European Reflections on New Indications for ...

Transplantation Publish Ahead of Print DOI: 10.1097/TP.0000000000002244

European Reflections on New Indications for

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Extracorporeal Photopheresis in Solid Organ Transplantation

Norbert Ahrens1†, Edward K. Geissler2, Volker Witt3, Marc Berneburg4, Daniel Wolff5,

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Stephan W. Hirt6, Bernhard Banas7, Hans J. Schlitt2 and James A. Hutchinson2

Institute for Clinical Chemistry and Laboratory Medicine, Transfusion Medicine, University

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Hospital Regensburg, Franz-Josef-Strauß-Allee-11, 93053 Regensburg, Germany

Department of Surgery, University Hospital Regensburg, Franz-Josef-Strauß-Allee-11, 93053

Regensburg, Germany 3

St. Anna Kinderspital, Medical University of Vienna, Kinderspitalgasse 6, 1090 Vienna,

Austria

Department of Dermatology, University Hospital Regensburg, Franz-Josef-Strauß-Allee-11,

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93053 Regensburg, Germany

Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-

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Allee-11, 93053 Regensburg, Germany 6

Department of Cardiothoracic Surgery, University Hospital Regensburg, Franz-Josef-Strauß-

Allee-11, 93053 Regensburg, Germany 7

Department of Nephrology, University Hospital Regensburg, Franz-Josef-Strauß-Allee-11,

93053 Regensburg, Germany

1 Copyright © Wolters Kluwer Health. Unauthorized reproduction of this article is prohibited.

† Corresponding author:

Dr. Norbert Ahrens Institute for Clinical Chemistry and Laboratory Medicine, Transfusion Medicine University Hospital Regensburg

93053 Regensburg, Germany Tel:

+49-941-944-6207

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email: [email protected]

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Franz-Josef-Strauß-Allee 11

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Author contributions

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Declarations: The authors have no other relevant conflict-of-interests to declare.

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NA, EKG, VW, MB, DW, SWH, BB, HJS and JAH participated in writing of this paper.

Abbreviations

AHA

American Heart Association

ASFA

American Society for Apheresis

ATMP

Advanced Therapeutic Medical Product


C57BL/6 mouse

Bc

BALB/c mouse

BOS

Bronchiolitis Obliterans Syndrome

CBMP

Cell-based medicinal product

cDC

Conventional dendritic cell

cGvHD

Chronic Graft-versus host disease

CNI

Calcineurin inhibitor

DC

Dendritic cell

DST

Donor-specific transfusion

DTR

Diptheria toxin receptor

ECP

Extracorporeal photopheresis

EEC

European Economic Community

EU

European Union

GvHD

Graft-versus host disease

HLA

Human Leucocyte Antigen

IDO

Indoleamine 2,3-dioxygenase

IL-10

Interleukin-10

ISHLT

International Society for Heart and Lung Transplantation

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B6

MHC

Major Histocompatibility Complex

8-MOP

8-methoxypsoralen

MRDC

Maturation-resistant DC

pDC

Plasmacytoid DC

PGE2

Prostaglandin-E2


Transforming Growth Factor beta

Treg

Regulatory T cell

USA

United States of America

UVA

Ultraviolet A

Vit.D3

Vitamin D3

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TGF-β


European Reflections on New Indications for Extracorporeal Photopheresis in Solid Organ Transplantation

In recent times, especially amongst our American colleagues, we have witnessed growing enthusiasm for applying extracoroporeal photopheresis (ECP) as a general or alloantigen-specific

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immunosuppressive therapy to novel indications in solid organ transplantation. ECP is a wellestablished second-line treatment for steroid-refractory acute and chronic GvHD, is

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recommended for prevention and treatment of cardiac transplant rejection, and is a valuable option for slowing functional deterioration of lung allografts in patients with treatment-resistant bronchiolitis obliterans syndrome. Hence, in a general sense, applying ECP as an induction,

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maintenance or rescue therapy in kidney, liver or other solid transplantation is a cogent suggestion. Nevertheless, it is doubtful whether current understanding of the pharmacological aspects of ECP is sufficient to devise optimal strategies for manufacture, quality-control and administration of ECP products to kidney or liver transplant recipients. There also remain many

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open questions about manufacturing ECP products, mechanisms of action, quality-control procedures, nonclinical pharmacological development and specific clinical indications that would likely be asked by European competent authorities when assessing a clinical trial

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application subject to Advanced Therapeutic Medical Product (ATMP) regulations.1 In our view, there is a very strong case for standardised reporting of technical and clinical procedures, implementing universal quality-control assays, and systematic reporting of clinical outcomes.

