Comparative efficacy of anti‐CD40 antibody–mediated costimulation blockade on long‐term survival of full‐thickness porcine corneal grafts in nonhuman primates

Porcine corneas may be good substitutes for human corneas in donor shortage. Therefore, we evaluated the efficacy and safety of an anti‐CD40 antibody–based regimen compared with an anti‐CD20 antibody–based regimen on the survival of full‐thickness corneas in pig‐to‐rhesus xenotransplant. Thirteen Chinese rhesuses underwent full‐thickness corneal xenotransplant. Six were administered anti‐CD40 antibody, and the others were administered anti‐CD20 antibody, basiliximab, and tacrolimus. Graft survival and changes in lymphocyte, donor‐specific and anti–Galα1,3Galβ1,4GlcNAc‐R (αGal) antibody, and aqueous complement levels were evaluated. Treatment with the anti‐CD40 antibody (>511, >422, >273, >203, >196, 41 days) and anti‐CD20 antibody (>470, 297, >260, >210, >184, 134, >97 days) resulted in long‐term survival of grafts. In the anti‐CD20 group, the number of activated B cells was significantly lower than that in the anti‐CD40 group, and the level of aqueous complements at 6 months was significantly higher than the preoperative level. There were no differences in the levels of T cells or donor‐specific and anti‐αGal antibodies between the 2 groups. In the anti‐CD20 group, 3 primates had adverse reactions. In conclusion, both the anti‐CD40 antibody– and the anti‐CD20 antibody–based protocols were effective for the long‐term survival of full‐thickness corneal xenografts, but the anti‐CD40 antibody–based treatment had fewer adverse effects.     

Corticosteroids and methotrexate as adjuvants to costimulation blockade in non‐human primate renal transplantation

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

No in vivo infection of triple immunosuppressed non‐human primates after inoculation with high titers of porcine endogenous retroviruses*

Abstract: Background: Porcine endogenous retroviruses (PERVs) released from pig tissue can infect selected human cells in vitro and therefore represent a safety risk for xenotransplantation using pig cells, tissues, or organs. Although PERVs infect cells of numerous species in vitro, attempts to establish reliable animal models failed until now. Absence of PERV transmission has been shown in first experimental and clinical xenotransplantations; however, these trials suffered from the absence of long‐term exposure (transplant survival) and profound immunosuppression. Methods: We conducted infectivity studies in rhesus monkeys, pig‐tailed monkeys, and baboons under chronic immunosuppression with cyclosporine A, methylprednisolone, and the rapamycin derivative. These species were selected because they are close to the human species and PERVs can be transmitted in vitro to cells of these species. In addition, the animals received twice, a C1 esterase inhibitor to block complement activation before inoculation of PERV. In order to overcome the complications of microchimerism, animals were inoculated with high titers of cell‐free PERV. In addition, to enable transmission via cell–cell contact, some animals also received virus‐producing cells. For inoculation the primate cell‐adapted strain PERV/5° was used which is characterized by a high infectious titer. Produced on human cells, this virus does not express alpha 1,3 Gal epitopes, does not contain porcine antigens on the viral surface and is therefore less immunogenic in non‐human primates compared with pig cell‐derived virus. Finally, we present evidence that PERV/5° productively infects cells from baboons and rhesus monkeys. Results: In a follow‐up period of 11 months, no antibody production against PERV and no integration of proviral DNA in blood cells was observed. Furthermore, no PERV sequences were detected in the DNA of different organs taken after necropsy. Conclusion: These results indicate that in a primate model, in the presence of chronic immunosuppression, neither the inoculation of cell‐free nor cell‐associated PERV using a virus already adapted to primate cells results in an infection; this is despite the fact that peripheral blood mononuclear cells of the same animals are infectible in vitro.     

Pre‐clinical results in pig‐to‐non‐human primate islet xenotransplantation using anti‐CD40 antibody (2C10R4)‐based immunosuppression

Background

Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti‐CD154 monoclonal antibody (mAb) in pig‐to‐non‐human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti‐CD40 (2C10R4) mAb‐based studies have not been reported.

Methods

Nine streptozotocin (STZ)‐induced diabetic rhesus monkeys were transplanted with adult porcine islets isolated from designated pathogen‐free (DPF) miniature pigs. They were treated with anti‐CD40 mAb‐based immunosuppressive regimen and were divided into 3 groups: anti‐CD40 only group (n = 2), belatacept group (anti‐CD40 mAb+belatacept, n = 2), and tacrolimus group (anti‐CD40 mAb+tacrolimus, n = 5). All monkeys received anti‐thymocyte globulin (ATG), cobra venom factor (CVF), adalimumab, and sirolimus. Blood glucose levels (BGL) and serum porcine C‐peptide concentrations were measured. Humoral and cellular immune responses were assessed by ELISA and ELISPOT, respectively. Liver biopsy and subsequent immunohistochemistry were conducted.

