Thank you for asking me to be part of your project on thoughts from the ancient modern history of xenotransplantation. Before I start, I ask your forgiveness for what are sure to be inaccuracies in my recall of detail and timing of events and my sketchy knowledge of events since my retirement 10 years ago.

I became involved in the late 80’s early 90’s when I moved from managing a clinical renal transplant service to a Clinical Immunology Department and wanted to continue my research interest in transplantation but without patients. At that time xeno was largely a surgical endeavour involving primates and it was unable to make any meaningful progress despite several hubristic surgeons who were willing, nay enthusiastic, to actually transplant a few unfortunate patients. (Think here of the litany of setbacks to progress of the field: #1: early surgical hubris).

David White, then of Cambridge, made the first really meaningful advance by showing that the complement cascade regulator Decay Accelerating Factor or DAF, could inhibit various in vitro models of hyperacute rejection (HAR) in the pig-human combination. To my mind this was beginning the modern era of xeno research. David and co went on to generate DAF transgenics and showed some inhibition of HAR and early graft survival. He was so enthusiastic that he stood on the stage at the Rome Transplantation Society meeting and called for a move to the clinic (#2: early scientific hubris). Despite the Pope who came to the meeting and addressed us in the hot sun, inter alia supporting xeno, there was no enthusiasm for immediate clinical application because the preclinical data did not support it. On its own, inhibition of the complement cascade was not sufficient because xenoantibodies continued to cause havoc.

Initially two scientific groups tackled antibody. David Cooper’s and our group showed by different routes that Gal was the main target. However, Uri Galili in a completely separate literature had already shown that Gal was the major target of human anti-pig antibodies. This prompted a series of efforts to remove the Gal target. The main effort was to inactivate the a1,3 galactosyltransferase gene in the pig. The other from Ian McKenzie’s group was to compete for the enzyme’s substrate using transgenic expression of human H-transferase. This was at least partially and often very effective but the appeal of elimination was greater. In those days, gene KO was performed in embryonic stem cells which were then used to generate hybrid blastocysts and finally hybrid animals which could then be bred on to become full KO animals. The only problem was that pig ES cells did not exist and we spent several years unsuccessfully trying to generate them. It was only when Dolly the cloned sheep came along that a technique that worked in pigs was found and there was a race to produce GalKO pigs. Our group won the patent race but not the actual race to first on the ground and within a couple of years at least 5 groups had made GalKO pigs. This was the second major advance toward clinical xenotransplantation. The GalKO and complement regulator transgenics were soon interbred and hyperacute rejection was overcome.

The next and overlapping era was that of the companies, the likes of Sandoz, Baxter and so on who had an interest in either immunosuppression or dialysis and so a position to protect. Their hope was a rapid translation to the clinic and / or to protect existing stakes such as dialysis, and they invested heavily in the various groups. The companies were very rough bedfellows and we were naïve virgins (setback #3). However, when the hope (or threat) of rapid clinical exploitation faded, they lost interest and the survivors went back to academic funding sore but experienced.

The cause of this fading of hope was two-fold, one from each side of the equation that needed solution for clinical application to be realistic. First, efficacy had to be shown. While there were plenty of signs of progress and indicators of imminent success in preclinical models, it was like reading a horror story. With each new page there was a new barrier, a new failure and new problem that required another genetic modification for solution. This is still the problem today with the two major clinical applications that were the main focus at that time; renal and cardiac transplantation – both solid organs. The focus was yet to seriously turn to pancreatic islet cells. The companies probably could have coped with this but when the other side of the equation raised its head, the bell started to toll. Safety had been discussed widely, pigs were dirty but could be cleaned up and isolated in expensive barrier facilities and the companies threw money at the problem and built them. But then came the stain that could not (at that time) be scrubbed clean: porcine endogenous retrovirus (setback #4). The horror was compounded by the fact that it was a retrovirus (think AIDS) and that one of the most prominent medical (and xeno) researchers of the time, Fritz Bach of Harvard, rose up to his full height and declared xenotransplantation to be dead (setback 4.1 – God had spoken, even the Pope’s prior blessing was now null and void). This was met with incredulity by xeno researchers many of the most prominent of whom were his friends and colleagues at Harvard Medical School. Bach’s intervention was denounced as treachery (and nest fouling) and he was until his death persona non grata to many around him. The companies were faced with efficacy still needing more money and an unknown amount more time and now a seeming insoluble problem with safety. They backed out of funding but still held tight to their grips on our IP via our patents…..just in case we were able to make something work. We bound up our wounds, took antibiotics and slowly recovered, finding some dignity and sufficient dollars in old fashioned academic funding. Interestingly the (continuing) progress of xeno to the clinic has been so slow that all the primary patents that these companies coveted so much are all now time expired, like me.

