Complement blockade with eculizumab to treat acute symptomatic humoral rejection after heart transplantation

Abstract Antibody‐mediated rejection (AMR) is a major barrier preventing successful discordant organ xenotransplantation, but it also occurs in allotransplantation due to anti‐HLA antibodies. Symptomatic acute AMR is rare after heart allograft but carries a high risk of mortality, especially >1 year after transplant. As complement activation may play a major role in mediating tissue injury in acute AMR, drugs blocking the terminal complement cascade like eculizumab may be useful, particularly since “standards of care” like plasmapheresis are not based on strong evidence. Eculizumab was successfully used to treat early acute kidney AMR, a typical condition of “active AMR,” but showed mitigated results in late AMR, where “chronic active” lesions are more prevalent. Here, we report the case of a heart recipient who presented with acute heart failure due to late acute AMR with eight de novo donor‐specific anti‐HLA antibodies (DSA), and who fully recovered allograft function and completely cleared DSA following plasmapheresis‐free upfront eculizumab administration in addition to thymoglobulin, intravenous immunoglobulins (IVIG), and rituximab. Several clinical (acute onset, abrupt and severe loss of graft function), biological (sudden high‐level production of DSA), and pathological features (microvascular injury, C4d deposits) of this cardiac recipient are shared with early kidney AMR and may indicate a strong role of complement in the pathogenesis of acute graft injury that may respond to drugs like eculizumab. Terminal complement blockade should be further explored to treat acute AMR in recipients of heart allografts and possibly also in recipients of discordant xenografts in the future.

mostly based on retrospective and uncontrolled studies collected from small heterogeneous patient populations (e.g., acute and chronic AMR, early and late AMR, and so on) with inconsistent and conflicting results. 2 The disparity observed in the treatment effects may, however, also arise from the variability of the possible interactions between donor-specific antibodies (DSA) and allograft endothelial cells (e.g., complement cascade activation, direct signaling via HLA molecules, Fc gamma receptor-dependent cellular effects), which may not be equally relevant in all AMR cases, and which are differently targeted by each particular therapy.
One of the most important pathogenic processes leading to tissue injury in allograft and xenograft acute AMR is the activation of the classical pathway of the complement cascade by endothelial-bound DSA, with ensuing C4d deposition, microvascular inflammation, thrombosis, and capillary obstruction. 6,7 In that setting, the use of drugs inhibiting complement-like eculizumab is appealing. Eculizumab is a humanized monoclonal antibody that binds to the C5 complement component, preventing its conversion to anaphylatoxin (C5a) and blocking the constitution of the cell membrane attack complex (C5b-9), which finally results in a powerful inhibition of the complement cascade.
In kidney allotransplantation, eculizumab has been successfully used to prevent acute AMR in sensitized high-risk recipients 8 or to treat early acute AMR together with plasmapheresis in sensitized recipients with abrupt posttransplant DSA rise and rapidly evolving allograft dysfunction, 9 a condition known to be complementdependent. Of note, our group also reported successful eculizumab administration instead of extracorporeal antibody removal, 10,11 with the idea that immediate direct neutralization of the pathogenetic effects of DSA by terminal complement blockade may favorably replace the more invasive, cumbersome, and slowly working plasmapheresis or immunoadsorption procedures. 6 Of importance also, therapeutic attempts with eculizumab were not all successful, for example, with mitigated results in late AMR 12 or when eculizumab was used as a rescue therapy after other treatments failed. 13 In heart transplantation (HTx), eculizumab given during 3 months after transplant was very recently shown to better prevent AMR than peri-operative plasmapheresis and intravenous immunoglobulins (IVIG) in highly sensitized recipients with positive virtual crossmatch. 14 However, the experience with eculizumab for established AMR is still preliminary and mainly restricted to refractory AMR after plasmapheresis. 15 To our best knowledge, the only report about eculizumab use is a retrospective analysis of 14 pediatric heart recipients. 16 Only 11 of them had endomyocardial biopsy (EMB) proven AMR, the remaining three receiving eculizumab for AMR prevention after HTx with a positive crossmatch. The global outcome was poor, with 50% early mortality.
