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Perspectives on the Optimal Genetically Engineered Pig in 2018 for Initial Clinical Trials of Kidney or Heart Xenotransplantation

Cooper, David K.C. MD, PhD, FRCS1; Ezzelarab, Mohamed MD2; Iwase, Hayato MD, PhD1; Hara, Hidetaka MD, PhD1

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doi: 10.1097/TP.0000000000002443
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Experimental organ xenotransplantation has made significant advances during the past few years,1-3 and increasing attention is being paid toward initiating clinical trials, particularly of pig kidney or heart transplantation,4,5 where the pathobiological problems, although slightly different,6 are less severe than after liver or lung transplantation. With life-supporting pig kidney transplantation in nonhuman primates (NHPs) extending to beyond 1 year,7-10 and heterotopic heart transplantation extending beyond 2 years,11,12 in our opinion, it may soon be ethical to move toward a clinical trial.

There are now a variety of genetically engineered pigs available or potentially available for use as the organ source for a clinical trial.2 Although there will undoubtedly be a greater variety in the future, the question can be asked as to what might realistically be the optimal pig among those currently available or that could become available in the very near future? This question revolves around 2 major genetic manipulations: (i) deletion of expression of xenoantigens against which humans are known to have natural (preformed) antibodies, and (ii) the transgenic expression of human complement- and/or coagulation-regulatory and/or anti-inflammatory proteins.2,13

We here put forward our own opinions on what genetically engineered pigs would be optimal as sources of kidneys or hearts if a clinical trial of xenotransplantation were to be initiated today. We would emphasize that the opinions we provide are personal, and that others in this field of research may have differing opinions. Furthermore, the data we provide have largely been selected to support our opinions.

DELETION OF PIG ANTIGENS AGAINST WHICH HUMANS HAVE NATURAL ANTIBODIES

There are currently 3 known xenoantigens: (i) galactose-α1,3-galactose (Gal), a product of the enzyme, α1,3-galactosyltransferase (GT),14,15 (ii) N-glycolylneuraminc acid (Neu5Gc), a product of the enzyme, cytidine monophosphate-N-acetylneuraminic acid hydroxylase,16-18 and (iii) Sda (a product of the enzyme, β-1,4N-acetylgalactosaminyltransferase).19

It should be noted that Old World NHPs express Neu5Gc, and therefore make antibodies only against Gal and Sda, whereas humans do not express any of the 3, and therefore make antibodies against all of them. In contrast, New World monkeys do express Gal, but do not express Neu5Gc and Sda, and so make antibodies against these 2 glycans [Hara et al, unpublished].

Knockout of even one of these, particularly of Gal,20,21 reduces in vitro IgM and IgG antibody binding to pig cells significantly (Figure 1),23,24 reduces serum cytotoxicity (Figure 2), and extends pig heart or kidney graft survival in immunosuppressed NHP recipients. In the initial studies, heterotopic pig heart graft survival was extended to almost 6 months,25,26 and (thymo)kidney graft survival to almost 3 months.27

FIGURE 1
FIGURE 1:
Human serum IgM (left) and IgG (right) binding to RBCs. RBCs were drawn from (i) a volunteer healthy human, (ii) a wild-type (WT) pig, (iii) an α1,3-galactosyltransferase gene-knockout (GTKO) pig, (iv) a GTKO pig that did not express Sda (GTKO/β4GalKO or double-knockout [DKO]), (iv) a DKO pig that did not express Neu5Gc (GTKO/β4GalKO/CMAHKO or triple-knockout [TKO] pig). Human RBCs (O blood type) were used as a negative control. Fourteen human sera were tested for antibody binding to pRBCs, and 6 human sera were tested for antibody binding to hRBCs. Human IgM and IgG binding to GTKO/β4GalKO/CMAHKO (TKO) pig RBCs was almost at the level of binding to human RBCs, and there was no detectable IgM or IgG binding to TKO RBCs. Relative GM = relative geometric mean, which was calculated by dividing the geometric mean value for each sample by the negative control (secondary antibody only). ns = not significant; **P < 0.01. (For details of methods, see reference22). CMAHKO, cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene knockout; RBCs, red blood cells.
FIGURE 2
FIGURE 2:
Protection of pAEC from human serum cytotoxicity by transgenic expression of a human CRP, CD46 The pAEC tested included (left-to-right) wild-type (WT, ie, genetically unmodified), GTKO, CD46-transgenic, and GTKO/CD46. Activated pAEC were exposed to IFN-γ for 48 h. Nonactivated (white bars) and activated (shaded bars) cells were then exposed to 25% human serum. Activation increased lysis of each cell type except GTKO/CD46 cells, which were completely resistant to serum cytotoxicity (reproduced with permission from Hara et al. Transpl Int. 2008;21:1163-1174; reference23). pAEC, pig aortic endothelial cells.