When I retired 10 years ago, PERV was the only significant known barrier to safety of xeno that could not be managed. At that time, and correct me if I’m out of date, as far as I know there is still no direct evidence of a human being infected with PERV let alone it causing any illness. So, all the concern is based on a theoretical possibility. However, I understand that a US company has edited out all the PERV sequences along with Gal and other transferases and inserted many complement and other regulators. This holds great promise for xeno. However, a minor word against premature adulation (potential setback #5). Building a stable pig herd for clinical use from a founder can be very difficult because of inbreeding issues, as hybrid vigour and fecundity suffer. Old-fashioned pig husbandry is still relevant.

I wish you all good luck and patience in what I hope are the last stages (of still indeterminate duration) before clinical application of xeno is a reality. The virtues in xeno are patience, constancy, self-reliance, doggedness, resilience, avoidance of hubris (and companies), a healthy dose of cynicism, self-deprecation, humility, humour and excellent science to justify academic funding. If anyone has these virtues, I have yet to meet her.

Because of the fear in some people’s minds that the COVID pandemic will permanently halt progress in xenotransplantation research due to concerns that a pig virus will be transferred to a human recipient of an organ or cell xenograft, I have been asked to comment on what episodes caused me worry or concern during the past more than 35 years of involvement in this field of research.

Let me state immediately that, although the shutdown associated with the COVID pandemic has certainly slowed my own group’s progress towards the clinic, I do not have any concerns that it will permanently prohibit clinical xenotransplantation. My reasons for believing this were published recently in a letter to the editor of ‘Xenotransplantation’, written in collaboration with Michael Knoll and Rita Bottino (Knoll 2020). Similarly, none of the previous ‘scares’ relating to potential infection, e.g., PERV, MERS, SARS, etc., shook my belief that xenotransplantation would go ahead as soon as we had solved the immunological problems that face us.

What has been of much more concern to me over the years has been the difficulty in obtaining sufficient funding for the expensive studies necessary for us to move forward. In the early days of xenotransplantation research, it was even more difficult than it is today to persuade the funding agencies to support xenotransplantation research. A grant application would be reviewed without major criticisms by a reviewer, but he/she would end their critique by asking, ‘But is it ever going to happen?’ and it would not be funded. They were correct, of course, to ask this question – it certainly will not happen if there is no financial support. The reviewer was obviously someone with either no vision or, more likely, a quite different vision for the future.

Nevertheless, one or two research groups managed to obtain considerable financial support from major companies. For example, in the naïve belief that a pig that expressed a single human complement-regulatory protein, CD55 (DAF), combined with cyclosporine therapy, would solve all of the problems and allow us to proceed immediately to clinical trials, Novartis reputedly invested hundreds of millions of dollars into the work of the UK biotech company, Imutran. Clearly, not enough enquiry had been made into the state of the science before this investment was made, suggesting that some business executives should not have been in the high positions they held in the pharmaceutical company.

Imutran’s first transgenic pig was a major step forward, and if Novartis has backed the company with perhaps $10 million per year for a decade or more, steady progress would have been made by Imutran’s excellent team of scientists, and Novartis might now be close to reaping the financial rewards of its modest investment.

