for C4d and IgG, and all of the renal allografts underwent chronic nephropathy [22]. Additionally, the identical ASKP1240 regimen resulted in chronic graft rejection after drug cessation in our previous liver transplantation study in cynomolgus monkeys [23]. In the current PITx study, we found that both cellular and humoral alloimmune responses were not elicited, even after ASKP1240 cessation in the two animals that accepted islet allografts (I-#5 and M-#4). The discrepancy regarding the efficacy of ASKP1240 in the current PITx and in Tx models in other organs is likely caused by different immunological characteristics of the transplants. Pancreatic islets mainly consist of β cells that only express MHC class I, although low expression of MHC class II may exist because of contaminating leukocytes [28-30].

Our IFN-γ ELIspot assay revealed that the cellular immune response against third-party but not donor antigens recovered after ASKP1240 cessation in animals that did not show signs of rejection (I-#5 and M-#4). These findings suggest that ASKP1240 treatment induced donor-specific tolerance in the two animals that accepted islet allografts. Alongside, the number of peripheral CD4+ T cells and CD20+ B cells increased in these animals (I-#5 and M-#4). Previous studies demonstrated that higher expression of CD4+CD25+Foxp3+ and γδ TCR+ T cells [31-33] or an increased number of peripheral B cells [34, 35] was associated with a state of clinical operational tolerance. In our flow cytometric analyses, however, the number of peripheral CD4+CD25+Foxp3+ T cells did not increase, and there was no clear evidence of regulatory T cell induction by the ASKP1240 treatment. With regard to B cell expansion, we have previously reported that suppression of germinal center formation in the spleen and lymph nodes was noted in some ASKP1240-treated animals, and we speculated that this suppression caused a rebound increase in the number of B cells in the periphery after ASKP1240 cessation in kidney transplant recipients [21, 22]; however, such an event was not reported for other anti-CD40 mAbs [17-20, 36]. The potential role of B cells in the tolerant state remains unclear, and further studies are necessary to define the role of B cell expansion after PITx.

Although the exact mechanisms responsible for islet allograft rejection remain unclear, previous studies have shown that cellular immune responses play a primary role in islet graft rejection [37, 38]. A histologic examination of islet allografts in the liver revealed that the cellular infiltrate surrounding the rejected islets was strongly positive for CD3+ T cells but not for CD20+ B cells, C4d or neutrophils [17, 39], which is consistent with recipient T cell alloreactivity assessed in mixed lymphocyte cultures [19]. Indeed, immunodepletion or modulation of T cells has emerged as a valuable adjunct treatment after PITx; it has been shown that higher insulin independence can be achieved by utilizing thymoglobulin [40], alemtuzumab [41, 42] or alternative nonFc-binding humanized anti-CD3mAb teplizumab (hOKT3-ala-ala) induction therapy [43]. Among several co-stimulatory signals, the CD40–CD154 pathway is a representative cascade that has been shown to play a central role in the activation of immune cells, including T cells, and in the priming of alloimmune responses [44, 45]. Blockade of this signaling pathway by various approaches has been shown to induce potent immunosuppression against cellular immunity and tolerance to allografts in experimental organ transplantation [46-48]. In PITx, Adams et al. [17] demonstrated that Chi220, which is a chimeric IgG1 CD40-specific mAb, dramatically prolonged islet allograft survival to 237, 237, 220, >185 and 172 days when combined with LEA29Y (belatacept). It has been shown that other anti-CD40 mAbs, i.e. 2C10 and 3A8, are also effective for preventing rejection of islet allografts in a nonhuman primate PITx model [18-20]. The efficacy of the blockade of CD40 signaling with regard to cellular immune responses was comparable to our results, in which both direct and indirect cellular alloimmune responses, as assessed by the IFN-γ ELIspot assay, were suppressed by ASKP1240 monotherapy during the treatment period; the effect continued to some degree even after ASKP1240 became absent. With regard to the prolongation of islet allograft survival time, ASKP1240 appeared to have a stronger effect than other anti-CD40 mAbs, e.g. Chi220 [17], 2C10 [20] or 3A8 [18, 19], but was equivalent to or less potent than anti-CD154 mAbs [12-14].