Antibodies to Gal and Neu5Gc are particularly low in patients with end-stage renal disease undergoing regular hemodialysis (Figure 3), as is antibody binding to pig cells.28 The reason for this is uncertain, but is more likely related to the immunocompromised state associated with renal failure rather than to the dialysis itself, as it is not thought likely that antibody is removed by hemodialysis. Patients undergoing chronic dialysis, therefore, should initially be at low risk of rejecting a pig graft by an antibody-mediated mechanism.

FIGURE 3
FIGURE 3:
Anti-Gal and anti-Neu5Gc IgM and IgG antibody levels in patients with ESRD. Anti-Gal and anti-Neu5Gc IgM/IgG were measured by ELISA in patients with ESRD (on hemodialysis) who had either (i) a high level (100%) of panel-reactive antibodies (cPRA, ie, antibodies against HLA; n = 10), or (ii) a negative cPRA, (ie, 0% anti-HLA antibodies n = 12), and in healthy human control subjects (n = 10). The patients with ESRD, whether highly sensitized to HLA or not, had significantly lower levels of anti-Gal and anti-Neu5Gc IgM (top panels) and IgG (bottom panels) antibodies than healthy control subjects. (ns = not significant; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (Reproduced with permission from Zhang Z, et al. Transplantation. 2018;102:e195-e204; reference28). ESRD, end-stage renal disease.

Although, to our knowledge, only one in vivo study using pigs that do not express 2 or 3 of these glycans has been reported,10 in vitro studies confirm that antibody binding and serum cytotoxicity are reduced further when 2 or all 3 glycans have been deleted from the pig cells.29-32

On this evidence, it seems reasonable to conclude that triple-knockout (TKO) pigs should form the basis of any clinical trial. There are some in the field of xenotransplantation research who feel that this is all that is required. Based on experience in allotransplantation, where the absence of any preformed antibody binding to the graft (and the prevention of the development of de novo antibody by effective immunosuppressive therapy) results in excellent outcomes, it is suggested that an absence of natural antibody should be associated with equally good outcomes after xenotransplantation (as long as the immunosuppressive therapy is totally effective).

However, we suggest that this hypothesis may be false because xenografts are not allografts. The mechanism by which a pig xenograft can be injured is much more complex than rejection of an allograft1 (Table 1). Although most of the same mechanisms occur after allotransplantation, they are of significantly less magnitude than after xenotransplantaton, and some are qualitatively different, for example, coagulation dysregulation. Factors, such as complement activation unrelated to antibody binding, molecular incompatibilities that contribute to coagulation dysregulation, the action of innate immune cells, and a systemic inflammatory response, may all play a much greater role in destruction of a xenograft than of an allograft. There is increasing evidence that all of these mechanisms can be activated in the absence of antibody. We here (below) draw attention to some of these points.

TABLE 1
TABLE 1:
Factors involved in pig organ graft failure after xenotransplantationa

We have observed one additional benefit of deletion of Gal expression, and that is that it is associated with a reduced T-cell response.33 The additional deletion of Neu5Gc expression reduces the T-cell response further (Figure 4), and this beneficial effect may be further enhanced if a TKO pig organ is the target for T cells.

FIGURE 4
FIGURE 4:
Proliferative response of human T cells (CD3+, CD4+, and CD8+ cells) to wild-type (WT), GTKO/CD46, and GTKO/CD46/CMAHKO pig PBMCs. PBMCs were isolated from healthy human volunteers (n = 9). The proliferation of CD3+, CD4+, and CD8+ T cells in human PBMC to pig PBMCs was identified and quantified by flow cytometry at the end of a 5-day CFSE-MLR. Deletion of Gal expression on the pig cells significantly reduced the proliferative response, and deletion of both Gal and Neu5Gc reduced it further. (*P < 0.05, **P < 0.01). (For details of methods, see reference28). PBMCs, peripheral blood mononuclear cells.