When the Novartis executives realized they had been overly-optimistic, coupled with their unjustified fear of PERV, they shutdown Imutran, and killed most of the remaining transgenic pigs – in my opinion, a second foolish business decision, compounding the first.

Similarly, at Tom Starzl’s urging, the University of Pittsburgh Medical Center (UPMC), the company that owns many of the hospitals in the Pittsburgh area, purchased the US pig biotech company, Revivicor, in 2004. When progress was slower than they had been led to anticipate, they withdrew their support precipitately. Revivicor was only days away from closure when saved by United Therapeutics in 2011.

Just as with Novartis, if UPMC had been patient enough to invest longer-term, it would today be in a position to soon dominate the field of transplantation. Given the immense wealth of both Novartis and UPMC, the necessary annual investments would have been ‘peanuts’ to them.

Fortunately, in 2005, the US NIH’s National Institute of Allergy and Infectious Diseases (NIAID) decided to support xenotransplantation research, enabling several groups to continue to make progress, which has been life-saving for the field.

I was never seduced by the lure of alternative technologies that were introduced during the past 30 years. For example, several gifted researchers were lured away from xenotransplantation by the perceived potential of stem cell technology. I was immensely skeptical that we would be able to produce a functioning kidney, heart, or liver from stem cells, at least not in my lifetime. With pigs, we already had organs that we knew would likely function well in humans. To achieve similar success from a stem cell seemed to me like the height of optimism. I had the same response to regenerative medicine, where we were promised that the decellularization of an unacceptable human organ or a pig organ, and its recellularization with cells from the potential specific recipient of the organ was going to be achievable. To me, this seemed unlikely within the next 50 years. And so, after careful consideration, and an unshakeable belief in the huge potential of xenotransplantation, it was easy for me to resist the attractions of a different field of research.

The ‘lows’ faced by the crises of funding were offset by the ‘highs’ experienced by the advances that have taken place over many years, e.g., the ever-improving techniques of genetic-engineering, the identification of the pig antigens against which humans have natural preformed antibodies, the introduction of agents that block the CD40/CD154 costimulation pathway, the identification and resolution of the dysfunctional coagulation and inflammatory responses that result from the presence of a pig graft, and, particularly, by the milestones achieved – the first islet transplant to maintain normoglycemia in a diabetic monkey for more than a year, the first pig kidney to function for more than 3 months, and then for 6 months, and even a year, the first heterotopic heart to function for a year, and the first orthotopic heart to function for 6 months. These highlights kept us believing in ultimate success.

There have been many disappointments, of course. Some of the genetic manipulations have been detrimental to the pig or have not had the anticipated effect on graft survival. At times, novel immunosuppressive regimens have proved inadequate. Some of these failures have been associated with our own lack of knowledge or forethought. Although some mistakes could possibly have been avoided by more careful planning, others were just part of a learning curve.

I once asked Norman Shumway, the major pioneer in the field of heart allotransplantation, whether he ever considered abandoning his research. He replied, “Not really. There was always just enough success - just enough gratification, if you will - that you could see that it probably would ultimately work and, if everybody kept working, we might get someplace.” That is exactly how I have felt about xenotransplantation.

However, Shumway worked in the laboratory for less than a decade before he was able to move into the clinic. We have had to struggle for very much longer, and still we are not quite ready to initiate a clinical trial. Nevertheless, it has been a very interesting and exciting experience, and I remain certain we will soon get there.

“We are at our very best, and we are happiest, when we are fully engaged in work we enjoy on the journey toward the goal we’ve established for ourselves.” Earl Nightingale.

Reference

Knoll MF, Cooper DKC, Bottino R. How the COVID-19 pandemic may impact public support for clinical xenotransplantation in the United States. Xenotransplantation. 2020; Jun 28:e12623.

