Xenogeneic organ transplantation

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Xenogeneic organ transplantation Stephen P Squinto Over the past few years, several major advances have occurred in the understanding of how the humoral and cellular immune system of humans recognizes and destroys transplanted cells, tissues and organs derived from animal sources. Consequently, armed with this new knowledge, several laboratories have now developed novel immunoprotective technologies that may allow xenotransplantation to be clinically feasible.

World primate model systems. If successful, the clinical transplantation of these engineered xenogeneic organs into humans can be anticipated. In this review, I will discuss the major advances that have occurred in the understanding of how the human immune system recognizes and destroys transplanted cells, tissues and organs derived from animal sources. The clinical need for organs

Address Alexion Pharmaceuticals Inc, 25 Science Park, New Haven, CT 06511, USA Current Opinion in Biotechnology 1996, 7:641-645 © Current Biology Ltd ISSN 0958-1669 Abbreviations DAF Gal gal-transferase GPI HAR H-transferase

decay accelerating factor galactose a1,3-galactosyl transferase glycophosphatidyl inositol hyperacute rejection c~1,2-fucosyltransferase

Introduction

T h e serious shortage of human organs available for transplantation has created an intense search for alternative sources of suitable organs. Recent attention to the topic of animal-to-human transplantation (xenotransplantation) has lead to public awareness through newspaper articles, television exposure, and news-format magazines. Public opinion surveys have revealed that individuals, when confronted with the potential loss of their own life, would generally accept a xenogeneic organ. Several groups have therefore recently developed rational strategies to address the severe organ-shortage problem that center around the development of genetically engineered transgenic animals as organ donors. Among the species considered as potential donors of xenogeneic organs, the major interest has focused on the pig. This is due to several advantageous qualities, including large litter size, age of sexual maturity, disease resistance, domestication, and, most importantly, striking similarities between swine and human organ size and physiology [1"]. Also, unlike primates, pigs are generally agreed to be an ethically acceptable alternative donor organ source [2,3]. T h e past two years have seen the development of transgenic pigs engineered to express human genes aimed at eliminating the human immune response to the foreign xenograft. T h e overall goal of this approach has been to eventually achieve the long-term survival of genetically engineered pig organs following transplantation into Old

Transplantation is clearly the treatment of choice for most patients suffering from end-stage organ failure. Significant progress in clinical allotransplantation has been achieved during the past decade. These pharmacological and surgical achievements have resulted in higher survival rates for the organ grafts as well as improved patient survival and quality of life. Additionally, organ transplantation provides significant cost savings for the health care system in the treatment of patients with severe and debilitating diseases. For example, it has been projected that the broader use of kidney transplantation instead of dialysis alone could save the Federal Government $2 billion annually. T h e profound medical benefits as well as the potential cost savings and the public benefit of the advances made in allogeneic transplantation have been severely limited, however, by a persistent and serious problem - - a severe shortage of donor organs. Many initiatives have been introduced to increase the donor organ supply, including publicity campaigns, distribution of donor cards, appointment of hospital-based transplant coordinators, implementation of procurement protocols, and educational programs for hospital personnel. Although these factors have helped to increase donor availability, they have not been sufficient to significantly increase donor supply. As a result, most patients who would benefit from organ transplantation do not even appear on a waiting list. T h e current need for organs (including kidney, heart, lung, and pancreas) is conservatively estimated to be at least 90000 per year in the United States alone [4]. The potential healthcare xenotransplantation

cost/benefit

of

An important consideration in the healthcare arena is the subject of healthcare costs. In any new therapeutic approach, a positive cost/benefit calculation is required. For clinical xenotransplantation, important efficiencies and reductions in certain costs should also be achievable. For patients with diseases in which organ dysfunction is acutely life-threatening, organ transplantation offers the opportunity to resume a high-quality, productive lifestyle with continued contribution to the overall economic growth of the nation. Patients with diseases that are

