Acardiac Surgeon's Efforts to Develop Xenotransplantation

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Chapter 3

THE NEXT GREAT MEDICAL REVOLUTION: A CARDIAC SURGEON’S EFFORTS TO DEVELOP XENOTRANSPLANTATION

David K. C. Cooper* Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, US

Keywords: Boston, Cape Town, history, medical, islets, Oklahoma City, organs, pig, genetically-engineered, Pittsburgh, xenotransplantation

ABBREVIATIONS

Gal galactose-α1,3-galactose; GTKO α1,3-galactosyltransferase gene-knockout; IBMIR instant blood-mediated inflammatory reaction; MSC mesenchymal stromal cell; NHH National Heart Hospital, London, UK; NHP nonhuman primate

* Corresponding Author Email: [email protected]. 28 David K. C. Cooper

“Success is the ability to go from one failure to another . . . with no loss of enthusiasm.” Winston Churchill

INTRODUCTION

As a medical student in the early 1960s I determined that I wished to follow a career in the relatively new field of cardiac surgery. I had been heavily influenced in this respect by the fact that at my medical school in London, Guy’s Hospital Medical School, two of the greatest hearts surgeons of that era were on the staff at the time - Sir Russell Brock (later Lord Brock) and Donald Ross. After graduation in 1963, I was fortunate to hold three internships at Guy’s - in general medicine, general surgery, and cardiothoracic surgery. Immediately following these appointments (in mid-1965), I took a temporary position as a ship’s surgeon in order to get some rest and recreation after what proved to be a period of sustained very hard work. I fulfilled this role in 1965 on a short cruise and on a six-week voyage on a scheduled liner to South Africa and back. While in Cape Town, at the suggestion of Donald Ross, a South African who had been an exact contemporary of Christiaan Barnard as a medical student at the University of Cape Town, I called on Professor Barnard, the senior cardiac surgeon there, and spent a few hours with him one afternoon. He took me on a ward round with his team, and I well remember seeing a patient with severe heart failure. When we moved away from the bedside, Barnard turned to me and said, “Of course, what this patient needs is a new heart.” At the time, I did not take this suggestion too seriously because I was aware that the results of kidney transplantation left much to be desired and I felt that it was unlikely anybody would perform a heart transplant in the near future. After a year teaching anatomy at Harvard Medical School (where I also gained my first experience of surgical research at the Peter Bent Brigham Hospital in the department headed by the legendary surgeon-scientist, Francis Moore), I spent a year in accident and emergency surgery in Cambridge in the UK. I then decided to pursue a PhD in surgical research at the University of London.

LONDON AND CAMBRIDGE

By this time (the summer of 1967), I firmly believed that transplantation was the next major development in cardiac surgery, and so my research at the Institute of Cardiology (attached to the National Heart Hospital [NHH]), supervised by Donald Ross, primarily involved methods of resuscitating and storing the heart for purposes of transplantation. I was also involved with some very early work in heart-lung transplantation in dogs directed by Donald Longmore, who was in charge of the cardiopulmonary bypass services at the NHH. The Next Great Medical Revolution 29

Not long after I had embarked on my research, Christiaan Barnard surprised everybody by carrying out the world’s first human-to-human heart transplant on December 3rd, 1967. Some 5 months later, I was privileged to watch the first heart transplant carried out in the UK (by Donald Ross at the NHH) on May 3rd, 1968. Having completed most of my experimental studies for the PhD, I spent three months in India as a volunteer at a hospital in the Punjab, and then returned to clinical surgery for two years in the UK to fulfill my general surgical requirements before sitting the examination for the Fellowship of the Royal College of Surgeons of England. I was fortunate to obtain an appointment at Addenbrooke’s Hospital in Cambridge where I came under the influence of Roy Calne (later Sir Roy Calne, FRS), one of the foremost pioneers in organ transplantation. Kidney transplantation was steadily being established (though the results remained poor), but liver transplantation was in its infancy at the time (1970-1971). I made a particular effort to participate in some of these operations and learn something of the immunosuppressive regimens that were being used. I was greatly impressed by Roy’s energy and drive. Following this surgical experience, I spent a year completing my PhD dissertation, and then moved into my chosen field of cardiothoracic surgery. Having completed my formal training, I was offered a temporary appointment at Papworth Hospital, where the designated senior registrar (equivalent to chief resident) was planning to spend a year in the United States. This one year extended to two years, and I was extremely fortunate to be a member of the team (led by Terence English, later Sir Terence) that carried out the first four heart transplants at Papworth in 1979, which gave me insight into the clinical management of these patients. At the end of my two years at Papworth, I was very keen to obtain an academic appointment in cardiothoracic surgery (where I would have the opportunity to combine clinical surgery with research), but this proved very difficult in the UK at that time, where there were only three academic appointments in the entire country. I therefore wrote to Christiaan Barnard, asking him whether I could join his team in a research capacity for a year. He generously offered me a position and, in the event, I remained in Cape Town for seven years, gradually being promoted to Associate Professor.

CAPE TOWN

When I arrived in Cape Town at the beginning of 1980 (where I was to spend some of the happiest years of my life), as well as working in the laboratory, Barnard asked me to take responsibility for the patients undergoing heart transplantation, which I was pleased to do. This proved immensely interesting, but challenging and stressful because about 50% of the patients died within a year of the transplant (as they were doing worldwide). I found Barnard a breath of fresh air as he allowed me considerable 30 David K. C. Cooper independence and also, although he demanded that the patients should be cared for conscientiously, he had a sense of humor that made working with him enjoyable. It was also in Cape Town that I had the great good fortune and pleasure of working with Winston Wicomb, PhD and Dimitri Novitzky, MD, PhD, two of the most innovative people I have ever met. While at the NHH I had written a short article in which I had stated that I thought the future of heart transplantation might lie in xenotransplantation, with the use of nonhuman primates (NHPs), such as chimpanzees, as potential sources of organs. I was probably influenced by the pioneering work of Keith Reemtsma and others in the 1960s [1]. In Cape Town, I was able to explore the possibility of xenotransplantation personally. When I arrived, I suggested this as an area of research to Barnard, but he was unenthusiastic. His opinion was that “we have enough problems in preventing rejection of a human heart without taking on the added burden of a xenograft.” Nevertheless, as NHPs were so readily available to us in Cape Town – where a baboon cost me $25 (whereas today, in 2018, in the U.S. the cost is in the region of $8,500) – I embarked on some experiments in the laboratory, transplanting African green (vervet) monkey hearts into baboons to immunologically mimic the baboon-to-human situation [2-4] (Figure 3.1). I also carried out heart allotransplantation in baboons across the ABO barrier, which had some similarities to xenotransplantation [5].

Figure 3.1. Research colleagues at the University of Cape Town in the early 1980s. (Left-to-right) Stuart Boyd, Frederick ’Boots’ Snyders, Prescott Madlingozi, Winston Wicomb, Sharon Smit, and Ferdinand Barends. Not photographed, but also valuable members of the team were Dimitri Novitzky, Joanna Martin, and John Roussouw.

Although I gave thought to the potential of NHPs as ‘donors’ for humans [6], it soon became obvious to me that, for various reasons, they would not be ideal. I therefore switched my attention to the pig as a potential source of organs for humans. To my The Next Great Medical Revolution 31 knowledge, although John Najarian’s group had investigated transplants between pigs and dogs (reviewed in [7], there had been no previous studies in the pig-to-NHP model except Roy Calne’s handful of pig liver transplants in baboons and chimpanzees in the late 1960s [8-10]. In the pig-to-baboon heart transplantation model, I soon found that hyperacute rejection occurred in every case [11]. I was fortunate to be collaborating with an excellent cardiac transplant pathologist, (the late) Alan Rose (Figure 3.2), who described the histopathology of hyperacute rejection in this model [12-14]. To overcome this hurdle, we perfused the recipient baboon’s blood through the donor pig kidneys (thus adsorbing anti-pig antibodies) before implanting the heart [15]. This led to some delay in rejection, but only to a maximum of four days, and was clearly not sufficient. To my knowledge, the only other person working in this model at that time was Guy Alexandre in Belgium, who, based on his innovative work in ABO-incompatible clinical kidney transplantation, was exploring pig renal xenotransplantation by using pre- transplant plasmapheresis to remove pig antibodies [16].

Figure 3.2. Some major collaborators. (Top, from left) Alan Rose, Egidio Romano, Simon Robson, Michel Awwad. (Bottom, from left) Henk-Jan Schuurman, David Ayares, Robin Pierson, Rita Bottino.