Here, we report the case of a heart allotransplant recipient with sudden acute heart failure due to late acute AMR with eight de novo quickly rising DSA who fully recovered allograft function following plasmapheresis-free eculizumab administration, and who completely cleared circulating DSA with thymoglobulin, IVIG, and rituximab subsequently given to suppress DSA production. We suggest that blocking the terminal complement pathway may become a useful strategy to treat cardiac acute AMR associated with abruptly occurring severe allograft dysfunction, intense DSA production, evident microvascular injury, and C4d deposition on EMB. In the future, similar therapeutic strategies may also apply to treat AMR in discordant xenotransplantation.

CASE REPORT
The patient is a 43-year-old male who presented with severe heart failure due to de novo dilated cardiomyopathy diagnosed 5 months infusion. Inotropic support terminated on day 5 and LVEF increased to 28%, 35%, 39%, and 47% on days 3, 6, 12, and 30, respectively, before normalization on day 85 (58%). DSA progressively decreased over 4 weeks and was undetectable for 6 months after treatment with no CAV at angiography ( Figure 1).
Importantly, the patient also received prophylactic phenoxymethylpenicillin to prevent Neisseria meningitidis infection between the first dose of eculizumab and CH-50 recovery as well as valganciclovir and trimethoprim-sulfamethoxazole to prevent Cytomegalovirus and Toxoplasma gondii reactivation during 3 months.
No significant infectious or oncologic complication occurred after rejection therapy.

DISCUSSION
In this case of acute pathologically proven AMR with severe allograft dysfunction, a poly-therapy consisting of steroids, thymoglob-ulin, IVIG, and upfront eculizumab was successfully used to block tissue injury and suppress DSA production. Although we acknowledge that all agents might have contributed to the successful outcome, the short delay observed between eculizumab infusion and hemodynamic improvement in a patient actually deteriorating after methylprednisolone and thymoglobulin administration suggests that eculizumab played a central role in AMR resolution.
Furthermore, as far as plasmapheresis or immunoadsorption was not used in this patient and as far as IVIG was only started on day 4, complement blockade with eculizumab was the sole undertaken intervention that directly targeted acute AMR before early improvement.
This finding underscores the key role complement can play in mediating tissue injury in AMR, both in allo-or xenotransplantation. Indeed, the currently contemplated pig-to-human kidney transplantation trials 1 would not be possible without introducing at least one human complement-regulatory transgene into the engineered pigs to provide some protection against AMR. 18 In both allo-and xenotransplantation, acute AMR is anyway a potentially dramatic event if not properly diagnosed and treated.
According to the current International Society for Heart and Lung or "definite," with "severe" AMR (pAMR3) being eventually considered when additional extensive tissue injury is also observed. 19 In our case, EMB was pAMR2, which we have previously shown to be more likely associated with DSA and overt graft dysfunction than pAMR1. 3 Nevertheless, the pAMR grading system is only modestly correlated to the outcome, with similar cardiovascular mortality among pAMR1 H+, pAMR1 I+, and pAMR2 patients, 20 and it does not provide definite guidance for patient management per se. 21  Nevertheless, elements of understanding may come from the field of kidney allotransplantation, where the timing of clinically apparent AMR is also closely related to outcome. Indeed, prompt therapy completely prevented graft loss and enabled graft function to recover to a near-normal level at 13 months in kidney recipients with early AMR, 9 whereas estimated glomerular filtration rate (eGFR) progressively and irremediably declined irrespective of the kind of therapy attempted in the large majority of patients with allograft dysfunction and late AMR in another cohort. 24 As opposed to heart transplantation, early renal AMR is restricted to a very specific phenotype of humoral rejection. Indeed, it occurs within the first 30 postoperative days as an anamnestic response of memory B-cells in presensitized patients with or without DSA at transplant time. Its clinical course is very aggressive, with an abrupt and intense rise of DSA accompanied by a fast eGFR decline eventually followed by oliguria and graft loss if untreated. By contrast, late AMR usually presents as a more indolent process, with usually no or only mild allograft dysfunction at diagnosis, progressive GFR loss over years, and it is associated with either preexisting or de novo DSA. At the biopsy level, early and late AMR also differ in both morphologic and immune-pathologic patterns. Whereas early AMR always displays typical features of "active AMR," late AMR may exhibit both "active" and/or "chronic active" lesions. "Active AMR" is characterized by acute tissue injury (e.g., microvascular inflammation, arthritis, acute thrombotic microangiopathy) and is interpreted as an initial and reversible process in the evolutive course of humoral rejection, while "chronic active AMR," depicted by transplant glomerulopathy, peritubular capillary basement membrane multilayering or arterial intimal fibrosis, feature more advanced and definitive lesions. 12,25 Of note, the current ISHLT working formulation does not discriminate between both entities in cardiac AMR 19 and this gap may account for some of the prognostic variations observed according to time from cardiac transplant.