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severely debilitating but not acutely life-threatening, currently consume excessive amounts of healthcare dollars over a prolonged period of time on maintenance therapies. Transplantation, in contrast, reduces healthcare expenditures. An example is the End Stage Renal Disease (ESRD) program on which an estimated $6billion of Federal monies were spent in 1992 [5]. T h e vast majority of these expenditures fall on the delivery of higher cost, lower quality of life care to patients on maintenance dialysis. The cost per quality-adjusted life year of kidney transplantation is 75% less than the cost of dialysis. It has been estimated that the broader implementation of renal transplantation, instead of dialysis, could save the Federal government in this program as much as $2billion annually [5]. In addition to the cost savings derived from the enhanced availability of donor organs that would allow transplant procedures to replace more costly modes of therapy, the production of standardized genetically engineered organ grafts will further reduce healthcare costs by making the entire process of organ procurement and distribution much more efficient. As of 1985, costs due to organ procurement accounted for more than 40% of the $40 000-$130 000 costs associated with organ transplantation. A standardized genetically engineered organ that could be made available whenever a patient required transplantation would eliminate such costly activities as maintaining a human donor on life support prior to removing the organ, transportation of the organ to the recipient often via charter aircraft, and hospitalization of recipients for extended periods while awaiting the availability of an appropriate organ. T h e replacement of the current organ procurement system with a more efficient and coordinated system involving standardized manufacturing, production planning, centralized distribution and inventory control as anticipated by the program will reduce these estimated acquisition costs substantially. In addition to contributing to the economic benefits derived from reduced acquisition costs, the move to regionalization of organ transplantation enabled by the program will yield additional savings in healthcare expenditures. Studies have demonstrated that the aggregate increase in the volume of transplant procedures at a single center could reduce transplant costs by as much as 70% [5]. In addition to the savings generated by this 'learning experience', it is anticipated that additional savings will also derive from more standard 'economies of scale' inherent in the program that have been observed in other industries in which volume is increased.

Pig-to-primate transplantation: the immunological barrier T h e major barrier to xenogeneic transplantation is the phenomenon of hyperacute rejection (HAR). Studies of the immunological response to xenotransplanted tissue have shown that these transplants inexorably fail due to HAR [6,7]. T h e hyperacute response is triggered by the deposition of preformed recipient antibodies

on the surface of the donor endothelium resulting in the subsequent activation of the classical pathway of the complement system [6]. Therefore, following the transplantation of a vascularized xenogeneic donor pig organ into a primate recipient, the massive inflammatory response that ensues from natural antibody activation of the classical complement cascade leads to the activation and destruction of the vascular endothelial cells and ultimately of the donor organ within minutes to hours after revascularization. Old World primates, including humans, have high levels of pre-existing circulating 'natural' antibodies that predominantly recognize carbohydrate determinants expressed on the surface of xenogeneic cells from discordant species [8]. Recent evidence indicates that most, if not all, of these antibodies react with the carbohydrate epitope Gal~l--+3Gal (Gal, galactose) [9,10], an epitope absent from Old World primates because of the lack of the functional cO,3-galactosyltransfetase enzyme [11,12]. T h e levels of these naturally circulating anti-Galal--+3Gal antibodies are approximately 1% of all the antibodies in any given individual [9]. T h e high titer of these antibodies is believed to arise from constant stimulation of the immune system to this epitope through naturally occurring infections and stimulation from the natural flora and fauna in the gastro-intestinal tract. In studies of in vivo animal model systems aimed at assessing HAR, 15 minutes after revascularization, xenoreactive antibodies are deposited on the surface of the donor endothelium [6]. T h e result of antibody deposition is the subsequent activation of serum complement proteins. Activation of the complement cascade results in the deposition of complement at the site of antibody reactivity, that is, the xenogeneic vascular endothelium. Following complement activation, there is significant aggregation and adhesion of platelets to the damaged endothelium as well as the formation of microthrombi and the migration of neutrophils and granulocytes into the interstitium [6]. Ultimately, endothelial cells are destroyed, which results in tissue ischemia and necrosis of the donor organ. Additional detrimental effects of antibody deposition include the cellular antibody-mediated effects that arise from Fc receptor-bearing cells interacting with deposited antibodies, resulting in acute vasculitis and vessel occlusion. Therefore, any strategy aimed at the successful transplantation of xenografts into humans must aim to abrogate the binding of preformed human antibodies to the xenograft as well as provide the xenograft with protection against activated human complement proteins.