OKLAHOMA CITY

In 1987, at the recommendation of Christiaan Barnard, who had retired and had relocated to Baptist Medical Center (now Integris Baptist Medical Center) in Oklahoma City in an advisory capacity, my Cape Town colleague, Dimitri Novitzky, and I were 32 David K. C. Cooper invited to join the staff of the hospital, where Nazih Zuhdi was establishing an organ transplant center. We helped establish a successful heart transplant program and, later, lung transplantation. Because of my studies on ABO-incompatible heart allo- and xeno-transplantation, I was approached by a group from the biotechnology company, Chembiomed, in Edmonton, Canada, who asked me to collaborate on some studies in this field. Based on the work of Venezuelan medical scientist, Egidio Romano [17] (Figure 3.2), these researchers had evidence to suggest that the intravenous infusion of synthetic A or B oligosaccharides would be bound by the respective anti-A or B antibodies in the blood and that this antibody-antigen complex would be cleared, thus reducing the antibody level in the blood and enabling an ABO-incompatible organ graft to be transplanted without fear of hyperacute rejection. Their theory was that, after an indeterminate period of oligosaccharide infusion, a state of ‘accommodation’ might develop [18]. In view of the model I had established of AB-incompatible heart transplantation in baboons, they invited me to explore the infusion of the relevant oligosaccharides. They had also developed immunoaffinity columns of the respective A and B saccharides for pre- transplant extracorporeal immunoadsorption of these antibodies from the blood. Although relatively skeptical that the continuous infusion of the A or B saccharides would prevent hyperacute rejection, I readily agreed to their proposal and, together with Yong Ye, Francisca Neethling, and others, we carried out a series of heterotopic heart allotransplants in baboons across the AB barrier. To my surprise and satisfaction, their hypothesis was correct and we were able to prevent antibody-mediated rejection, the grafts surviving until an adaptive cellular response developed [19].

Figure 3.3. Two of my early and invaluable collaborators - Eugene Koren (far left) and Rafael Oriol (far right) - with Takaaki Kobayashi and Douglas Smith (who ran the tissue typing laboratory in Oklahoma City, and had a longstanding interest in swine leukocyte antigens). (Photograph courtesy of Takaaki Kobayashi.) The Next Great Medical Revolution 33

With the invaluable financial support of Baptist Medical Center, I was continuing my studies of pig-to-baboon heart transplantation, and this experience with ABO- incompatible allotransplantation led me to believe that, if we could identify the carbohydrate antigenic targets for anti-pig antibodies, then the infusion of the relevant oligosaccharide (or the extracorporeal immunoadsorption of those antibodies) might lead to prolonged survival of a pig heart in a baboon. I naively thought that this might be the only step we needed to take in order to obtain long-term pig graft survival (again because it was hoped that accommodation would take place). I was fortunate to begin to collaborate with an experienced medical scientist at the Oklahoma Medical Research Foundation, Eugene Koren (Figures 3.3 and 3.4). At his suggestion, we perfused human plasma through pig organs ex vivo, eluted the anti-pig antibodies that had become bound to the pig vascular endothelium, and sent these to Chembiomed for testing against a large panel of synthetic oligosaccharides. It was very exciting when Heather Good (Figure 3.4) reported to me that the major target for anti-pig antibodies was galactose-α1,3-galactose (Gal) (Figure 3.5), although there were some minor targets as well [20-22].

Figure 3.4. Members of the collaborative group that in 1991 definitively identified galactose-α1,3- galactose as a major target for human anti-pig antibodies. (From the left) Francesca Neethling (Baptist Medical Center [BMC], partly hidden), Yong Ye (BMC), and Marek Niekrasz (University of Oklahoma). (From the right) Heather Good (Chembiomed), Gene Koren (Oklahoma Medical Research Foundation), me (BMC), and Andrew Malcolm (Chembiomed). The photograph was taken at the 1st International Congress on Xenotransplantation held in Minneapolis in August 1991, where this work was first presented. 34 David K. C. Cooper

Figure 3.5. Diagram of the comparative structures of the human O, A, and B blood group glycans and the pig α-Gal glycan.

At that time, I had never heard of Gal, but the Chembiomed people told me that they had carried out some studies for Uri Galili, and that he had demonstrated that all humans had anti-Gal antibodies and, furthermore, that pigs were among the non-primate mammals that expressed Gal. I asked them why they had not told me this before we carried out our experiments, but they were unable to explain this. I rapidly read all I could of Uri Galili’s work [23], and it became certain to me that we had identified the correct target for anti-pig antibodies. We presented the results of our study at the First International Congress on Xenotransplantation, held in Minneapolis in August of 1991, having submitted the abstract some months previously [20, 22], which was undoubtedly the first time that the importance of Gal in xenotransplantation had been reported (discussed in [24]). Others soon confirmed its importance [25] (which had not been demonstrated by Galili because xenotransplantation was not an area of interest to him). Rafael Oriol, a consultant for Chembiomed who was an expert in ABO immunogenetics, elegantly confirmed the expression of Gal on various tissues of the pig [26] and Francesca Neethling investigated its role in the primate immune response in vitro [27-29]. The Next Great Medical Revolution 35

Figure 3.6. Photograph taken after the dinner organized by Takaaki Kobayashi in Osaka in 2013 (by which time some of the attendees had departed). (Seated, from the left) Takaaki Kobayashi, me, Leo Buhler, Kenji Kuwaki. (Standing, from the left) Minoru Fujita, Rita Bottino, Satoshi Gojo, Kazuhiko Teranishi, Whayoung Lee, Hidetaka Hara, Yifan Dai, Tadatsura Koshika, Hao-Chih Tai.

It was at the Minneapolis congress that Eugene Koren, Rafael Oriol (Figure 3.3), and I determined that the ideal approach to xenotransplantation would be to genetically engineer the pig so that it did not express Gal (an α1,3-galactosyltransferase gene- knockout [GTKO] pig) [30]. At that time, this was possible in mice and, in fact, was soon carried out [31, 32], but was not possible in pigs until the development of the technology that brought about the first cloned mammal, Dolly the sheep [33], and subsequently the first cloned pig (34) and the first genetically-modified cloned pig [35,36]. A homozygous GTKO pig was not available until 2003 [37, 38]. In the meantime, with the invaluable help of veterinarian, Gary White, and his colleagues, particularly Marek Niekrasz (Figure 3.4), at the animal facility at the Oklahoma University Health Sciences Center [OUHSC], where we learnt of the tether and jacket system in the management of baboons [39], we spent a great deal of time in carrying out immunoadsorptions of antibody using immunoaffinity columns of synthetic Gal oligosaccharides [40, 41] or infusing these Gal saccharides into baboons [42, 43]. Initially, there was insufficient synthetic Gal available for us to attempt continuous intravenous infusion but, from my reading, I realized that melibiose had some similarities to Gal. Our initial studies, therefore, involved the intravenous infusion of melibiose into baboons, testing their serum, and also in carrying out pig heart transplants [44]. We did not measure the level of melibiose in the blood of the baboon, but when urine from the baboon fell to the floor, the soles of our shoes temporarily stuck to the floor through the stickiness of the sugar.

36 David K. C. Cooper

Although the extracorporeal immunoadsorption of anti-Gal antibodies clearly delayed rejection of a transplanted pig organ, this was only temporary, and when antibodies returned to the blood, a delayed form of antibody-mediated rejection occurred [45]. Shigeki Taniguchi even investigated hearts from animals that did not express Gal, e.g., ostriches, without success [46]. With Takaaki Kobayashi (Figures 3.3 and 3.6) and Shigeki Taniguchi, we also attempted to prolong pig heart graft survival in baboons by the use of cobra venom factor (kindly provided by David White) and other anti-complement agents, with some success, achieving wild-type pig heart graft survival of three weeks, which was the world’s longest xenograft survival at that time [47, 48]. We subsequently demonstrated that sensitization to a xenograft did not seem to increase the risk of subsequent allotransplantation or vice versa [49-51] (reviewed in [52]), though others have subsequently shown that prior allosensitization may be detrimental. One interesting episode of my time in Oklahoma City followed the studies by our senior liver transplant surgeon, Luis Mieles, who developed experience of auxiliary liver allograft and xenograft support in baboons [53]. We believed that temporary support of a patient in fulminant hepatic failure could be achieved by a baboon liver until a cadaveric human liver became available. To ensure that the ‘bridging’ of a patient in this way was not contrary to any government guidelines, I explained our plan over the telephone to an official of the FDA. “That’s interesting,” he said. “Let us know what happens.” There appeared to be no guidelines or concerns about transplanting a baboon organ into a patient in those days. Unfortunately, local opposition within the hospital resulted in this plan never coming to fruition. If my memory serves me correctly, these plans preceded Tom Starzl’s clinical attempts at baboon liver transplantation in Pittsburgh in 1993 [54].