Manufacturing procedures: The essence of all technical and clinical procedures in ECP is UVA-irradiation of 8-methoxypsoralen (8-MOP)-treated autologous peripheral blood leucocyte


suspensions (apheresate) to induce apoptotic cell death in lymphocytes and, perhaps, polarisation of nonapoptotic monocytes towards a regulatory phenotype. In the USA, Therakos is the sole supplier of medically approved, fully automated closed inline ECP devices, which were introduced in three generations: UVAR, UVAR XTS and CELLEX.2 By contrast, a variety of ECP methods are established as medical therapies in Europe, including ECP with Therakos

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systems and a variety of closed or open offline systems, which involve manual processing steps (Fig. 1). The precise technical differences between the various manufacturing methods are

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beyond the scope of this article;3 however, it is important to appreciate that alternative methods result in ECP products that differ substantially with regard to cellular composition, total cell numbers, apoptotic cell content, presence of different types of psoralen photoadducts, and

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excipients.4,5 Inevitably, such methodological variations confound analyses of clinical outcomes between different sites and across time, a problem often compounded by inadequate technical descriptions in published articles. In our view, there is a pressing need for minimal information standards in ECP6,7 to facilitate aggregation of data on clinical outcomes.8 Descriptions of ECP

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procedures often lack information about treatment volumes, irradiation times and blood counts from apheresates: sometimes reports even omit details of irradiation devices. Furthermore, the quality of ECP products is often poorly reported, especially their content of apoptotic cells,

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activated thrombocytes and viable monocytes.

Mechanisms of action: ECP has been described as a ‘bidirectional therapy’ because it ameliorates T cell-mediated pathologies whilst also preserving protective immunity and possibly stimulating antitumour responses.9 In our view, this description is somewhat unhelpful since, although ‘immunosuppression’ and ‘immunoactivation’ are opposing reactions in a clinical


sense, their underlying immunological mechanisms are not mutually antagonistic. Over the years, various explanations for the tolerogenic effects of ECP have been advanced,10 but these are not necessarily competing ideas. Conceptually, the immunosuppressive activities of ECP treatment can be divided into (1) effects mediated by living cells rendered immunosuppressive

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by the ECP process and (2) effects mediated by dead or dying cells.

Many groups have characterized ECP-induced changes in monocytes that confer in vitro T cell-

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suppressive activities; naturally, these observations led to claims that ECP is fundamentally a tolerogenic dendritic cell (DC)-based therapy. Of particular mention, Edelson and colleagues have described an integrin-12 and platelet-dependent13 conversion of blood monocytes into DC-

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like cells14 following ECP treatment. The resultant DC-like cells, which are perhaps better classified as activated monocytes or macrophages,15,16 are capable of phagocytosing, processing and presenting exogenous model antigens to CD4+ T cells in vitro,14 as well as exhibiting a remarkable and unexplained capacity for cross-presentation of soluble, exogenous antigen to

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CD8+ T cells.17 Substantial advances in myeloid regulatory cell therapy for solid transplantation have been made over the last 10 years, both in terms of manufacturing development and understanding their pharmacodynamic effects.18 Of all current methods for producing tolerogenic

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DC-based therapies,19 we know of no protocol to generate stably suppressive cells from monocytes in less than 3 days;20 by contrast, processing by ECP takes hours at most, albeit at the cost of far greater product heterogeneity. This leads to the interesting proposition that ex vivomodified monocytes in ECP products serve as precursors to tolerogenic or tolerogenic DC, which arise through further differentiation steps in vivo. In support of this hypothesis, ECPmodified monocytes share certain characteristics with naturally occuring ‘licensed monocytes.’20


The profoundly immunosuppressive effects of apoptotic cells administered by intravenous injection are now very well understood:11 apoptotic cells are rapidly cleared by recipient macrophages and DC which then up-regulate suppressor factors (eg, TGF-ß, IL-10, IDO and PGE2) and down-regulate costimulatory molecules; in turn, such ‘in situ-tolerised’ macrophages and DC suppress T cell effector activity and support Treg function. Notably, Morelli and

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colleagues have shown that recipient conventional dendritic cells (cDC) were indispensible for the tolerising effect of donor-derived immature DC in C57BL/6 (B6) mice given a BALB/c (Bc)

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heart transplant. Using CD11c-DTR bone marrow chimeras, they showed that pre-transplant administration of Vit.D3-treated, ‘maturation-resistant’ donor-strain DC (MRDC) could not prolong cardiac allograft survival in the absence of recipient cDC.21 Donor-strain MRDC were

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quickly eliminated by innate mechanisms, leading to their uptake by recipient cDC as apoptotic remnants. Such donor alloantigen-loaded cDC were then able to control alloreactive T cell responses through abortive activation. Surprisingly, using the same experimental system, syngeneic MRDC pulsed with Bc antigens were also found to exert their allograft-protective