EXPRESSION OF HUMAN PROTECTIVE TRANSGENES

Complement Activation

Complement can be activated by the classical pathway, in which case an absence of human antibody binding to a pig graft should prevent activation of this pathway. However, complement can also be activated through the alternative or lectin pathways in the absence of antibody binding.34 Such factors as ischemia-reperfusion injury35-42 and an infectious episode43-47 may activate complement and cause graft injury. Compared with human complement-regulatory proteins (CRPs), pig CRPs are relatively ineffective in protecting against human complement activation (unless overexpressed48), but the introduction into the pig of a human complement-regulatory transgene provides much greater protection.23,49

In the very early days of xenotransplantation research, the expression of human (h) CD55 (decay-accelerating factor) extended pig graft survival from minutes, hours, or days to weeks or months.50,51 Importantly, in these studies, no steps had been taken to either delete any pig xenoantigens, which in any case was impossible at that time, or reduce recipient NHP antipig antibody levels. The expression of hCD55 could extend pig kidney graft survival approximately to the same extent as deletion of expression of Gal, fully demonstrating its beneficial effect.

When GTKO pig grafts were transplanted into NHPs, the additional expression of a human CRP (hCRP) reduced the incidence of early graft failure.52-54 However, NHP antibody still bound to the GTKO pig cells, and perhaps hCRP expression would not have provided extra protection if there had been no antibody binding, as might occur if a TKO graft had been transplanted.

Nevertheless, on the basis of the available evidence, we would suggest that expression of at least 1 hCRP would be beneficial, and certainly not detrimental. Furthermore, Miyagawa has produced evidence suggesting that expression of 2 hCRPs is more effective than 1.55,56

Just as absence of expression of Gal is associated with a reduced T-cell response,33 expression of a hCRP may similarly be associated with a reduced T-cell response.57

Coagulation Dysfunction

When a GTKO pig organ, with or without expression of a hCRP, was transplanted into an NHP, molecular incompatibilities between the coagulation systems of pig and primate resulted in the development of thrombotic microangiopathy in the graft and consumptive coagulopathy in the recipient. This again may be associated with antibody, or possibly complement, activation of the vascular endothelium of the graft. It thus may not occur if there is no antibody activation of the vascular endothelial cells, but this assumption depends entirely on the complete absence of natural antibody in the host and, furthermore, on the absolute efficacy of the immunosuppressive regimen in preventing any de novo antibody production. This was proven to be difficult even in allotransplantation.58

It should also be noted that Lin has demonstrated in vitro that coagulation can be activated by human platelets and/or innate immune cells, for example, monocytes, in the absence of antibody.59-61

In contrast, the expression of a human coagulation-regulatory protein, for example, thrombomodulin, endothelial protein C receptor, or both, delays graft failure significantly.8,9,62 Expression of tissue factor pathway inhibitor, CD39, and/or CD73 may also be effective in this respect.

To illustrate this point, we would draw attention to our own experience in which 2 GTKO.hCD46 pig kidney grafts that expressed human coagulation-regulatory proteins functioned well in baboons for >7 and > 8 months, respectively, until infectious complications necessitated termination of the experiments, whereas 2 baboons with kidney transplants from GTKO.hCD46 pigs (initially thought to be expressing human thrombomodulin, but later found not to be expressing this human coagulation-regulatory protein) developed consumptive coagulopathy within 12 days.9 Except for the genetic modifications in the pigs, all other aspects of the experiments, for example, immunosuppressive therapy, were identical.

This study also demonstrated that, even when a low level of serum antipig antibody was present in the 2 long-surviving baboons, graft rejection could be prevented by the expression of the human transgenes. This would be a safeguard in patients in whom a low level of antibody was erroneously not detected or in whom a low level of de novo antibody develops.

Any thrombin activation can be detrimental to the outcome of the transplant. For example, thrombin activates the human cellular response to pig cells in vitro (Figure 5).63 This was an important observation as it indicates that cellular rejection can be stimulated by mechanisms that are not considered part of the adaptive immune response.

FIGURE 5
FIGURE 5:
Thrombin activates the human cellular response to GTKO pig PBMCs (upper panel) The degree of activation of GTKO pig PBMCs by thrombin was comparable to that resulting from stimulation of the cells by pIFN-γ. Thrombin-stimulated activation of the human cellular response was reduced by the addition of hirudin, confirming that thrombin was the stimulatory factor. (Lower panel) In thrombin-activated pig aortic endothelial cells, CD86 (costimulation pathway) mRNA was upregulated in a dose-dependent manner, though this upregulation was reduced by hirudin (modified from Ezzelarab C, et al. Xenotransplantation. 2012;19:311-316; reference63). pIFN-γ, porcine interferon-gamma.

Systemic Inflammation

A systemic inflammatory response has been well described after pig organ (and even artery patch) transplantation in NHPs and is illustrated by a sustained (>3 months) rise in C-reactive protein in the recipient (Figure 6).64-67 There is evidence that it precedes the onset of disturbances in coagulation (Figure 7).65,66 The inflammatory response is associated with deposition of C-reactive protein in the transplanted pig kidney (Figure 8). Whether this is related to antibody binding to the graft remains uncertain. However, it is well known that inflammation and the innate immune response augment the adaptive immune response to a graft (Figure 9), and contribute to activation of coagulation dysfunction,22,63,65,66,68 and therefore any genetic manipulation that might reduce inflammation of the graft, for example, expression of hemeoxygenase-1 (HO-1) or A20, may be beneficial to the long-term survival of the graft.