Results

All animals restored normoglycemia immediately after porcine islet transplantation and finished the follow‐up without any severe adverse effects except for one animal (R092). Most animals maintained their body weight. Median survival, as defined by a serum porcine C‐peptide concentration of >0.15 ng/mL, was 31, 27, and 60 days for anti‐CD40 only, belatacept, and tacrolimus groups, respectively. Anti‐αGal IgG levels in serum and the number of interferon‐γ secreting T cells in peripheral blood mononuclear cells did not increase in most animals.

Conclusion

These results showed that anti‐CD40 mAb combined with tacrolimus was effective in prolonging porcine islet graft survival, but anti‐CD40 mAb was not as effective as anti‐CD154 mAb in terms of preventing early islet loss.     

A Phase 1, Randomized Ascending Single‐Dose Study of Antagonist Anti‐Human CD40 ASKP1240 in Healthy Subjects

This first‐in‐human, phase I study evaluated the safety, tolerability, pharmacokinetic and pharmacodynamic profile of ASKP1240 in healthy subjects. Twelve sequential groups (each 6 active and 3 placebo) were randomly assigned to placebo or single ascending doses of intravenous ASKP1240 (0.00003–10 mg/kg). ASKP1240 exhibited nonlinear pharmacokinetics, with mean maximal serum concentrations and area under the serum concentration–time curves ranging from 0.7 to 251.6 μg/mL and 6.5 to 55409.6 h·μg/mL following doses 0.1 mg/kg–10 mg/kg, respectively. CD40 receptor occupancy by ASKP1240, which was dose‐dependent, reached a maximum at doses above 0.01 mg/kg. ASKP1240 was well tolerated, with no evidence of cytokine release syndrome or thromboembolic events. Treatment emergent antibodies to ASKP1240 were detected in 5/70 (7.1%) ASKP1240 recipients. In conclusion, antagonism of the CD40/CD154 interaction with ASKP1240 was safe and well tolerated at the doses tested.     

Differential outcomes in 3 types of acute antibody‐mediated rejection

Abstract: Introduction: The aim of this study is to investigate the prevalence, predictors, and clinical outcomes of acute antibody‐mediated rejection (AAMR). Methods: Retrospective analysis of 833 adult patients who received kidney transplantation between 1/1/2001 and 8/15/2007. Results: The prevalence of AAMR and acute cellular rejection was 2% and 8.2%, respectively. Eight patients had type I, seven type II, and two type III AAMR. All patients had at least one strong donor‐specific anti‐HLA antibodies (DSA) with a median fluorescence intensity (MFI) value of over 6000 and the mean number of strong DSAs was 2.0 ± 0.7. Fifteen of 17 patients received pre‐transplant desensitization treatment. During a median 28 months of follow‐up (range: 12–38 months), two patients died (88% patient survival), and nine lost their allografts (35% graft survival). While all type I AAMR patients responded to treatment, all type III patients, and four of seven patients with type II AAMR lost their allografts earlier, and three type II AAMR patients later due to transplant glomerulopathy. Conclusions: AAMR is mainly seen in patients with pre‐transplant strong DSAs. There is a striking difference in clinical outcomes of AAMR that types II and III AAMR patients have poor prognosis compared to type I AAMR patients.     

Fc‐Disabled Anti‐Mouse CD40L Antibodies Retain Efficacy in Promoting Transplantation Tolerance

CD40L antibodies have proven to be powerful immunosuppressive agents in nonhuman primates but unfortunately perturb blood coagulation. Neither the therapeutic nor the prothrombotic mechanism of anti‐CD40L is defined sufficiently to determine whether these effects can be uncoupled. Recent evidence suggests that the Fc region of anti‐CD40L antibodies interacting with Fc receptors plays an important role in stabilizing platelet aggregates. An Fc‐disabled, aglycosylated anti‐CD40L heavy chain variant was therefore created to determine whether it might still be useful in promoting transplantation tolerance. In a number of mouse models an engineered aglycosyl anti‐CD40L recapitulated the effects of the intact anti‐CD40L antibody in tolerance protocols involving transplantation of allogeneic bone marrow and skin. In contrast, another anti‐CD40L variant with a conventional rat γ2b heavy chain was less effective in ensuring long‐term skin graft survival, possibly associated with its faster clearance from the circulation. These results show that short pulses of anti‐CD40L antibody therapy may still be useful in tolerance protocols even when the Fc region is disabled.     