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effects through delivery of alloantigen to recipient cDC. Transfer of MHC antigens from adoptively transferred DC to recipient cDC is mediated by extracellular vesicles with characteristics of exosomes,22,23 which accounts for both indirect and semidirect antigen

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presentation.24 These powerful experiments have led many researchers to doubt whether the in vitro immunoregulatory activities of myeloid regulatory cells have any relevance whatsoever to their therapeutic properties in vivo. Applying these lessons to ECP, it highly likely that in situ tolerisation of recipient DC by subcellular particles from apoptotic cells contributes to the therapeutic effect of ECP;25 indeed, it is plausible that in situ tolerisation of recipient cDC is the principal mechanism by which ECP suppresses pathological T cell responses in vivo.


Quality-control procedures: Quality control procedures for ECP products should be updated to reflect our current understanding of ECP pharmacodynamics. In Europe, ECP products manufactured using open offline methods are regarded as somatic cell-based medicinal products (CBMP) that are governed by a legislative framework enacted through EU Regulation 1394/2007/EEC on ATMPs26 and an amendment of Directive 2001/83/EEC relating to medicinal

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products for human use.27 At once, this legislation both recognizes the inherent difficulties of characterising cell-based therapies as pharmacological agents, but also imposes exacting

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standards for preclinical characterization of cell products, comparable to those applied to conventional pharmaceuticals. Critically, these regulations require the composition and purity28 of a cell product to be precisely specified, as well as the pharmacological potency of every batch

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to be quantified. From the preceding discussion, it is evident that no single product description could be applied to the great variety of ECP products, especially because there is no single, definite mechanism of pharmacological action. Consequently, there is no consensus as to what in-process and pharmaceutical release assays should be performed for ECP products. In our

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view, there is a critical need for standardized quality-control assays that allow harmonized measurements of (1) the cellular composition of ECP products, (2) induction of apoptosis in lymphocytes,29,30 (3) monocyte polarization, (4) psoralen photoadducts,31 (5) T cell-

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suppression,32 and (6) capacity for antigen cross-presentation. Ideally, these in vitro assays should be complemented by standardised potency assays in humanized mice.33

Various groups have characterized changes in immunological profiles of patients undergoing ECP treatment, but these changes are generally small and only weakly predictive of treatment outcome. There are many reports of increased circulating Tregs in patients undergoing ECP for


various indications, including chronic GvHD (cGvHD)34,35 and solid organ transplant rejection.36,37 Unfortunately, gross methodological inconsistencies make these reports difficult to compare: from the literature alone, it is not possible to reliably judge the magnitude and duration of ECP-induced Treg-enrichment.38 Elevated pretreatment (but not posttreatment) expression of CCR7 by CXCR3+ CCR4+ CD4+ and CXCR3+ CCR4+ CD8+ T cells discriminates between ECP-

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responsive and nonresponsive cGvHD patients, hinting that retention of central memory T cells in secondary lymphoid organs after ECP may be mechanistically important, although not

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necessarily antigen-specific.39 Others have claimed that ECP causes a persistent normalization of cDC:pDC ratios in peripheral blood,40 which is most likely an epiphenomenon of successful treatment of the underlying disease. In our view, there is an urgent need to identify markers of

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pharmacodynamic effect, which should be measured in a standardized way,38 to enable assessment of ECP efficacy across different instruments, clinical sites and time.

ECP in solid organ transplantation: In lung transplantation,41 ECP is principally used in the

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management of refractory bronchiolitis obliterans syndrome (BOS), a form of chronic, obstructive allograft dysfunction that eventually affects the majority of lung transplant recipients. In such cases, ECP can lead to stabilization, or slowed deterioration, of allograft function,

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sometimes associated with histopathological improvement. The benefits of ECP in chronic, restrictive lung allograft dysfunction are less certain.42,43 There is some limited evidence that ECP is effective in treatment of acute or acute-on-chronic pulmonary allograft rejection; however, use of ECP as induction or maintenance therapy in lung transplantation has not been adequately explored. In cardiac transplantation,44 several small trials conducted in the ‘90s employed ECP as an adjunct immunosuppressive therapy for treatment of acute rejection or as a


prophylactic intervention. Based on these data, guidelines issued by the American Society for Apheresis (ASFA) make a strong recommendation that ECP is an appropriate therapy for prevention and treatment of cardiac allograft rejection;45 furthermore, the American Heart Association (AHA) and International Society for Heart and Lung Transplantation (ISHLT) endorse ECP as therapy for refractory antibody-mediated cardiac transplant rejection with a view

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to abrogating the underlying pathlogic T cell activation and augmenting regulatory T cell

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activity.25,46

Over the years, ECP has also been used in liver,47,48 kidney49-53 and facial54 transplantation to prevent or treat allogeneic rejection, or to manage transplant-related complications, such as