FIGURE 6
FIGURE 6:
Response of serum C-reactive protein to the presence of a GTKO/CD46 pig artery patch grafts sutured into the wall of the abdominal aorta of the recipient baboons After the transplant, there was an immediate and sustained increase (for >3 months) in C-reactive protein, even when the recipient received therapy with either CVF (pretransplantation) to deplete complement, or AAT (pretransplantation and posttransplantation). However, pretransplant and posttransplant treatment with the IL-6-receptor blockade agent, tocilizumab (Actemra), prevented the rise in C-reactive protein (for details, see reference64). CVF, cobra venom factor; AAT, alpha-1-antitrypsin.
FIGURE 7
FIGURE 7:
After pig kidney transplantation in baboons, CRP increases before the development of a consumptive coagulopathy. Platelet counts, and fold increases in CRP and D-dimer were calculated in a baboon recipient of a pig renal xenograft. High levels of CRP were detected as early as 3 days after kidney xenotransplantation, prior to the development of CC, as indicated by reduced platelet counts and elevated D-dimer (reproduced with permission from Ezzelarab and Cooper66). CC, consumptive coagulopathy.
FIGURE 8
FIGURE 8:
CRP deposition in a pig kidney transplanted into a baboon, an indicator of the inflammatory response to the graft (left panel) At 30 minutes after reperfusion, no CRP deposition was detected. In 2 different kidneys at the time of euthanasia (middle and right panels), CRP deposition was detected in the glomeruli (arrow heads, right panel) and tubules (arrows, middle, and right panels). Our data suggested that both the xenograft and the recipient contributed to the C-RP production (we could detect minimal CRP in NHPs undergoing heart allotransplantation [not shown], illustrating the difference in response to a xenograft from an allograft) (reproduced with permission from Ezzelarab et al.65).
FIGURE 9
FIGURE 9:
Inflammation increases the proliferative response of human PBMC to WT and GTKO pAEC. In vitro study of the effect of inflammation (exposure to IFN-γ) on the human PBMC response to pAECs. When nonactivated (by IFN), the response to WT pAECs was greater than to GTKO cells (P < 0.05). There was a significant increase in the PBMC response when the pAEC were activated by IFN (P < 0.01), the response to WT cells again being significantly greater than to GTKO cells (P < 0.05). This study illustrates how inflammation can greatly increase the immune response to a xenograft. (CPM, counts per minute; SI, stimulation index) (reproduced with permission from Wilhite et al.33).

There is little or no rise in C-reactive protein in allotransplantation models in NHPs,65,66 perhaps supporting our hypothesis that the pathobiological response to a xenograft is different from that to an allograft, although this could be related to an absence of antibody binding to the allograft vascular endothelium.

Although the administration of an anti-inflammatory agent, for example, tocilizumab, to the recipient of the graft may suppress the systemic inflammatory response (as evidenced by reduced levels of C-reactive protein or serum amyloid A),64,67,69 recent evidence indicates that tocilizumab, although binding to primate IL-6 receptors, does not bind to IL-6 receptors on the pig graft22 [Gao et al, manuscript submitted; Hara et al, unpublished data], and therefore has no protective effect on the graft.

An important observation made recently by our group indicates that, when pig vascular endothelial cells expressing only natural pig thrombomodulin (which also has an anti-inflammatory effect) are activated by tumor necrosis factor-alpha (TNF-α), the expression of thrombomodulin is significantly downregulated (Figure 10).68 This suggests that, when a pig organ is exposed to inflammation (which we have documented is universal after a pig organ transplant into an NHP), thrombotic microangiopathy is likely to develop. In contrast, in pig vascular endothelial cells transgenic for human thrombomodulin, thrombomodulin is not downregulated when the cells are activated by TNF-α, but is significantly upregulated (Figure 10). We presume that expression of the transgenically expressed human thrombomodulin remains stable as a result of how the transgene was introduced (in this case using an ICAM-2 promoter), though the exact mechanism remains uncertain. Nevertheless, this benefit from the transgenic procedure may be a major reason why pig heart transplants have performed so well after transplantation in NHPs,12 and why the absence of the anti-inflammatory effect of a human transgene, such as thrombomodulin, resulted in the early development of consumptive coagulopathy in the recipient of the pig kidney graft (reviewed above).9