Long‐Term Hepatic Allograft Acceptance Based on CD40 Blockade by ASKP1240 in Nonhuman Primates

Blockade of the CD40–CD154 costimulatory signal is an attractive strategy for immunosuppression and tolerance induction in organ transplantation. Treatment with anti‐CD154 monoclonal antibodies (mAbs) results in potent immunosuppression in nonhuman primates (NHPs). Despite plans for future clinical use, further development of these treatments was halted by complications. As an alternative approach, we have been focusing on the inhibition of the counter receptor, CD40 and have shown that a novel human anti‐CD40 mAb, ASKP1240, markedly prolongs renal allograft survival in NHPs, although allografts eventually underwent chronic allograft nephropathy. On the basis of our previous findings that a CD40–CD154 costimulation blockade induces tolerance to hepatic, but not cardiac, allografts in rodents, we tested here our hypothesis that a blockade of CD40 by ASKP1240 allows acceptance of hepatic allografts in NHPs. A 2‐week ASKP1240 induction treatment prolonged liver allograft survival in NHPs; however, the graft function deteriorated due to chronic rejection. In contrast, a 6‐month ASKP1240 maintenance monotherapy efficiently suppressed both cellular and humoral alloimmune responses and prevented rejection on the hepatic allograft. No serious side effects, including thromboembolic complications, were noted in the ASKP1240‐treated monkeys. We conclude that CD40 blockade by ASKP1240 would be a desirable immunosuppressant for clinical liver transplantation.     

Influence of preoperative anti‐HLA antibodies on short‐ and long‐term graft survival in recipients with or without rituximab treatment

We investigated the relationship between preoperative anti‐HLA antibodies (donor‐specific antibody, DSA) and the graft survival rate in recipients who had or had not received rituximab (Rit) treatment. The subjects were categorized into four groups as follows: DSA+Rit?, n = 39; DSA?Rit?, n = 121; DSA+Rit+, n = 74; and DSA?Rit+, n = 47. We examined the influence of preoperative DSA on the incidence of graft rejection and the survival rate of recipients who had or who had not received rituximab before transplantation. The 6‐month acute rejection rates based on graft biopsies were 39%, 19%, 15%, and 0% for the DSA+Rit?, DSA?Rit?, DSA+Rit+, and DSA?Rit+ groups. The rates of chronic antibody‐mediated rejection after more than 6 months were 50%, 22%, 18%, and 0%. The 5‐year graft survival rate was significantly lower in the DSA+Rit? group (84%) than in the other groups (95% for DSA?Rit?, 98% for DSA+Rit+, and 91% for DSA?Rit+). The rate of the appearance of de novo anti‐HLA antibodies was higher in the groups that did not receive rituximab treatment. The rate of graft loss associated with chronic antibody‐mediated rejection was also higher in the DSA+Rit? group than in the other groups (P = 0.01). The presence of DSA and the administration of rituximab had strong impacts on not only short‐term graft rejection, but also long‐term graft rejection and its association with the graft survival time.     

Determining donor‐specific antibody C1q‐binding ability improves the prediction of antibody‐mediated rejection in human leucocyte antigen‐incompatible kidney transplantation

Detrimental impact of preformed donor‐specific antibodies (DSAs) against human leucocyte antigens on outcomes after kidney transplantation are well documented, however, the value of their capacity to bind complement for predicting antibody‐mediated rejection (AMR) and graft survival still needs to be confirmed. We aimed to study DSA characteristics (strength and C1q binding) that might distinguish harmful DSA from clinically irrelevant ones. We retrospectively studied 60 kidney‐transplanted patients with preformed DSA detected by single antigen bead (SAB) assays (IgG and C1q kits), from a cohort of 517 kidney graft recipients (124 with detectable anti‐HLA antibodies). Patients were divided into DSA strength (MFI < vs. ≥ 15 000) and C1q‐binding ability. AMR frequency was high (30%) and it increased with DSA strength (P = 0.002) and C1q+ DSA (P < 0.001). The performance of DSA C1q‐binding ability as a predictor of AMR was better than DSA strength (diagnostic odds ratio 16.3 vs. 6.4, respectively). Furthermore, a multivariable logistic regression showed that C1q+ DSA was a risk factor for AMR (OR = 16.80, P = 0.001), while high MFI DSAs were not. Graft survival was lower in high MFI C1q+ DSA in comparison with patients with C1q? high or low MFI DSA (at 6 years, 38%, 83% and 80%, respectively; P = 0.001). Both DSA strength and C1q‐binding ability assessment seem valuable for improving pretransplant risk assessment. Since DSA C1q‐binding ability was a better predictor of AMR and correlated with graft survival, C1q‐SAB may be a particularly useful tool.