Solutions to the immunological challenge The problem of natural antibody reactivity and subsequent complement activation that occurs during xenogeneic organ transplantation in the pig-to-primate model has prompted the recent development of several strategies aimed at preventing natural anti-Gakxl--+3Gal antibody

Xenogeneic organ transplantation Squinto

reactivity and inhibiting human complement. Most of these efforts to prevent HAR have focused on modifying the immune system of the host. For example, strategies for prolonging discordant xenograft survival have included depletion of the naturally occurring xenoreactive antibodies with plasma exchange, plasmapheresis or donor organ absorption, and inhibition of complement with either cobra venom factor or soluble complement receptor type 1 [13,14]. Other groups are proposing to treat patients with suprapharmacologic doses of antibody-binding carbohydrates potentially in combination with splenectomy [15]. These approaches to block antibody-induced HAR have the following critical drawbacks and, therefore, make them clinically unattractive: they only transiently reduce antibody titer; they do not prevent rebound production of organ-binding antibodies; they result in globally decreased and compromised immune function; and they could potentially result in increased susceptibility of the host to infection. Rather than developing systemic immunosuppressive approaches to abrogating HAR, my colleagues and I and others [16,17°,18 °] have chosen to take advantage of an opportunity unique to xenotransplantation, that is, the ability to genetically modify the donor organ. One such strategy employed by several groups has been to achieve high-level expression of species-restricted human complement inhibitor proteins in vascularized pig organs via transgenic engineering [16,17°,18°]. In vitro studies on the consequences of antibody-depen-

dent complement activation at the surface of the vascular cells have demonstrated that deposition of the terminal complement proteins C5b-9 directly stimulate cell activation as well as procoagulant and proinflammatory responses by activating pathways that are normally coupled to receptor-initiated signal transduction mechanisms [19-21]. These include the intracellular influx of extracellular calcium and the activation of protein kinase C. In human endothelial cells, these signal transduction events are accompanied by the increased secretion of von Willebrand factor, the increased surface expression of GMP-140, and the increased vesiculation of the endothelial cell membrane exposing binding sites for coagulation factor Va, leading to increased membrane-catalyzed prothrombinase activity. Importantly, all of these responses were dependent on the presence of the terminal complement component C9 and did not occur when complement formation was activated through the formation of C5b-8 only [21]. These results strongly suggest that the complete assembly of the membrane attack complex (C5b-9) is required for complement-induced cell activation and lysis. Studies on the susceptibility of non-primate cells to complement-mediated lysis have shown that these cells are readily lysed by human complement even though they are generally resistant to lysis by complement derived from a homologous source. This phenomenon,

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termed homologous species restriction of complementmediated lysis, extends to human cells as well and several experiments have revealed that species-selective membrane factors exist that serve to protect cells from homologous complement-mediated damage [19]. A plasma membrane constituent reported to exhibit this speciesrestricted complement-inhibitory activity is the leukocyte antigen CD59 [19]. CD59 is a membrane glycoprotein of 18-21 kDa that is tethered to the plasma membrane by a glycophosphatidyl inositol (GPI) anchor. Several groups have identified cDNA clones for human CD59, have successfully expressed human CD59 in nonhuman cells and have demonstrated that these cells are protected from lysis by human complement but are not protected from lysis by guinea pig complement [22,23]. These in vitro studies have confirmed the C5b-9 inhibitory activity and species restriction of recombinant human CD59. These studies also have suggested that the expression of human CD59 in a heterologous transgenic animal could protect the cells, tissues and organs from hyperacute rejection mediated by human complement. My colleagues and I and others have demonstrated expression of human CD59 in several tissues of a transgenic pig, including the vascular endothelium, and showed that cells from this animal are resistant to complement-mediated cell lysis in vitro [16,17°,18°]. Recently, we have conducted experiments that demonstrate the efficacy of high-level CD59 vascular expression in the transgenic organ when the organ is perfused ex vivo with human blood [24] and transplanted orthotopically into Old World primates. Although prolonged survival was observed, incomplete protection resulted in the eventual rejection of the organ within hours to days after transplantation. Nontransgenic control pig organs were typically hyperacutely rejected within 1-2 h after transplantation into Old World primates. As observed in these experiments and also with systemic complement depletion, organ failure appears to be related to acute antibody-dependent vasculitis. Another engineering strategy that has been proposed aims to eliminate the Gal~l---~3Gal epitope from the donor xenograft. However, unlike the successful expression of human complement inhibitor proteins in transgenic pigs, this strategy has been largely theoretical to date. That is, the generation of Galc~l---~3Gal negative pigs might be accomplished by disrupting the porcine gal-transferase gene via homologous recombination. However, this approach requires the availability of porcine embryonic stem cells, which makes it currently impractical. My colleagues and I chose to develop a strategy to down-regulate the expression of the Gal~l---~3Gal epitope in porcine xenografts that would have a practical application toward the generation of Galal---~3Gal-deficient transgenic pigs. Our strategy takes advantage of intracellular competition between the c~l,3-galactosyl transferase (gal-transferase) and the ~l,2-fucosyltransferase