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GvHD after solid organ transplantation55,56 or calcineurin inhibitor (CNI)-nephrotoxicity after liver transplantation. However, despite relatively widespread availability of the technique since the ‘90s, ECP has gained little traction in kidney, liver and other solid transplantation, possibly due to its relatively high cost, the requirement for patients to attend clinic frequently, and

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scepticism about its immunological effects. Solid organ transplant patients are usually treated in cycles on two consecutive days that are started with high frequency and that are subsequently tapered down. There is considerable uncertainity about the applied frequencies that vary between

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centers.44,57,58 There is currently no place for ECP as a routine induction, maintenance or rescue therapy in kidney or liver transplantation; therefore, attention has focused on more exotic indications, such as tolerance induction, desensitisation and retarding kidney allograft fibrosis. In our view, current evidence for these proposed applications needs to be strengthened. Based on preclinical experiments in small animal models, some researchers advocate the perioperative administration of donor-derived (or partly HLA-matched 3rd party) ECP products as a


tolerogenic induction therapy. This strategy has obvious immunological parallels with the abandoned practice of donor-specific transfusion (DST)59 that was credited with fewer acute rejection episodes and improved allograft survival when donor and recipient shared at least one HLA-DR antigen.60 Unfortunately, DST was also associated with an unacceptably high rate of sensitization, so the practice was largely abandoned after the introduction of cyclosporin. In kidney

transplant

recipients

receiving

DST

without

concomitant

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prospective

immunosuppression, sensitisation rates ranged from 8% to 29%, but this could be reduced by

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cotreatment with azathioprine to 7% to 16%.61 In our view, the risk of alloimmunisation when administering donor-derived or random 3rd-party ECP products outweighs the probable benefits of inducing allospecific regulation in kidney transplant recipients. The rationale for using ECP as

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a pretransplant desensitizing therapy is not entirely explicit. Although treatment of acute rejection in cardiac transplant recipients was associated with reduced circulating alloantibody levels,62 perhaps through inhibition of antibody production by germinal center B cells or plasmablasts,63 it is unclear how ECP should eradicate memory B cells or long-lived plasma

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cells.64,65 Notably, predominantly antibody-mediated autoimmune diseases, including myasthenia gravis,66 idiopathic thrombocytopaenic purpura and autoimmune haemolytic anaemia are not amenable to ECP. As it currently stands, the hope that ECP could prolong allograft survival in

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cases of chronic kidney transplant dysfunction is not supported by available clinical or preclinical evidence.

Conclusions: Over many years, ECP has proven to be a very safe procedure with infrequent acute complications, no long term side effects and few contraindications.67 The clinical benefits of ECP in GvHD and cardiothoracic transplantation are well documented; nevertheless, ECP


remains a relatively specialized, intensive and expensive therapy. Optimal conditions for generating ECP products are not completely defined and the immunological effects of ECP in patients are poorly understood; moreover, optimal cell doses, frequency of treatment and comedications have not been systematically investigated in solid organ transplantation. In our view, given the present deficit of knowledge, using ECP in kidney or liver transplant recipients

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would be difficult to justify without substantial advances in preclinical and manufacturing development. In the future, through a more complete understanding of the immunological effects

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of ECP in patients, it may be possible to extend its applications in transplantation. In the first instance, this means dissecting the relative contributions of apoptotic cells versus ‘tolerised’ monocyte-derived cells to the tolerogenic effects of ECP products after administration. With this

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understanding, it may be possible to engineer simpler, more consistent cell-based therapies with accurately specified pharmacological qualities. In our view, current ECP procedures must eventually give way to better standardized manufacturing processes that result in more homeogenous, fully-defined cell products: it is not certain that 8-MOP/UVA-treatment will be

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necessary to achieve these goals. Accordingly, the pathway to ECP-like therapy in kidney or liver transplantation is likely to be long, and its final embodiment is likely to be very different

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from present-day ECP products.


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Legends

Figure 1.

Systems to perform extracorporeal photopheresis (ECP). (a) Closed inline systems (Therakos) combine apheresis and UV irradiation in one device, and remain connected to the patient for the whole of the procedure. In Europe, closed inline systems are

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regulated as medical therapies. (b) Open offline systems use separate devices for apheresis and UV irradiation in processes that require detachment of the leukapheresate

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from the patient. In Europe, open offline methods are mostly regarded as ATMP manufacturing processes. (c) Open inline systems combine separate devices operating at the bedside, which allows unbroken communication between the apheresate and

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patient. Because the apheresate is not detached from the patient, such processes may be regarded as medical treatments that do not require a manufacturing permit and are

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outside the scope of pharmaceutical authority surveillance.


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Figure 1