BOSTON

As I was no longer completely happy at Baptist Medical Center and had resigned my position, I was delighted when David Sachs approached me about joining him in the Transplantation Biology Research Center (TBRC) at the Massachusetts General Hospital/Harvard Medical School in 1996 (at which time I decided to retire from clinical surgery). My junior colleague, Francesca Neethling, originally from Cape Town, kindly moved with me from Oklahoma City, and was an immense help in establishing our laboratory. David Sachs was attempting to induce tolerance in baboons to wild-type pig kidney grafts by hematopoietic cell chimerism and, with Simon Robson (Figure 3.2) and Jay Fishman, we were fortunate to receive NIH funding to continue these studies. This involved extensive pre-transplant treatment that included irradiation, which was The Next Great Medical Revolution 37 associated with considerable morbidity, and so I suggested we explore the induction of hematopoietic chimerism and kidney xenotransplantation separately. Over the next several years, I was fortunate to work with a number of excellent research fellows from Europe and Asia (Figure 3.6). With regard to the induction of tolerance, our experience was that, even after the infusion of very large numbers of pig hematopoietic stem cells, chimerism was lost within minutes, probably from macrophage phagocytosis [55], a problem that still has not been resolved today. Through the generosity of David White and his colleagues at Imutran in Cambridge in the UK, we were fortunate to obtain pigs that expressed a human complement- regulatory protein, CD55 (DAF). Transplantation of one of these organs delayed antibody-mediated rejection, but, in our hands, did not prolong graft survival for more than a few weeks [56]. It was in Boston that I first fully realized the problem of coagulation dysfunction in the pig-to-NHP model. Surprisingly, I had not seen this in Oklahoma City, possibly because I just did not recognize it or, most likely, because rejection occurred so rapidly that consumptive coagulopathy did not have time to develop. In Boston, largely through the work of Frank Ierino [57] and Tomasz Kozlowski [58], the problem of consumptive coagulopathy became very clear to us. It was a pleasure to collaborate with Simon Robson, who had previously provided evidence to suggest that coagulation dysfunction would be problematic after pig organ xenotransplantation [59].

Figure 3.7. Some of the research team at the Transplantation Biology Research Center at the Massachusetts General Hospital/Harvard Medical School in the late 1990s. Ian Alwayn is in the left foreground, with Alan Watts to his right, with Leo Buhler sititng opposite. Leo is flanked by Geoff Oravec and Anette Wu. (Alan Watts supervised more than 300 antibody immunoadsorptions in baboons.) 38 David K. C. Cooper

When joined by Leo Buhler (Figure 3.7) and Ian Alwayn (Figure 3.7) (the latter being the first of several excellent research fellows who joined me from Erasmus Medical Center in Rotterdam, whose studies I jointly supervised with Jan Ijzermans), it became clear to us that conventional immunosuppressive therapy consisting of cyclosporine, mycophenolate mofetil, and corticosteroids, did not prevent an adaptive immune response (including an elicited anti-pig antibody response) to a pig organ graft [60, 61]. This stimulated us to explore some of the new co-stimulation-blockade agents that were becoming available, in particular, anti-CD154 (CD40L) mAb, which successfully prevented the adaptive immune response [61]. To my knowledge, this was the first use of costimulation blockade therapy in a pig-to-primate model. Our impression was that consumptive coagulopathy developed more rapidly after kidney transplantation than after heart transplantation, and Christoph Knosalla, whose dedication and persistence did much to get us through this very difficult and frustrating period, investigated this at the molecular level [62]. Those who followed him in the laboratory benefited from his immense efforts during this critical period. Eventually, in 2004, through the genetic engineering work of our colleagues at Immerge Biotherapeutics and the University of Missouri [38], we were able to test GTKO pig organ transplants in baboons, using an immunosuppressive regimen based on anti-CD154mAb. In these studies, Michel Awwad (Figure 3.2) gave us great support (particularly in identifying baboons with low anti-pig antibody levels) and Henk Schuurman (Figure 3.2) proved an immensely hardworking and valued collaborator. Thanks largely to the excellent work of Kenji Kuwaki (Figure 3.6), this combination of a GTKO pig organ and costimulation-blockade (in baboons selected for low anti-pig antibody levels) prolonged pig heterotopic heart graft survival for several weeks, and in one case for six months (the longest survival recorded to that date) [63, 64]. Kaz Yamada demonstrated that life-supporting GTKO pig kidney graft survival (combined with thymus transplantation) could be prolonged to a maximum of 83 days [65], which was comparable to the results obtained by Emanuele Cozzi and his colleagues at Imutran (and in Padua) where, using a hCD55 transgenic kidney, graft survival had extended to 90 days [66, 67]. Although his work was mainly directed to spleen allotransplantation, Frank Dor, from Erasmus in Rotterdam, was an invaluable member of our team at the time, and contributed greatly by mentoring new research fellows and technicians. Nevertheless, in both the heart and kidney transplants, a thrombotic microangiopathy developed [63, 64, 68-73], which ultimately resulted in a consumptive coagulopathy. Rapid excision of the heart graft at this time reversed the coagulopathy, confirming that its cause was the presence of the graft [74]. Despite the successful production of GTKO pigs and these encouraging results, both BioTransplant and Immerge went out of business when Novartis ceased its financial support.

The Next Great Medical Revolution 39

The International Xenotransplantation Association (IXA)

It was during my time in Boston that I helped establish the IXA (in 1998), becoming its founding honorary secretary. In 1999-2001 had the great privilege of being its second president (Figures 3.8 and 3.9). The proudest moment of my academic life was when I was awarded honorary membership of the IXA in 2009 (Figure 3.10), but perhaps the happiest moment was a dinner in Osaka in 2013, organized largely by Takaaki Kobayashi, where I was joined by several of my previous research fellows and other colleagues from Japan and elsewhere (Figure 3.6); several who could not be with us sent their good wishes in the form of videos or messages. I remain very grateful to Takaaki for putting this memorable reunion together.

Figure 3.8. With the organizers (Joe Leventhal on the left and Jonathan Fryer on the right) of the 6th International Congress on Xenotransplantation held in Chicago in 2001. The 9/11 twin towers terrorist tragedy had just taken place in the USA, and many of those planning to attend the congress cancelled at the last minute but, thanks to Joe and Jonathan, the congress was very successful. I remain very grateful to them for overcoming the hurdles they faced.

Figure 3.9. As president of the IXA in 2001, at the Chicago congress I had the privilege of presenting John Najarian with Honorary Membership (the second person so honored). 40 David K. C. Cooper

Figure 3.10. At the 10th International Congress on Xenotransplantation (held jointly with IPITA) in Venice in 2009, I was awarded Honorary Membership of the IXA by Robin Pierson (president, left) and Emanuele Cozzi (organizer of the congress).

PITTSBURGH

Soon after this (in 2004), at the invitation of Tom Starzl (Figure 3.11), I relocated to the Thomas E. Starzl Transplantation Institute (STI) at the University of Pittsburgh where, in collaboration with David Ayares (Figure 3.2) and his colleagues at Revivicor in Blacksburg, VA (which company had received a major venture capital investment from the University of Pittsburgh Medical Center, a company that owned more than 20 hospitals in the Pittsburgh area), we were able to continue studies in the GTKO pig-to- baboon model [37] (Figure 3.12). Unfortunately, despite initial assurance of their long- term commitment, the administration of UPMC did not have sufficient vision and patience to pursue an interest in xenotransplantation long-term, and Revivicor was purchased by United Therapeutics (Martine Rothblatt) in July 2011. As chairman of the Revivicor scientific advisory board (2004-2011), I had encouraged David Ayares to offer help to several other groups worldwide by providing them with genetically-engineered pig cells. For example, when the German consortium established by Bruno Reichart was experiencing difficulty, Revivicor provided them with GTKO pig cells. With pigs to be provided by Revivicor, we formed a consortium with Robin Pierson (Figure 3.2) and his team and applied successfully for a grant restricted to xenotransplantation studies in NHPs. Fortunately, we have been funded by NIH continuously for almost the past 15 years.

The Next Great Medical Revolution 41

Figure 3.11. Some members of the Thomas E. Starzl Transplantation Institute (STI) research team at the University of Pittsburgh with Dr Starzl in 2014. (Seated, from the left) Mohamed Ezzelarab, me, Dr Starzl, Hidetaka Hara, Hayato Iwase. (Standing, from the left) Hong Liu, Huidong Zhou, Whayoung Lee, Cassandra Long (who, as lab manager, was key to the team’s smooth functioning, and to whom I am immensely grateful), and Yuko Miyagawa.