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(H-transferase) [25"]. We demonstrated that these enzymes can be coexpressed within a porcine cell and transgenically in mouse tissues and organs and that the H-transferase is dominant over the gal-transferase. T h e result of this enzyme competition is a remodeling of carbohydrate structures on the cell surface of the xenograft such that the non-immunogenic H-epitope (the product of the H-transferase reaction) substitutes for the expression of the xenoreactive Galccl---~3Gal epitope (the product of the gal-transferase). The H-transferase carbohydrate product, the H-epitope, corresponds to the universal donor blood group type O blood group antigen. Therefore, our engineering strategy serves two purposes: the elimination of the predominant xenoreactive epitope and the concomitant replacement with a universally accepted antigen. Consequently, xenografts expressing the H-epitope in place of the Galal---~3Gal epitope are no longer targets for human natural antibody binding and are significantly resistant to HAR. We have recently generated H-transferase-containing pigs and are breeding them to complement inhibitor-expressing transgenic pigs to achieve immunoprotected xenogeneic organs.

In addition to developing a practical approach to downregulating the xenoreactive carbohydrate residues on the porcine graft, my colleagues and I have also developed a novel and robust approach to blocking human complement attack on xenograft. Our data and the results of others have suggested that long-term xenograft survival will most likely require both the down-regulation of the Galc~l---)3Gal residue as well as the expression of multiple human complement inhibitor proteins. T h e technical difficulties associated with engineering multiple complement inhibitors to be expressed on the vascular endothelium of a transgenic animal are substantial. To overcome this problem, we have utilized genetic engineering approaches to develop a potent bifunctional novel complement inhibitor, termed DC, to block complement at two distinct points in the complement cascade. T h e DC molecule contains functional domains from DAF (decay accelerating factor), a C3/C5 convertase inhibitor, and CD59, a terminal complement inhibitor [26]. In vitro experiments demonstrate that the DC molecule inhibits complementmediated cell lysis when challenged with human serum and that it retains both DAF and CD59 function. T h e development of the DC chimeric complement inhibitor and subsequent demonstration that it retains both the activity of the terminal complement inhibitor CD59 and the activity of the human C3 convertase inhibitor DAF (CD55) permits the development of a single transgenic animal which would otherwise require a double transgenic expressing two complement inhibitor genes in the appropriate tissues at sufficiently high levels. We have recently been successful in generating transgenic mice that express high levels of the DC inhibitor. The generation of DC transgenic pigs is in progress.

Achieving high-level cell-surface expression of the DC bifunctional complement inhibitor and high-level expression of the H-transferase enzyme in the vascular endothelium of the transgenic pig organs will effectively eliminate both the antibody and complement components of the massive inflammatory response to the xenograft. T h e combination of the novel strategies discussed here provides a rational approach to the elimination of HAR and represents a critical first step toward making animal organ xenotransplantation a viable alternative to genetically matched human organ allotransplantation. Conclusions