FIGURE 10
FIGURE 10:
Inflammation downregulates expression of natural pig TBM, but not of transgenic human TBM. Transgenic expression of human TBM (right), but not of natural pig TBM (left), on pig vascular endothelial cells protects against inflammation. The expression of natural pig TBM was down-regulated after exposure to TNF-α confirmed by real-time PCR (*P < 0.05, **P < 0.01), whereas the expression of transgenic human TBM was upregulated confirmed by flow cytometry. Transgenically expressed human TBM would appear to be resistant to downregulation by inflammation (the expression of pig TBM in GTKO/CD46 pAECs and human TBM in human TBM-transgenic pAECs was measured by real-time PCR and flow cytometry [clone 1A4, BD Biosciences, San Jose, CA], respectively. The PCR primers sequences used were: pTBM: Sense 5′-GAA GCT ATG AGG TCC AGC CC-3′; Antisense 5′-CAG ACA GAC AGC GAA GAG CA-3′.) (details in Lee et al. Ocul Immunol Inflamm. 2016;24:579-593). TBM, thrombomodulin.

COMMENT

One topic not discussed above is that of expression of swine leukocyte antigens (SLA) on the graft. Although little work has been carried out to explore this topic in NHPs, immunologically naive NHPs do not appear to have antibodies directed against SLA. However, there is increasing evidence that at least some patients with anti-HLA antibodies, that is, with panel-reactive antibodies, may have antibodies that cross-react with SLA expressed on the pig graft.24,70,71 Although genetic engineering techniques are being developed to overcome this problem, largely by Tector and his colleagues,70-72 in the initial clinical trials it might be wise to exclude any patients with anti-HLA antibodies.

As the absence of antibody binding to the graft is of such fundamental importance, TKO pigs will be the basic sources of organs for clinical xenotransplantation. However, it would seem only sensible, if not mandatory, for the graft to express several human transgenes for which there is considerable in vivo and/or in vitro evidence of benefit. These include both hCRPs and human coagulation-regulatory proteins, some of which also have anti-inflammatory functions, and transgenes, such as HO-1, that has an anti-inflammatory protective effect. Furthermore, the introduction of human CD47 has a suppressive effect on the function of monocytes and macrophages and also on T cell activity,73,74 and the expression of HLA-E or HLA-G may also suppress innate cell function, particularly of natural killer cells.75-78 A TKO pig expressing CD46, CD55, thrombomodulin, and endothelial protein C receptor would be close to optimal, and the additional expression of HO-1 and/or CD47 would likely be beneficial. It is very unlikely that any group will be able to assess in vivo the importance of each of the 9 genetic modifications individually, but it is hoped that national regulatory authorities will accept in vitro evidence that indicates the benefit of each deletion and transgene.5

The above genetic engineering approach is directed mainly to overcoming the innate immune response, which has been most problematic to date. It thus relies on the administration of immunosuppressive agents to the recipient NHP (or human patient) to prevent an adaptive immune response. In this respect, agents that block the CD40/CD154 costimulation pathway have proved most successful.7-9,11,12,25-27,79-81

However, additional genetic manipulations that reduce the T cell response, for example, expression of a mutant SLA class II transactivator (CIITA) gene (that results in reduced expression of SLA class II),82,83 knockout of SLA class I,72 or expression of CD47,73,74 are all likely to be beneficial in allowing the intensity of exogenous immunosuppressive therapy to be reduced. Alternatively, or additionally, human CTLA4-Ig can be expressed in the pig organ.84

This would suppress both the direct and indirect T cell responses, and produce a local immunosuppressive effect. In contrast, pig CTLA4-Ig is less effective at binding to human CD80/86.85

In conclusion, absent or minimal antibody binding is clearly important, but the ability to protect the graft further by insertion of selected human transgenes will almost certainly provide additional protection.

We have not commented on genetic manipulations that can be made to prevent porcine endogenous retrovirus (PERV) activation, in part because we are not convinced that this is necessary. However, if proved essential, activation of PERV can be inhibited86-88 or PERV can be deleted.89

Whether deletion of 62 PERV genes, in addition to the genetic manipulations discussed above, will affect the viability or function of the pig cells remains uncertain. Finally, although pig kidneys and heart have functioned satisfactorily for months after transplantation into NHPs, genetic manipulations may prove beneficial to resolve any functional discrepancies between pig and primate, if these are identified. For example, if remains uncertain whether porcine erythropoietin functions satisfactorily in primates, but, if this proves to be the case, the introduction of the transgene for human erythropoietin may be required. This topic and other physiological aspects of kidney xenotransplantation have recently been discussed elsewhere.90

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