In a recent book on Xenotransplantation edited by Pr. D Cooper (Recollections of Pioneers in Xenotransplantation Research), I was invited to write a chapter on our (modest) contribution in this field. This invitation gives me the opportunity to take some distance to the daily “battle field” of research in xenotransplantation, decades after my first apparition -using “Xenotransplantation” as a key word- on PubMed, to portrait what could be the actual place of xenotransplantation in clinic in the future. In my contribution entitled “A journey in xenotransplantation science, from Mélies illumination to medical wisdom”, despite the recent preclinical achievement in preclinical primate models and all amazing possible progresses offered by new technologies (Perv free donors, reduction of pig tissue immunogenicity, chimeric animals hosting human tissues, etc.), I confessed I still had difficulties to envision xenotransplantation of vascularized organs to enter the clinic for replacing allograft long term purposes, mostly due to the tremendous immunological barrier that xenotransplantation is facing. Rather, I suggested that more attention could be paid to the less mediatic approach of using animal engineered tissues (as “biological” heart valves) or animal molecules (as it has been the case for decades for insulin and more recently for monoclonal or polyclonal antibodies) now widely used in clinic. Moreover, the success of such approach initially based on a pragmatic behavior, now benefits of the developments of the xenotransplantation science mentioned above.

The COVID-19 pandemic of course questions researchers engaged in the field of xenotransplantation. The major concern, actually inherent to the topic itself, is the safety issue regarding animal derived viral (Perv) or molecular (“Mad cow”) risks, a concern that has actually triggered a schism in our community, particularly following Fritz Bach ethical warning on the risk of introduction of xenotransplantation in the clinic. A major benefit of this crisis has been the extremely active policy of regulatory offices of states and academic institutions (and particularly of IXA) facing this potential risks. In this respect, decades long discipline of all the research community working on xenotransplantation has been remarkable. Interestingly, in the same time, the research efforts spurred by xenotransplantation challenge itself – such as for the progress in the genetic engineering of large animals, have been able in few years to solve the major identified alarm that triggered the debate of “ethic in xenotransplantation”: the risk related to Perv.

However, the COVID-19 pandemic, as past pandemics of severe outbreaks from animal viruses, occurred in uncontrolled arena which differ from the arena of laboratories working on “xenotransplantation science”. Moreover, the perspective of “soft xenotransplantation” I defended in the book edited by D. Cooper is highlighted by observations directly issued from research in the field of “xenotransplantation science” and which may provide powerful tools to combat COVID-19 disease. As an example IgGs anti- CoV-2 Spike able to neutralize interaction of CoV-2 with their cellular ACE2 receptor present in COVID-19 convalescent plasma are a promising therapeutic tool to prevent severe COVID-19 disease. Interestingly, whereas a large scale production of such neutralizing anti CoV-2 human antibodies may be challenging, animals engineered for producing low immunogenicity polyclonal IgGs (as done by alteration of genes coding for the synthesis of aGal or Neu5Gc xeno antigens in pigs, or by introduction of genes encoding human IgG in the bovine genome) can be utilized to prepare large amounts of neutralizing polyclonal antibodies for passive therapy of the disease. Moreover, we have recently shown that disparate evolution constraints have resulted in a lack of physical and functional interaction between pig IgGs and human cellular Fc-Receptors (1). This absence of pig IgG binding to human Fc-R should prevent the risk of CD-16 stimulation of patient lung macrophages by pig anti-CoV IgGs at a critical stage of the disease which, in the context of a passive humoral therapy, would represent a serious safety hazard. Another example of “xenotransplantation science” derived research is a monoclonal inhibiting the C5a component resulting from the human complement activation cascade which may also decrease the uncontrolled inflammation of the innate immune response in COVID-19 disease. Thus, facing the devastating consequences of the COVID-19 pandemic on human health, economy, social disparities, research institutions and in fact almost all components of our societies, “xenotransplantation science” may modestly offer opportunities to prevent or combat the severe forms of the disease using targeted strategies. Furthermore the pandemic may also contribute to curve the future of xenotransplantation toward more “soft” clinical applications.

Preclinical cardiac orthotopic xenotransplantation experiments during the COVID19-pandemic: hygienic measures