T h e major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of HAR, which results from the deposition of high-titer preformed antibodies that activate serum complement on the lumenal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Reviewed here are newly developed strategies to overcome HAR in the pig-to-primate transplant setting, which include the genetic incorporation into transgenic organs and high-level expression of both a novel human bifunctional complement inhibitor and a human blood group enzyme. T h e expression of the human blood group enzyme is designed to significantly reduce the natural antibody reactivity to the discordant pig tissue whereas the expression of the complement inhibitor will result in inhibition of complement-mediated cell activation and lysis. High-level cell-surface expression of the complement inhibitor and high-level expression of the human blood group enzyme in vascular endothelium will effectively eliminate both the antibody and complement components of the massive inflammatory response to the xenogeneic tissue. Elimination of HAR will establish inroads into understanding the cellular immune response towards the discordant tissue. Hypothetically, it is quite conceivable that standard immunosuppressive regimens that are routinely practised with allotransplantation will also be effective drug therapies for xenotransplantation. Therefore, it is critical to develop a system that tests these possibilities in order to solve an ever-growing need for donor organs.

References

and

recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: • *•

of special interest of outstanding interest

1. Cozzi E, White DJG: The generation of transgenic pigs as • potential organ donors for humans. Nat Med 1995, 1:964-966. Cozzi and White describe a rather significant effort to generate transgenic pigs that express sufficiently high levels of the human complement inhibitor, CD55 or DAF. Detailed analysis of the transgenic effort to screen large

Xenogeneic organ transplantation Squinto

numbers of founder animals from which a herd is derived is described. This is the first report demonstrating the efficiency with which transgenic pigs expressing high levels of DAF can be achieved. 2.

Nuffield Council on Bioethics: Animal-to-human transplants: the ethics of xenotransplantation. London: Nuffield Council on Bioethics; 1996.

3.

The Committee on Xenograft Transplantation: Xenotransplantation: Science, Ethics, and Public Policy. Washington DC: Institute of Medicine, National Academy Press; 1996.

4.

The United Network for Organ Sharing: Scientific Research Report. Richmond, VA: United Network for Organ Sharing; 1995. Woods JL: Health Services Research. Washington, DC: United States Public Health Service; 1992:219-238.

20.

Hamilton KK, Hattori R, Esmon CT, Sims PJ: Complement proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex. J Biol Chem 1990, 265:3809-3813.

21.

Galili U, Clark MR, Shohet SB, Buehler J, Macher BA: Evolutionary relationship between the natural anti-Gal antibody and the Galocl -->3Gal epitope in primates. Proc Natl Acad Sci USA 1987, 84:1369-1373.

Hattori R, Hamilton KK, McEver RP, Sims PJ: Complement proteins C5b-9 induce secretion of high molecular weight multimers of von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface. J Biol Chem 1989, 264:9053-9060.

22.

Sandrin MS, Vaughan HA, Dabkoski PL, McKenzie IFC: Anti-pig IgM antibodies in human serum reacts predominately with the Gal((xl,3)Gal epitopes. Proc Natl Acad Sci USA 1993, 90:11391-11395.

Zhao J, Rollins SA, Maher SE, Bothwell ALM, Sims PJ: Amplified gene expression in CD59-transfected Chinese hamster ovary cells confers protection against the membrane attack complex of human complement. J Biol Chem 1991, 266:13418-13422.

23.

Larsen RD, Riverra-Marrero CA, Ernst LK, Cummings RD, Lowe JB: Frameshift and non-sense mutations in a human genomic sequence homologous to a murine UDP-GahBGall,4-D-GIcNAcc~l,3-galactosyl-transferase cDNA. J Bio/ Chern 1990, 265:7055-7061.

Kennedy SP, Rollins SA, Burton WV, Sims PJ, Bothwell ALM, Squinto SP, Zavoico GG: Protection of porcine aortic endothelial cells from complement-mediated cell lysis and activation by recombinant human CD59. Transplantation 1994, 57:1494-1501.

24.

Kroshus TJ, Bolman RM III, Dalmasso AP, Rollins SA, Williams Squinto SP, Fodor WL: Expression of human CD59 in transgenic pig organs enhances survival in an ex vivo xenogeneic perfusion model. Transplantation 1996, 61:1513-1521.

Oriol R, Ye ¥, Koren E, Cooper DKC: Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute rejection in pig-to-man xenotransplantation. Transplantation 1993, 56:1433-1442.