Many previous studies have shown that development of anti-donor antibodies is a risk factor for graft failure in kidney, heart and lung transplantation [49]. Transplanted islets have not been considered to be at risk for antibody-mediated injury because pancreatic islets are cells and not a vascularized organ. However, it is increasingly being recognized that B cells play a substantial role in graft damage, not only through their derived antibodies but also through interactions between T cells and other immune cells. Campbell et al. [50] reported that the presence of anti-HLA Abs before transplantation was associated with a significant loss of C-peptide after clinical PITx and that flow cross match alone was unable to predict outcome. Furthermore, pancreatic islet transplant recipients developed de novo DSA after clinical and nonhuman primate PITx [19, 51]. We observed a similar trend in our nonhuman primate PITx model, as all animals (I-#3, I-#4 and M-#3) that showed islet rejection had developed DSA before graft rejection, although further studies are necessary to define the role of DSA in islet allograft rejection.

Because CD40 is constitutively expressed on dendritic cells, macrophages and B cells, signaling via CD40 has been shown to be crucial for B cell activation and immunoglobulin class switching [52, 53]. Indeed, we have demonstrated in the present study that ASKP1240 abrogated the formation of DSA, both IgM and IgG isoforms, and anti-ASKP1240 antibodies, at least during the treatment course. These results were consistent with our previous findings for liver and kidney transplantation using ASKP1240. In contrast, Badell et al. [19] reported that 60% of PITx monkeys receiving the 3A8-based regimen had developed DSA at the time of rejection. Considering that addition of CTLA4Ig to this 3A8-based regimen strongly prevented generation of DSA and markedly prolonged allograft survival [19], it seems likely that DSA is mainly T cell-dependent, and that controlling donor-reactive helper T cell responses in addition to DSA formation is a key issue to achieve long-term engraftment of allogeneic islet grafts. Because ASKP1240 can potently inhibit both cellular and humoral immune responses, CD40 blockade by ASKP1240 may be advantageous for immunosuppression after PITx.

Clinical trials on anti-CD154 mAbs were terminated because of thromboembolic complications; therefore, side effects following ASKP1240 treatment have always been a major concern. In the present study, no histopathological evidence of thromboembolism was observed in any animal during ASKP1240 treatment, after drug cessation, or at the time of autopsy (data not shown). In addition, no abnormal findings were seen in peripheral blood hematology and chemistry or in tissues such as the brain, heart, lung, intestine or kidneys (data not shown). Perioperative complications, such as internal hernia, peritonitis and gastric dysmobility, were noted in some of our PITx recipients. These complications were related to the surgical procedure, as total pancreatectomy is an invasive method for DM induction that requires complex surgical procedures and postoperative management [24, 25].

Finally, we conclude that the fully human anti-CD40 mAb ASKP1240 induced potent immunosuppressive effects for pancreatic islet allografts without causing serious side effects. CD40 blockade by ASKP1240 suppressed cellular and humoral alloimmune responses and prevented rejection for the duration of therapy; furthermore, it allowed long-term acceptance of islet allografts in two cynomolgus monkeys. ASKP1240 seems to be a promising agent for immunosuppression after PITx.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

The authors thank Mr. R. Mitsuo and Mr. T. Nakamura for helping to perform the study and Ms. M. Arako, Mr. J. Kimura and others at SNBL for the excellent animal care. This work was supported in part by grants from the Kyowa Hakko Kirin Co., Ltd. and Astellas Pharma Inc., and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#233903090 to K.Y.).

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Additional supporting information may be found in the online version of this article at the publisher's web-site.

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ajt12330-sm-0001-SupFigs-S1.pdf379K

Figure S1. Histological features of transplanted islet allografts. Representative macroscopic appearance of islet allografts from the long-term graft accepted and rejected animals is shown. Histological examination of the liver from a long-term graft accepted animal (M-#4) demonstrated the presence of islet allografts in the liver portal area (H&E), and no evidence of cellular infiltration, including that of CD3+, CD4+ or CD8+ cells, into or surrounding the islet allografts. In contrast, in the liver from the graft-rejected animal (M-#3), focal CD3+, CD4+ and CD8+ cellular infiltrates surrounding islet allografts were noted.

Figure S2. Cellular immune responses against third-party antigens. The frequency of directly (A) and indirectly (B) stimulated IFN-γ-secreting T cells against third-party antigens in the induction (left panels) and maintenance (right panels) treatment groups are shown. The results are presented as the fold change relative to the number before PITx. Both direct and indirect cellular responses against third-party antigens were elevated in all animals showing allograft rejection (I-#3, I-#4 and M-#3). In contrast, in recipient animals that accepted islets without allograft rejection (I-#5 and M-#4), these cellular responses were suppressed during the ASKP1240 treatment period but tended to recover after the discontinuation of ASKP1240 administration.

ajt12330-sm-0002-SupInfo-S1.doc73K

Supporting Information.

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