We first explored the detrimental effect of anti-nonGal antibody (i.e., antibody directed to targets other than Gal) on GTKO pig cells in vitro [75-78]. As first suggested to me by Simon Robson, we hypothesized (as did others) that binding of anti-nonGal antibodies, and possibly complement fractions, to the pig vascular endothelium resulted in low-grade activation that initiated a local procoagulant state [79]. Eventually, this led to thrombotic microangiopathy and, ultimately, to consumptive coagulopathy. However, studies by Chih Che Lin, in collaboration with Tony Dorling in the UK, showed that coagulation dysfunction was more complicated than we had anticipated in that the mere exposure of primate platelets to pig vascular endothelial cells initiated their aggregation in the absence of antibody or complement [80, 81]. GTKO pigs expressing a human complement-regulatory protein, CD46 - the CD46 transgenic pigs having been originally produced by Ian McKenzie and his colleagues in Australia - became available through Revivicor and appeared beneficial in extending graft survival further [82-84], but did not address the problem of coagulation dysfunction. When pigs that also expressed a human coagulation-regulatory protein (e.g., GTKO/CD46/thrombomodulin pigs) became available, prolonged pig graft survival became more consistent [85-87]. In both the pig artery patch and heart transplant models, we demonstrated that CTLA4-Ig (in the form of abatacept or belatacept) was unsuccessful in preventing an adaptive immune response, but that the combination of anti-CD40mAb and belatacept 42 David K. C. Cooper was successful [85]. Our colleagues at the National Heart, Lung and Blood Institute (the NHLBI of the NIH) (using Revivicor pigs and an immunosuppressive regimen originally based on ours) demonstrated that a high dose of anti-CD40mAb alone was sufficient to achieve long-term graft survival [88-90]. Indeed, they subsequently reported two baboons with heterotopic heart grafts that functioned more than one year, in one case for more than two years, with graft failure only occurring when all immunosuppressive therapy was discontinued. These results were highly encouraging, and so in a life-supporting kidney transplant model, using kidneys from a GTKO/CD46 pig expressing human coagulation-regulatory proteins, we used a high-dose anti-CD40mAb-based regimen similar, but not identical, to that used by the NHLBI group. We achieved kidney graft survival of almost five months – the longest pig kidney graft survival reported to that date [86]. However, soon after, the Emory/Indiana group reported longer survival of two rhesus monkeys with pig kidney grafts that functioned, in one case, for almost ten months [91] (Adams AB, personal communication). There were several differences between the Emory/Indiana and Pittsburgh experiments, including (i) the use of a monkey versus a baboon, (ii) the use of anti-CD154mAb rather than anti-CD40mAb, and (iii) the selection of monkeys with low anti-pig antibody levels. It was notable that the successes at Emory were only achieved when anti-CD154mAb was used (rather than belatacept), and only when monkeys were selected with low anti-pig antibody levels. However, surprisingly, this good result was achieved using a kidney from a pig that did not express a human coagulation-regulatory protein.

INFLAMMATION

The systemic inflammatory response that develops after pig organ or even artery patch transplantation in baboons had become increasingly obvious to us, largely through the astute observations of the very innovative Mohamed Ezzelarab (Figure 3.11) [92, 93]. Inflammation augments the immune response and we believed needed to be controlled if truly long-term pig graft survival was to be obtained. The inclusion of an anti-IL-6R blockade agent in our regimen was perhaps the first step towards addressing this barrier [93], though pigs expressing HO-1 or A20 may be preferable. Hayato Iwase, who proved meticulous in his care of the baboons, subsequently reported pig kidney graft survival of almost nine months with no signs of rejection [87]. Our ability to extend life-supporting kidney graft survival for longer periods enabled us to make observations on pig renal function in the baboon [87,94].

The Next Great Medical Revolution 43

Figure 3.12. Some of the research team at the STI in about 2011. (Left-to-right) Goutham Kumar, Tadatsura Koshika, Burcin Ekser, Dirk van der Windt, Hidetaka Hara, me, Cassandra Long, Martin Wijkstrom, Eefjie Dons, Tyler Wilhite, Minoru Fujita.

PIG LIVER XENOTRANSPLANTATION

In Pittsburgh, working with Bruno Gridelli and Burcin Ekser (Figure 3.12) (who proved immensely productive), we explored GTKO/CD46 pig orthotopic liver transplantation in baboons using conventional immunosuppressive therapy [95-97]. The immediate development of thrombocytopenia (in the absence of hyperacute rejection) indicated that there are additional problems to be overcome if the pig liver is to provide even a bridge to allotransplantation in patients with fulminant hepatic failure. However, these studies proved very valuable, and demonstrated that pig hepatic function was surprisingly good in a baboon [96].

SUPPRESSION OF THE T CELL RESPONSE

The absence of the expression of Gal and the presence of a human complement- regulatory protein had additional beneficial effects that we did not anticipate. The first was that they resulted in a weaker adaptive immune response (as well as protection from the humoral response) [98, 99], suggesting that this may eventually enable successful organ xenotransplantation when administering conventional immunosuppressive therapy rather than costimulation blockade-based therapy, which is not yet clinically available. Hayato Iwase (Figure 3.11), also found that these genetic manipulations reduced platelet aggregation to pig aortic endothelial cells [100], indicating an effect in preventing 44 David K. C. Cooper thrombotic microangiopathy. The expression of a human coagulation-regulatory protein, of course, reduced platelet aggregation further [100]. The T cell response, which continues to be investigated by my long-time and now indispensable colleague, Hidetaka Hara (Figures 3.11 and 3.12), can be reduced by more direct methods, e.g., expression of CTLA4-Ig (or LEA29Y) in the pig [101] or of a mutant class II transactivator (CIITA-DN) that suppresses SLA class II expression and prevents its upregulation [102,103]. However, the T cell response is complex; Mohamed Ezzelarab has demonstrated that human T cells also respond to thrombin-activated pig cells [104].

NEONATAL B CELL TOLERANCE

Pleunie Rood, who was my first fellow in Pittsburgh and helped me greatly in establishing the research program at the STI, demonstrated that infant baboons and humans did not develop anti-pig antibodies until 3-6 months after birth [105, 106], confirming observations that Francesca Neethling had made in the 1990s in collaboration with Robert Michler’s group [107, 108]. Furthermore, anti-nonGal antibodies remained low throughout the first year. In a very demanding animal model, Eefjie Dons (Figure 2.12) demonstrated that anti- pig antibody production could be prevented in infant baboons treated with an anti- CD154mAb [106], suggesting a method by which B cell tolerance to pig antigens could probably be developed. Unfortunately, we were unable to obtain funding to extend these observations, which might have enabled successful pig heart transplantation (and even tolerance induction) in infants and young children, e.g., those with complex congenital heart disease.

PIG ISLET XENOTRANSPLANTATION

At the TBRC, Leo Buhler had carried out the first pig islet transplant in a NHP using a costimulation blockade-based regimen [109]. In Pittsburgh, in collaboration with islet expert, Rita Bottino (Figure 3.2), and Massimo Trucco (now at the Allegheny Health Network), we carried out several series of experiments of pig islet xenotransplantation in streptozotocin-induced diabetic cynomolgus monkeys [110-112]. Using islets from CD46 pigs (provided to Revivicor by Ian McKenzie and Bruce Loveland), Dirk van der Windt (Figure 3.12) and Rita demonstrated normoglycemia for a period >1 year in the absence of the need for insulin therapy [111], which was the first time such prolonged survival had been reported. Furthermore, when we obtained GTKO/CD46 pigs expressing human The Next Great Medical Revolution 45 coagulation-regulatory proteins [113], this excellent outcome was repeated by Rita Bottino [112]. However, we could not achieve this success on a consistent basis, most likely through the development of primary graft failure related to the instant blood- mediated inflammatory reaction (IBMIR). Nevertheless, we were convinced that pig islet transplantation has an immense clinical potential, and today is close to clinical trials. The studies by Chung-Gyu Park and his colleagues in Seoul have been particularly encouraging. Rita set up an in vitro assay to investigate IBMIR, and provided evidence that, rather than being simply a non-specific response to exposure of the islets to primate blood, it is more associated with antibody and complement deposition, suggesting it may be a form of hyperacute rejection [114, 115]. Mohammed Ezzelarab became interested in the potential of mesenchymal stromal cells (MSCs) derived from the organ- or islet-source genetically-engineered pigs as a means of augmenting the survival of a graft [116]. He felt this would be particularly important in relation to islet transplantation as the MSCs should (i) increase revascularization of the graft, (ii) have an anti-inflammatory effect, and (iii) reduce the adaptive immune response. He carried out several in vitro experiments to investigate this possibility [117-119], but we have not had sufficient grant funding to test genetically- engineered pig MSC therapy in vivo.

Figure 3.13. The very productive research group at the Shenzhen Second People’s Hospital (First Affiliated Hospital of Shenzhen University) in about 2016. Seated on the far left is Lisha Mou, who supervises most of the research by the group. Third from the left is Yifan Dai (Nanjing Medical University), who set up the collaboration between the Shenzhen and the STI groups. Seated to my left is Zhiming Cai, the president of the hospital who established the research group, and to his left is Hidetaka Hara, and then Dengke Pan (Institute of Animal Sciences, Beijing). 46 David K. C. Cooper

One important aspect of pig islet xenotransplantation to which Anna Casu drew attention was the differences in glucose metabolism between pigs and primates [120, 121]. Through Yifan Dai (formerly of Revivicor and then the University of Pittsburgh, before he returned to China) (Figure 3.6), we developed a productive collaboration with a new group based in Shenzhen in China (Figure 3.13), and pursued several lines of research with them, with a particular interest in islet xenotransplantation. In addition, my group developed a long-standing collaboration with Professor Yi Wang of the University of South China in Hengyang, who directed several excellent young research fellows to work with us.