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12.

Sandrin MS, Vaughan HA, McKenzie IFC: Identification of Gal(x(1-->3)Gal as the major epitope for pig-to-human vascularized xenografts. Transplant Rev 1994, 8:134-149.

13.

Leventhal JR, Dalmasso AP, Cromwell JW~Platt JL, Manivel CJ, Bolman RM III, Matas AJ: Prolongation of cardiac xenograft survival by depletion of complement. Transplantation 1993, 55:857-866.

14.

Pruitt SK, Kirk AD, Bollinger RR, Marsh HC Jr, Collins BH, Levin JL, Mault JR, Heinle JS, Ibrahim S, Rudolph AR eta/.: The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts. Transplantation 1994, 57:363-370.

15.

Cooper DKC, Koren E, Oriol R: Oligosaccharides and discordant xenotransplantation /mmunol Rev 1994, 141:31-58.

16.

Fodor WL, Williams BL, Matis LA, Madri JA, Rollins SA, Knight JW, Velander W, Squinto SP: Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection. Proc Nat/ Acad Sci USA 1994, 91:11153-11157.

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18. Fodor WL, Squinto SP: Engineering of transgenic pigs for • xenogeneic organ transplantation. Xenobiotica 1995, 3:23-25. Fodor and I summarize our efforts to engineer transgenic pigs to express the human complement inhibitor CD59. Also described is the rather ubiquitious expression of CD59 in tissues and organs of F1 generation transgenic pigs using a class I minigene to drive expression. Rollins SA, Zhao J, Ninomiya H, Sims PJ: Inhibition of homologous complement by CD59 is mediated by a speciesselective recognition conferred through binding of C8 within C5b-8 or C9 within C5b-9. J Biol Chem 1991,146:2345-2352.

Sommerville CA, D'Apice AJF: Future directions in transplantation: xenotransplantation. Kidney/nt 1993, 44(suppl 42):112-121.

10.

protect swine-to-primate cardiac xenografts from humoral injury. Nat Med 1995, 1:423-427. These authors describe a 'painting' technology used to generate transgenic pigs expressing human complement inhibitors. The globin promoter is used to drive expression of GPI-anchored human complement inhibitor proteins in the ethryocytes of the transgenic pigs. The GPI-containing proteins can 'jump' to the endothelial cells of the pig organs and provide transient protection against human complement. This is the first evidence that human complement inhibitors can provide transient protection from human complement following pig-to-primate organ transplantation.

19.

Platt JL, Lindman BJ, Chen H, Spitalnik S, Bach F: Endothelial antigens recognized by xenoreactive antibodies. Transplantation 1990, 50:817-822.

9.

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McCurry KR, Kooyman DL, Alvarado CG, Cotterell AH, Martin MJ, Logan JS, Platt JL: Human complement regulatory proteins

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Sandrin MS, Fodor WL, Mouhtouris E, Osman N, Cohney S, Rollins SA, Guilmette ER, Setter E, Squinto SP, McKenzie IFC: Enzymatic remodelling of the carbohydrate surface of a xenogeneic cell substantially reduces human antibody binding and complement-mediated cytolysis. Nat Med 1995, 1:1261-1267. These authors describe a novel gene engineering approach that results in an enzymatic remodeling of the carbohydrate composition of xenografts and xenogeneic cells. This approach utilizes intracellular enzyme competition to replace the highly antigenic c~l,3-galactosyl-terminal carbohydrate with a human carbohydrate structure that is inert with regard to the human immune system. The authors demonstrate that the H-transferase enzyme can effectively outcompete the gal-transferase enzyme and replace the (x-Gal carbohydrate epitope with a nonimmunogenic (xl,2-fucosyl residue. As a consequence of this engineering strategy, both in vitro with xenogeneic cells and in vivo in transgenic mice, xenografts are not recognized by human antibodies and survive when challenged with human serum. 26.

Fodor WL, Rollins SA, Guilmette ER, Setter E, Squinto SP: A novel bifunctional chimeric complement inhibitor that regulates C3 convertase and formation of the membrane attack complex. J Immunol 1995, 155:4135-4138.