In addition, early and late kidney AMR also differ in their immunephenotypic pattern, with almost invariable C4d deposition in early disease but frequent C4d negative specimen in late cases. 25  Notwithstanding post-transplant timing, our acute AMR case shares many characteristics with early kidney AMR that may hypothetically suggest complement-dependent tissue injury and sensitivity to therapeutic terminal complement blockade. First, DSA increased extremely abruptly and intensely, with a transition from undetectability to 46,249 MFI in total after 32 days. Although pretransplant screening did not reveal any relevant anti-HLA antibodies to the implanted organ in our patient, we cannot exclude cryptic pre-sensitization and late anamnestic response at the time of steroid tapering, especially since pretransplant LVAD use confers a significant risk factor for anti-HLA antibodies production, 28  Finally, the treatments implemented beyond eculizumab and aimed at DSA suppression also likely contributed to the remarkable long-term outcome of our patient, with no relapsing AMR and no subsequent CAV.
Indeed, DSA strikingly decreased over 4 weeks, enabling eculizumab discontinuation after only two doses, and were undetectable 6 months after treatment initiation. The control of DSA production is usually attempted with agents targeting B-(e.g., IVIG and Rituximab) and Tcells (e.g., corticosteroids and thymoglobulin) in different combinations despite few supporting data and conflicting results. 17 As illustrated here, time from DSA onset to detection may be an important factor determining therapeutic response and early DSA may be easier to suppress. 17 Prospective multicenter randomized-controlled studies will be needed in the future to assess the optimal timing of eculizumab administration, to compare its efficacy and safety with more established therapeutic modalities like plasmapheresis, and to precise which clinical profile can best predict treatment benefit. Meanwhile, we believe that our experience added to the recent knowledge on prevention and treatment of AMR with eculizumab may help to reduce the high fatality burden of this syndrome in heart transplantation.
We also believe that experience with eculizumab in allotransplantation may be relevant in discordant xenotransplantation, particularly in the near upcoming trials of pig-to-human kidney transplantation.
Although the use of organs from triple KO pigs (GalT-KO, CMAH-KO, and B4GalNT2-KO) is anticipated to lessen the risk of hyper-acute rejection in humans, 29 delayed active or acute AMR is still possible through de novo production of DSA directed against various antigens like swine leucocytes antigens class I. 1,30 Furthermore, overexpression of transgenes coding for human complement regulatory proteins like CD46 does not completely prevent complement-dependent injury in pig hearts exposed to human blood. 31 Like in allotransplantation, the implementation of strategies combining immediate tissue injury blockade with desensitization will certainly be necessary to overcome AMR and improve xenotransplant survival. In that sense, eculizumab but also other new complement-blocking or complement-depleting compounds such as C1 esterase inhibitors, C3b inhibitors, and cobra venom factor will potentially have a role to play in the prevention and/or treatment of xenotransplant AMR 1 .