PIG CORNEAL XENOTRANSPLANTATION

Keen to develop a field that might become ready for clinical trials at an early stage, Hidetaka Hara and Whayoung Lee (Figure 3.11) began investigations – initially in vitro - of pig corneal transplantation in NHPs [122-125]. We then carried out full-thickness corneal transplants (penetrating keratoplasty) as well as endothelial keratoplasty (the first time this latter technique had been attempted in a pig-to-NHP model), but found that the development of retrocorneal membranes (but not of rejection) limited success [126]. It is uncertain why these developed so consistently, but our initial thoughts are that they result from discrepancies in the thickness between the pig cornea and the recipient cornea; they are also seen in corneal allotransplants in infants and young children. However, corneal transplantation has an immense potential and it has only been lack of funding that has prevented us from exploring this field further [123, 127]. We have been greatly encouraged by the work of Mee Kum Kim and her colleagues in South Korea (reviewed in [128]).

TRANSFUSION OF PIG PACKED RED BLOOD CELLS

This is another field we have explored, particularly by Hidetaka Hara. In due course, I believe that all clinical transfusions of packed red blood cells will be from genetically- engineered pigs that are housed under isolation conditions, which will provide a safer source of blood transfusion than humans [129, 130].

BIOPROSTHETIC HEART VALVES

Increasing evidence from our laboratory and from others indicates that valves taken from GTKO pigs would survive longer in patients than wild-type pig valves [131], a topic explored by our Canadian collaborator, Rizwan Manji [132, 133]. Valves from The Next Great Medical Revolution 47

GTKO pigs that express neither N-glycolylneuraminic acid (cytidine monophosphate-N- acetylneuraminic acid hydroxylase gene-knockout) [134-136] nor the antigen encoded by β(1,4)N-acetylgalactosaminyltransferase (βGalNT2) [137, 138] will survive even longer [139]. I feel sure that pigs not expressing these three antigens will form the basis for clinical trials of organ or cell transplantation. While the immunological and pathobiological hurdles of xenotransplantation were being investigated and slowly but steadily overcome [45, 140], consideration had to be given to three other topics of importance – (i) the potential of xenozoonosis, (ii) the selection of patients for the first clinical trials, and (iii) the regulatory aspects of this potential new form of therapy.

THE POTENTIAL PROBLEM OF XENOZOONOSIS

Being convinced at a very early stage (the late 1980s) of the clinical potential of xenotransplantation, in Oklahoma City Yong Ye and I and colleagues began to consider what microorganisms would need to be eradicated from the pigs [141]. A few years later, I was fortunate to be a member of a committee set up by Novartis to consider this matter in detail [142]; the recommendations of this committee remain valid today. While at the TBRC, my group had the good fortune to collaborate with Jay Fishman and Nicolas Mueller on aspects of pig microorganisms in relation to xenotransplantation [143-149]. We demonstrated the detrimental effect of pig cytomegalovirus (CMV) on graft outcome, establishing that all organ-source pigs should be CMV-negative, which can be achieved by early-weaning of the piglets. These observations have been ‘rediscovered’ by others more recently.

SELECTION OF PATIENTS FOR THE FIRST CLINICAL TRIALS OF XENOTRANSPLANTATION

Selection will depend to some extent on how successful the first trials are anticipated to be, and this in turn will relate to the results achieved in preclinical models. When there is a perceived moderate risk of failure or complication, selection will need to be particularly vigorous. If the chance of success is considered to be high (as I believe it now is), then a wider group of patients could be included. We have considered these points in a number of publications [150-153]. Based on our experimental data, we have also considered such topics as to how xenograft function (and impending graft failure) could be monitored [154].

48 David K. C. Cooper

GUIDELINES AND REGULATORY ASPECTS OF XENOTRANSPLANTATION

The regulatory aspects to some extent are influenced by the conclusions drawn from the above two points – potential for infection and the selection of patients [153, 155]. The ethical aspects of xenotransplantation have long been of interest to me [156, 157].

BIRMINGHAM

Although we were very happy in Pittsburgh, in 2016 Hidetaka Hara, Hayato Iwase, and I relocated our laboratory to the University of Alabama at Birmingham. The great attraction was that UAB had recently received funds from Martine Rothblatt (who had purchased Revivicor) to build a biosecure pig facility that would enable a clinical trial to be initiated. My hope is that we can transfer xenotransplantation successfully into the clinic, which would give me the immense pleasure of seeing the research of so many of us come to fruition.

“History tells us that procedures that were inconceivable yesterday, and barely achievable today, often become routine tomorrow.” Thomas Starzl

ACKNOWLEDGMENTS

It has been an immense privilege and pleasure for me to collaborate with so many research fellows and faculty members at all of the centers at which I have worked. I would particularly draw attention to Takaaki Kobayashi, who was a recent president of the International Xenotransplantation Association (IXA), and Leo Buhler, who is the current editor of Xenotransplantation and president of the IXA. Both have become highly valued friends. I had a particularly valuable collaboration with Jan Ijzermans at Erasmus University Rotterdam, with whom I jointly supervised a number of excellent PhD candidates (Ian Alwayn, Frank Dor, Pleunie Rood, Dirk van der Windt, and Eefjie Dons). Working with all of them has been a privilege and I cannot overestimate the contributions they have made to the work I have briefly reviewed here. I am greatly indebted to Mohammed Ezzelarab, Hidetaka Hara, and Hayato Iwase, who, having joined me as research fellows, became my colleagues on the faculty at the Thomas E. Starzl Transplantation Institute and, in two cases, at UAB, and who have contributed immensely to our work in the past decade, as has my Pittsburgh colleague, Rita Bottino. The Next Great Medical Revolution 49

Little of our recent work would have been possible without the collaboration of David Ayares, Carol Phelps, and their colleagues at Revivicor, who for several years have provided us with genetically-engineered pigs not available anywhere else in the world. Since the early 1990s, my belief has been that genetic engineering of the source pig is key to the success of xenotransplantation. Until relatively recently, this was not always realized by others, although they perhaps may not admit it now. In addition, my group’s long and enjoyable collaboration with Robin Pierson and his colleagues at the University of Maryland at Baltimore has proved very productive, particularly in applying successfully together for NIH funding. I have also had the opportunity of collaborating with colleagues abroad, particularly with Yifan Dai in Nanjing, with whom I and my colleagues in Pittsburgh helped to establish a xenotransplantation research program in Shenzhen, headed by Zhiming Cai and Lisha Mou. I continue to have a valuable collaboration with Yi Wang of the University of South China in Hengyang, with whom I have jointly supervised several postdoctoral students (Hao Zhou, Maolin Jiang, Jiang Li, Huidong Zhou, Bingsi Gao, Tao Li, Qi Li, Juan Li, Liaoran Zhou). Working in the field of xenotransplantation research has not only been intellectually stimulating and hugely enjoyable, largely because I have always been totally convinced of its immense clinical applicability, but has enabled me to make many good friends and see many parts of the world that I perhaps would not otherwise have visited. The number of friends is too large to mention, but I would single out Ian McKenzie, Mauro Sandrin, Tony d’Apice, and Peter Cowan in Australia, Emanuele Cozzi, the late Carl-Gustav Groth, and Jean-Paul Soulillou in Europe, and Curie Ahn, Chung-Gyu Park, and Mee Kum Kim in South Korea. In addition, many of my former research fellows have become good friends. One of the most frustrating aspects of working in any field of research is insufficient funding to pursue the studies that you feel will bring eventual success. I was very grateful to Baptist Medical Center for funding my research in Oklahoma City, and to Biotransplant and Immerge, as well as the NIH, at the TBRC, and to the NIH and Revivicor when I was at the STI. I am convinced that, with sufficient funding, we will solve the remaining problems relating to pig kidney, heart, corneal, and islet xenotransplantation within the next two years, and those relating to pig liver transplantation within the next five years, enabling clinical trials to be initiated.

REFERENCES

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[17] Romano, E.L., Soyano, A., Linares, J. Preliminary human study of synthetic trisaccharide representing blood substance A. Transplant Proc. 1987; 19: 4475- 4478. [18] Bach, F.H., Turman, M.A., Vercellotti, G.M., Platt, J.L., Dalmasso, A.P. Accommodation: a working paradigm for progressing toward clinical discordant xenografting. Transplant Proc. 1991; 23: 205-207. [19] Cooper, D.K.C., Ye, Y., Niekrasz, M., et al. Specific intravenous carbohydrate therapy. A new concept in inhibiting antibody-mediated rejection--experience with ABO- incompatible cardiac allografting in the baboon. Transplantation. 1993;56:769-777. [20] Good, A.H., Cooper, D.K.C, Malcolm, A.J., et al. Identification of carbohydrate structures that bind human antiporcine antibodies: implications for discordant xenografting in humans. Transplant Proc. 1992; 24: 559-562. [21] Cooper, D.K.C. Depletion of natural antibodies in non-human primates--a step towards successful discordant xenografting in humans. Clin Transplant. 1992; 6: 178-183. [22] Cooper, D.K.C., Good, A.H., Koren, E., et al. Identification of -galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transpl Immunol 1993; 1: 198-205. [23] Galili, U. Evolution of alpha 1,3galactosyltransferase and of the alpha-Gal epitope. Subcell Biochem. 1999; 32: 1-23. [24] Cooper, D.K.C. Identification of alpha Gal as the major target for human anti-pig antibodies. Xenotransplantation. 2009; 16: 47-49. [25] Sandrin, M.S., Vaughan, H.A., Dabkowski, P.L., McKenzie, I.F. Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes. Proc Natl Acad Sci USA. 1993; 90: 11391-11395. [26] Oriol, R., Ye, Y., Koren, E., Cooper, D.K.C. Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplantation. 1993; 56: 1433-1442. [27] Neethling, F.A., Koren, E., Ye, Y., et al. Protection of pig kidney (PK15) cells from the cytotoxic effect of anti-pig antibodies by alpha-galactosyl oligosaccharides. Transplantation. 1994; 57: 959-963. [28] Neethling, F.A., Joziasse, D., Bovin, N., Cooper, D.K.C., Oriol R. The reducing end of alpha Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera. Transpl Int. 1996; 9: 98-101. [29] Neethling, F.A., Cooper, D.K.C. Serum cytotoxicity to pig cells and anti-alphaGal antibody level and specificity in humans and baboons. Transplantation. 1999; 67: 658-665. 52 David K. C. Cooper

[30] Cooper, D.K.C, Koren, E., Oriol, R. Genetically engineered pigs. Lancet. 1993b; 342: 682-683. [31] Thall, A.D., Maly, P., Lowe, J.B. Oocyte Gal alpha 1,3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. J Biol Chem. 1995; 270: 21437-21440. [32] Tearle, R.G., Tange, M.J., Zannettino, Z.L., et al. The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation. Transplantation. 1996; 61: 13-19. [33] Campbell, K.H., McWhir, J., Ritchie, W.A., Wilmut, I. Sheep cloned by nuclear transfer from a cultured cell line. Nature. 1996; 380: 64-66. [34] Polejaeva, I.A., Chen, S.H., Vaught, T.D., et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature. 2000; 407: 86-90. [35] Lai, L., Kolber-Simmonds, D., Park, K.W., et al. Production of alpha-1,3- galactosyltransferase knockout pigs by nuclear transfer cloning. Science. 2002; 295(5557): 1089-1092 [36] Dai, Y., Vaught, T.D., Boone, J., et al. Targeted disruption of the alpha1,3- galactosyltransferase gene in cloned pigs. Nat Biotech. 2002; 20: 251-255. [37] Phelps, C.J., Koike, C., Vaught, T.D., et al. Production of alpha 1,3- galactosyltransferase-deficient pigs. Science. 2003; 299: 411-414. [38] Kolber-Simonds, D., Lai, L., Watt, S.R., et al. Production of alpha-1,3- galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci USA. 2004;.101:.7335-7340. [39] Cooper, D.K.C., Ye, Y., Niekrasz, M. Heart transplantation in primates. In: Handbook of Animal Models in Transplantation Research. D. V. Cramer, L. G. Podesta and L. Makowka (eds). Boca Raton, CRC Press. 1994. pp.173-200. [40] Taniguchi, S., Neethling, F.A., Korchagina, E.Y., et al. In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column. Transplantation. 1996; 62: 1379-1384. [41] Cooper, D.K.C., Cairns, T.D., Taube, D.H. Extracorporeal immunoadsorption of alphaGal antibodies. Xeno. 1996;4:27-29. [42] Simon, P.M., Neethling, F.A., Taniguchi, S., et al. Intravenous infusion of Galalpha1-3Gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts. Transplantation. 1998; 65: 346-353. [43] Romano, E., Neethling, F.A., Nilsson, K., et al. Intravenous synthetic alphaGal saccharides delay hyperacute rejection following pig-to-baboon heart transplantation. Xenotransplantation. 1999; 6: 36-42. [44] Ye, Y., Neethling, F.A., Niekrasz, M., et al. Evidence that intravenously administered alpha-galactosyl carbohydrates reduce baboon serum cytotoxicity to pig kidney cells (PK15) and transplanted pig hearts. Transplantation. 1994; 58: 330-337. The Next Great Medical Revolution 53

[45] Cooper, D.K.C., Ezzelarab, M.B., Hara, H., et al. The pathobiology of pig-to- primate xenotransplantation: a historical review. Xenotransplantation. 2016; 23: 83-105. [46] Taniguchi, S., Neethling, F.A., Oriol, R., et al. Ratites (ostrich, emu) as potential heart donors for humans: immunologic considerations. Transplant Proc. 1996; 28: 561. [47] Kobayashi, T., Neethling, F.A., Taniguchi, S., et al. Investigation of the anti- complement agents, FUT-175 and K76COOH, in discordant xenotransplantation. Xenotransplantation. 1996; 3: 237-245. [48] Kobayashi, T., Taniguchi, S., Neethling, F.A., et al. Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation. 1997; 64: 1255-1261. [49] Ye, Y., Luo, Y., Kobayashi, T., et al. Secondary organ allografting after a primary "bridging" xenotransplant. Transplantation. 1995; 60: 19-22. [50] Baertschiger, R.M., Dor, F.J., Prabharasuth, D., Kuwaki K., Cooper, D.K.C. Absence of humoral and cellular alloreactivity in baboons sensitized to pig antigens. Xenotransplantation. 2004; 11: 27-32. [51] Hara, H., Ezzelarab, M., Rood, P.P., et al. Allosensitized humans are at no greater risk of humoral rejection of GT-KO pig organs than other humans. Xenotransplantation. 2006; 13: 357-365. [52] Cooper, D.K.C., Tseng, Y.L., Saidman, S.L. Alloantibody and xenoantibody cross- reactivity in transplantation. Transplantation. 2004; 77: 1-5. [53] Mieles, L., Ye, Y., Luo, Y., et al. Auxiliary liver allografting and xenografting in the nonhuman primate. Transplantation. 1995; 59: 1670-1676. [54] Starzl, T.E., Fung, J., Tzakis, A., et al. Baboon-to-human liver transplantation. Lancet. 1993; 341: 65-71. [55] Tseng, Y.L., Sachs, D.H., Cooper, D.K.C. Porcine hematopoietic progenitor cell transplantation in nonhuman primates: a review of progress. Transplantation. 2005; 79: 1-9. [56] Buhler, L., Yamada, K., Kitamura, H., et al. Pig kidney transplantation in baboons: anti-Gal(alpha)1-3Gal IgM alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation. Transplantation. 2001; 72: 1743-1752. [57] Ierino, F.L., Kozlowski, T., Siegel, J.B., et al. Disseminated intravascular coagulation in association with the delayed rejection of pig-to-baboon renal xenografts. Transplantation. 1998; 66: 1439-1450. [58] Kozlowski, T., Shimizu, A., Lambrigts, D., et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation. 1999; 67: 18-30. 54 David K. C. Cooper

[59] Robson, S.C., Cooper, D.K.C., d'Apice, A.J. Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation. 2000; 7: 166- 176. [60] Alwayn, I.P., Basker, M., Buhler, L., Cooper, D.K.C. The problem of anti-pig antibodies in pig-to-primate xenografting: current and novel methods of depletion and/or suppression of production of anti-pig antibodies. Xenotransplantation. 1999; 6: 157-168. [61] Buhler, L., Awwad, M., Basker, M., et al. High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response. Transplantation. 2000; 69: 2296-2304. [62] Knosalla, C., Yazawa, K., Behdad, A., et al. Renal and cardiac endothelial heterogeneity impact acute vascular rejection in pig-to-baboon xenotransplantation. Am J Transplant. 2009; 9: 1006-1016. [63] Kuwaki, K., Tseng, Y.L., Dor, F.J., et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med. 2005; 11: 29-31. [64] Tseng, Y.L., Kuwaki, K., Dor, F.J., et al. alpha1,3-Galactosyltransferase gene- knockout pig heart transplantation in baboons with survival approaching 6 months. Transplantation. 2005; 80: 1493-1500. [65] Yamada, K., Yazawa, K., Shimizu, A., et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med. 2005; 11: 32-34. [66] Cozzi, E., Vial, C., Ostlie, D., et al. Maintenance triple immunosuppression with cyclosporin A, mycophenolate sodium and steroids allows prolonged survival of primate recipients of hDAF porcine renal xenografts. Xenotransplantation. 2003; 10: 300-310. [67] Baldan, N., Rigotti, P., Calabrese, F., et al. Ureteral stenosis in HDAF pig-to- primate renal xenotransplantation: a phenomenon related to immunological events? Am. J. Transplant. 2004; 4: 475-481. [68] Kuwaki, K., Knosalla, C., Dor, F.J., et al. Suppression of natural and elicited antibodies in pig-to-baboon heart transplantation using a human anti-human CD154 mAb-based regimen. Am J Transplant. 2004; 4: 363-372. [69] Houser, S.L., Kuwaki, K., Knosalla, C., et al. Thrombotic microangiopathy and graft arteriopathy in pig hearts following transplantation into baboons. Xenotransplantation. 2004; 11: 416-425. [70] Shimizu, A., Yamada, K., Yamamoto, S., et al. Thrombotic microangiopathic glomerulopathy in human decay accelerating factor-transgenic swine-to-baboon kidney xenografts. J Am Soc Nephrol. 2005; 16: 2732-2745. The Next Great Medical Revolution 55

[71] Hisashi, Y., Yamada, K., Kuwaki, K., et al. Rejection of cardiac xenografts transplanted from alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pigs to baboons. Am J Transplant. 2008; 8: 2516-2526. [72] Shimizu, A., Hisashi, Y., Kuwaki, K., et al. Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from alpha1,3- galactosyltransferase gene-knockout pigs in baboons. Am J Pathol. 2008; 172: 1471-1481. [73] Shimizu, A., Yamada, K., Robson, S.C., Sachs D.H., Colvin, R.B. Pathologic characteristics of transplanted kidney xenografts. J Am Soc Nephrol. 2012; 23: 225-235. [74] Buhler, L., Basker, M., Alwayn, I.P., et al. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation. 2000; 70: 1323-1331. [75] Rood, P.P., Hara, H., Ezzelarab, M., et al. Preformed antibodies to alpha1,3- galactosyltransferase gene-knockout (GT-KO) pig cells in humans, baboons, and monkeys: implications for xenotransplantation. Transplant Proc. 2005; 37: 3514- 3515. [76] Rood, P.P., Hara, H., Busch, J.L., et al. Incidence and cytotoxicity of antibodies in cynomolgus monkeys directed to nonGal antigens, and their relevance for experimental models. Transpl Int. 2006; 19: 158-165. [77] Ezzelarab, M., Hara, H., Busch, J., et al. Antibodies directed to pig non-Gal antigens in naive and sensitized baboons. Xenotransplantation. 2006; 13: 400-407. [78] Hara, H., Long, C., Lin, Y.J., et al. In vitro investigation of pig cells for resistance to human antibody-mediated rejection. Transpl Int. 2008; 21: 1163-1174. [79] Gollackner, B., Goh, S.K., Qawi, I., et al. Acute vascular rejection of xenografts: roles of natural and elicited xenoreactive antibodies in activation of vascular endothelial cells and induction of procoagulant activity. Transplantation. 2004; 77: 1735-1741. [80] Lin, C.C., Chen, D., McVey, J.H., Cooper, D.K.C., Dorling, A. Expression of tissue factor and initiation of clotting by human platelets and monocytes after incubation with porcine endothelial cells. Transplantation. 2008; 86: 702-709. [81] Lin, C.C., Ezzelarab, M., Shapiro, R., et al. Recipient tissue factor expression is associated with consumptive coagulopathy in pig-to-primate kidney xenotransplantation. Am J Transplant. 2010; 10: 1556-1568. [82] Ezzelarab, M., Garcia, B., Azimzadeh, A., et al. The innate immune response and activation of coagulation in alpha1,3-galactosyltransferase gene-knockout xenograft recipients. Transplantation. 2009; 87:805-812. [83] Azimzadeh, A., Kelishadi, S., Ezzelarab, M., et al. Early graft failure of GTKO pig organs in baboons is reduced in hCPRP expression. Xenotransplantation. 2009; 16: 356 (Abstract). 56 David K. C. Cooper

[84] Azimzadeh, A., Kelishadi, S., Ezzelarab, M.B., et al. Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement-regulatory protein. Xenotransplantation. 2015; 22: 310-316. [85] Iwase, H., Ekser, B., Satyananda, V., et al. Pig-to-baboon heart transplantation - first experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens. Xenotransplantation. 2015; 22: 211-220. [86] Iwase, H., Liu, H., Wijkstrom, M., et al. Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date. Xenotransplantation. 2015; 22: 302-309. [87] Iwase, H., Hara, H., Ezzelarab, M., et al. Immunological and physiologic observations in baboons with life-supporting genetically-engineered pig kidney grafts. Xenotransplantation. 2017; 24: doi: 10.1111/xen.12293 [88] Mohiuddin, M.M., Singh, A.K., Corcoran, P.C., et al. Role of anti-CD40 antibody- mediated costimulation blockade on non-Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model. Xenotransplantation. 2014; 21: 35-45. [89] Mohiuddin, M.M., Singh, A.K., Corcoran, P.C., et al. One-year heterotopic cardiac xenograft survival in a pig to baboon model. Am J Transplant. 2014; 14: 488- 489. [90] Mohiuddin, M.M., Singh, A.K., Corcoran, P.C., et al. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to- primate cardiac xenograft. Nat Commun. 2016;7: 11138. PMID: 27045379 [91] Higginbotham, L., Mathews, D., Breeden, C.A., et al. Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model. Xenotransplantation. 2015; 22: 221-230. [92] Ezzelarab, M.B., Ekser, B., Azimzadeh, A., et al. Systemic inflammation in xenograft recipients precedes activation of coagulation. Xenotransplantation. 2015; 22: 32-47. [93] Iwase, H., Ekser, B., Zhou, H., et al. Further evidence for a sustained systemic inflammatory response in xenograft recipients (SIXR). Xenotransplantation. 2015; 22: 399-405. Remove bold print from [93] [94] Iwase, H., Klein, E., Cooper, D.K.C. Physiological aspects of pig kidney transplantation in primates. Comparative Medicine. 2018. In press. [95] Ekser, B., Long, C., Echeverri, G.J., et al. Impact of thrombocytopenia on survival of baboons with genetically modified pig liver transplants: clinical relevance. Am J Transplant. 2010; 10: 273-285. [96] Ekser, B., Echeverri, G.J., Hassett, A.C., et al. Hepatic function after genetically engineered pig liver transplantation in baboons. Transplantation. 2010; 90: 483- 493. The Next Great Medical Revolution 57

[97] Ekser, B., Burlak, C., Waldman, J.P., et al. Immunobiology of liver xenotransplantation. Expert Rev Clin Immunol. 2012; 8: 621-634. [98] Wilhite, T., Ezzelarab, C., Hara, H., et al. The effect of Gal expression on pig cells on the human T-cell xenoresponse. Xenotransplantation. 2012; 19: 56-63. [99] Ezzelarab, M.B., Ayares, D., Cooper, D.K.C Transgenic expression of human CD46: does it reduce the primate T-cell response to pig endothelial cells? Xenotransplantation. 2015; 22: 487-489. [100] Iwase, H., Ekser, B., Hara, H., et al. Regulation of human platelet aggregation by genetically modified pig endothelial cells and thrombin inhibition. Xenotransplantation. 2014; 21: 72-83 [101] Phelps, C.J., Ball, S.F., Vaught, T.D., et al. Production and characterization of transgenic pigs expressing porcine CTLA4-Ig. Xenotransplantation. 2009; 16: 477- 485. [102] Hara, H., Witt, W., Crossley, T., et al. Human dominant-negative class II transactivator transgenic pigs - effect on the human anti-pig T-cell immune response and immune status. Immunology. 2013; 140: 39-46. [103] Iwase, H., Ekser, B., Satyananda, V., et al. Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs. Transpl Immunol. 2015; 32: 99-108. [104] Ezzelarab, C., Ayares, D., Cooper, D.K.C., Ezzelarab, M.B. Human T-cell proliferation in response to thrombin-activated GTKO pig endothelial cells. Xenotransplantation. 2012; 19: 311-316. [105] Rood, P.P., Tai, H.C., Hara, H., et al. Late onset of development of natural anti- nonGal antibodies in infant humans and baboons: implications for xenotransplantation in infants. Transpl Int. 2007; 20: 1050-1058. [106] Dons, E.M., Montoya, C., Long, C.E., et al. T-cell-based immunosuppressive therapy inhibits the development of natural antibodies in infant baboons. Transplantation. 2012; 93: 769-776. [107] Neethling, F., Cooper, D.K.C., Xu, H., Michler, R.E. Newborn baboon serum anti- alpha galactosyl antibody levels and cytotoxicity to cultured pig kidney (PK15) cells. Transplantation. 1995; 60: 520-521. [108] Minanov, O.P., Itescu, S., Neethling, F.A., et al. Anti-GaL IgG antibodies in sera of newborn humans and baboons and its significance in pig xenotransplantation. Transplantation. 1997; 63: 182-186. [109] Buhler, L., Deng, S., O'Neil, J., et al. Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade. Xenotransplantation. 2002; 9: 3-13. [110] Rood, P.P., Bottino, R., Balamurugan, A.N., et al. Reduction of early graft loss after intraportal porcine islet transplantation in monkeys. Transplantation. 2007; 83: 202-210. 58 David K. C. Cooper

[111] van der Windt, D.J., Bottino, R., Casu. A., et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. Am J Transplant. 2009; 9: 2716-2726. [112] Bottino, R., Wijkstrom, M., van der Windt, D.J., et al. Pig-to-monkey islet xenotransplantation using multi-transgenic pigs. Am J Transplant. 2014; 14: 2275- 2287. [113] Wijkstrom, M., Bottino, R., Iwase, H., et al. Glucose metabolism in pigs expressing human genes under an insulin promoter. Xenotransplantation. 2015; 22: 70-79. [114] van der Windt, D.J., Marigliano, M., He, J., et al. Early islet damage after direct exposure of pig islets to blood: has humoral immunity been underestimated? Cell Transplant. 2012; 21: 1791-1802. [115] Nagaraju, S., Bertera, S., Tanaka, T., et al. In vitro exposure of pig neonatal islet- like cell clusters to human blood. Xenotransplantation. 2015; 22: 317-324. [116] Ezzelarab, M., Ayares, D., Cooper, D.K.C. The potential of genetically-modified pig mesenchymal stromal cells in xenotransplantation. Xenotransplantation. 2010; 17: 3-5. [117] Ezzelarab, M., Ezzelarab, C., Wilhite, T., et al. Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses. Xenotransplantation. 2011; 18: 183-195. [118] Kumar, G., Hara, H., Long, C., et al. Adipose-derived mesenchymal stromal cells from genetically modified pigs: immunogenicity and immune modulatory properties. Cytotherapy. 2012; 14: 494-504. [119] Li, J., Ezzelarab, M.B., Ayares, D., Cooper, D.K.C The potential role of genetically- modified pig mesenchymal stromal cells in xenotransplantation. Stem Cell Rev. 2014; 10: 79-85. [120] Casu, A., Bottino, R., Balamurugan, A.N., et al. Metabolic aspects of pig-to- monkey (Macaca fascicularis) islet transplantation: implications for translation into clinical practice. Diabetologia. 2008; 51: 120-129. [121] Casu, A., Echeverri, G.J., Bottino, R., et al. Insulin secretion and glucose metabolism in alpha 1,3-galactosyltransferase knock-out pigs compared to wild- type pigs. Xenotransplantation. 2010; 17: 131-139. [122] Hara, H., Cooper, D.K.C. The immunology of corneal xenotransplantation: a review of the literature. Xenotransplantation. 2010; 17: 338-349. [123] Hara, H., Cooper, D.KC. Xenotransplantation--the future of corneal transplantation? Cornea. 2011; 30: 371-378. [124] Lee, W., Miyagawa, Y., Long, C., Cooper, D.K.C., Hara, H. A comparison of three methods of decellularization of pig corneas to reduce immunogenicity. Int J Ophthalmol. 2014; 7: 587-593. The Next Great Medical Revolution 59

[125] Lee, W., Miyagawa, Y., Long, C., et al. Expression of NeuGc on pig corneas and its potential significance in pig corneal xenotransplantation. Cornea. 2016; 35: 105-113. [126] Lee, W., Mammen, A., Dhaliwal, D.K., et al. Development of retrocorneal membrane following pig-to-monkey penetrating keratoplasty. Xenotransplantation. 2017; 24(1): doi: 10.1111/xen.12276. [127] Lamm, V., Hara, H., Mammen, A., Dhaliwal, D., Cooper, D.K.C. Corneal blindness and xenotransplantation. Xenotransplantation. 2014; 21: 99-114. [128] Kim, M.K., Hara, H. Current status of corneal xenotransplantation. Int J Surg. 2015; 23: 255-260. [129] Cooper, D.K.C. Porcine red blood cells as a source of blood transfusion in humans. Xenotransplantation. 2003; 10: 384-386. [130] Cooper, D.K.C., Hara, H., Yazer, M. Genetically engineered pigs as a source for clinical red blood cell transfusion. Clin Lab Med. 2010; 30: 365-380. [131] Cooper, D.K.C. How important is the anti-Gal antibody response following the implantation of a porcine bioprosthesis? J Heart Valve Dis. 2009; 18: 671-672. [132] Manji, R.A., Menkis, A.H., Ekser, B., Cooper, D.K.C Porcine bioprosthetic heart valves: The next generation. Am Heart J. 2012; 164: 177-185. [133] Manji, R.A., Ekser, B., Menkis, A.H., Cooper, D.K.C. Bioprosthetic heart valves of the future. Xenotransplantation. 2014; 21: 1-10. [134] Bouhours, D., Pourcel, C., Bouhours, J.E. Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (Gal alpha 1-3Gal), blood group H determinant and N- glycolylneuraminic acid. Glycoconj J. 1996; 13: 947-953. [135] Zhu, A., Hurst, R. Anti-N-glycolylneuraminic acid antibodies identified in healthy human serum. Xenotransplantation. 2002; 9: 376-381. [136] Lutz, A.J., Li, P., Estrada, J.L., et al. Double knockout pigs deficient in N- glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation. 2013; 20: 27-35. [137] Byrne, G.W., Du, Z., Stalboerger, P., Kogelberg, H., McGregor, C.G. Cloning and expression of porcine beta1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen. Xenotransplantation. 2014; 21: 543-554. [138] Estrada, J.L., Martens, G., Li, P., et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes. Xenotransplantation. 2015; 22: 194-202. [139] Lee, W., Hara, H., Ezzelarab, M.B., et al. Initial in vitro studies on tissues and cells from GTKO/hCD46/NeuGcKOpigs. Xenotransplantation. 2016; 23: 137-150. [140] Cooper, D.K.C, Ekser, B., Ramsoondar, J., Phelps, C., Ayares, D. The role of genetically-engineered pigs in xenotransplantation research. J Pathol. 2016; 238: 288-299. 60 David K. C. Cooper

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[155] Cooper, D.K.C., Pierson III, R.N., Hering, B.J., et al. Regulation of clinical xenotransplantation – time for a reappraisal?. Transplantation. 2017; 102: 1766- 1769. Doi:10.1097/TP. [156] Cooper, D.K.C. Ethical aspects of xenotransplantation of current importance. Xenotransplantation 1996;3:264-274. [157] Smetanka, C., Cooper, D.K.C. The ethics debate in relation to xenotransplantation. Rev Sci Tech. 2005; 24: 335-342.

DAVID K. C. COOPER - BRIEF BIOGRAPHY

David Cooper is a Professor of Surgery in the Department of Surgery at the University of Alabama at Birmingham (UAB) in the USA. He studied medicine at Guy’s Hospital Medical School in London (now merged with King’s College London), and subsequently trained in general and cardiothoracic surgery in Cambridge and London, interrupting this training to carry out research for the PhD degree. He was present at the first heart transplant in the UK in 1968, and was a member of the team that initiated the heart transplant program at Papworth Hospital in Cambridge in 1979. Between 1972 and 1980, he was a Fellow and Director of Studies in Medicine at Magdalene College, Cambridge. In 1980 he took up an appointment in cardiac surgery at Groote Schuur Hospital at the University of Cape Town where, under Professor Christiaan Barnard (who, in 1967 had carried out the world’s first heart transplant), he had responsibility for patients undergoing heart transplantation. With Winston Wicomb and Dimitri Novitzky, he developed a hypothermic perfusion device to store donor hearts (which was used clinically), and investigated the detrimental effects of brain death on donor organs before establishing thyroid hormone therapy in the management of potential organ donors. 62 David K. C. Cooper

In 1987, he relocated to the Oklahoma Transplantation Institute in the USA where he continued to work in both the clinical and research fields. With colleagues, he identified the importance of the Gal antigen in xenotransplantation. After 17 years as a surgeon- scientist, he decided to concentrate on research, initially at the Massachusetts General Hospital/Harvard Medical School in Boston, subsequently at the Thomas E. Starzl Transplantation Institute at the University of Pittsburgh, and now at UAB. His major interest is the xenotransplantation of organs, islets, and corneas in pig-to-nonhuman primate models, with the aim of using genetically-engineered pigs as a solution to the organ shortage for clinical transplantation. The Royal College of Surgeons of England appointed him to a Hunterian Professorship (1983), Arris and Gale Lectureship (1988), Amott Lectureship (2002), and awarded him the 1997 Jacksonian Prize and Medal. Professor Cooper has published almost 900 medical and scientific papers and chapters, has authored or edited 11 books, and has given more than 300 invited presentations worldwide. Between 1994-1997, he was Honorary Secretary/Treasurer of the International Society for Heart and Lung Transplantation. He was the Founding Honorary Secretary of the International Xenotransplantation Association IXA) in 1997, its President in 1999- 2001, and the Editor-in-Chief of Xenotransplantation from 2001-2007. In 2009, he was elected to Honorary Membership of the IXA. He has received several awards for his research, and in 2016 he was made a Fellow of his alma mater, King’s College London.