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What Therapeutic Regimen Will Be Optimal for Initial Clinical Trials of Pig Organ Transplantation?

Bikhet, Mohamed MD1; Iwase, Hayato MD, PhD1; Yamamoto, Takayuki MD, PhD1; Jagdale, Abhijit MD1; Foote, Jeremy B. PhD2; Ezzelarab, Mohamed MD3; Anderson, Douglas J. MD, MS4; Locke, Jayme E. MD, MPH4; Eckhoff, Devin E. MD4; Hara, Hidetaka MD, PhD1; Cooper, David K.C. MD, PhD, FRCS1

Author Information
doi: 10.1097/TP.0000000000003622
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Abstract

INTRODUCTION

There has been considerable experience in immunosuppressive (IS) therapy administered to nonhuman primates (NHPs) with genetically engineered pig kidney or heart grafts.1-11 Recipient survival has exceeded 6 or even 12 mo in several reports. Although these encouraging results have largely been achieved in NHPs with low preformed anti-pig antibody levels, many humans have no preformed antibodies against cells from the most advanced genetically engineered pigs.12 Given the much greater difficulty in maintaining an immunosuppressed NHP than an immunosuppressed human patient, consistent survival of >6 mo of a life-supporting pig organ in a NHP is considered by many to be sufficient to warrant consideration of a clinical trial.

With the results of pig organ xenotransplantation steadily improving, in part related to the availability of more advanced genetically engineered pigs, consideration is now being given to the first clinical trials of xenotransplantation. In some cases, for example, pig kidney transplantation, they may be planned as destination therapy13 (A. Jagdale, unpublished data, 2020). In others, for example, pig heart transplantation in infants with life-threatening congenital heart disease, these may be directed towards bridging to allotransplantation.14

Largely on the basis of our laboratory’s experience, extending back many years, and that of others reported in the literature, we here discuss what regimen might provide acceptable and successful immunosuppression in the first patients to undergo pig kidney or heart transplantation. Variations in our suggested regimen may be equally successful, but we hope our comments will focus attention on this topic.

We and others have significant experimental experience indicating that conventional calcineurin-based IS is not successful in preventing immune injury in pig-to-NHP cell or organ transplantation models.10,15-17 We have therefore concentrated our attention on blockade of the CD40/CD154 costimulation pathway, first introduced into xenotransplantation in 2000 by Bühler et al,15 who were also the first to demonstrate the inadequacy of conventional IS. Details of the IS and anti-inflammatory therapy we have administered for several years in the pig-to-NHP model, with little change, are provided in Table 1. The major mechanistic effects and side effects of the key agents in this regimen are summarized in Appendix.

TABLE 1. - Immunosuppressive, anti-inflammatory, and adjunctive drugs used in pig-to-baboon kidney transplantation experiments during the past several years
Agent Dose (duration)
Induction
 Thymoglobulin (ATG; Genzyme, Cambridge, MA) 10 mg/kg IV (day −3; to reduce the CD3+T-cell count to <500/mm3)
 Anti-CD20 mAb (rituximab; Genentech, South San Francisco, CA) 10 mg/kg IV (day −2)
 CVF (Complement Technology, Tyler, TX) OR C1-esterase inhibitor (Berinert; CSL Behring, King of Prussia, PA) 100 U/kg (days −1 and 0)17.5 U/kg IV (days 0, 1, 7 and 14)
Maintenance
 Anti-CD40 mAb (2C10R4, a chimeric rhesus IgG4; NIH NHP Resource Center, Boston, MA) 50 mg/kg (days −1, 0, 4, 7, 14, and weekly)
 Rapamycin (Rapa; LC Laboratories, Woburn, MA) 0.01–0.04 mg/kg IM ×2/d (target trough 6–10 ng/mL), beginning on day −4.
 Methylprednisolone (Astellas, Deerfield, IL) 5 mg/kg/d on day 0, tapering to 0.125 mg/kg/d by day 7.
Anti-inflammatory
 Etanercept (TNF-α antagonist; Amgen, Thousand Oaks, CA) 1 mg/kg (day 0), 0.5 mg/kg IV (days 3, 7, 10)
Adjunctive
 Triiodothyronine (levothyroxine; Xgen, Big Flats, NY) Aspirin (Bayer, Deland, FL) 4 µg IV ×2/d on days 0–540 mg PO (alternate days), beginning on day 4
 LMWH (Eisai, Woodcliff Lake, NJ) 700 IU/d SC, beginning of day 1
 Famotidine (APP Pharmaceuticals, Schaumburg, IL) 0.25 mg/kg/d ×2 (days −7 to 14)
 Erythropoietin (Amgen) 500 U IV ×2–3 weekly, beginning on day −4
 Ganciclovir (Genentech) 5 mg/kg/d IV, from day −4 to day 14 and when the baboon is sedated for blood draws (×2 weekly)
 Valganciclovir (Genentech) 15 mg/kg/d PO, beginning on day 15
 Sulfamethoxazole and trimethoprim (Teva, North Wales, PA) 10 mg/kg IV daily, on days 4–14
 Sulfamethoxazole and trimethoprim oral suspension (Akorn, Lake Forest, IL) 75 mg/m2 PO ×2/d. ×3 weekly, beginning on day 15
CVF, cobra venom factor; IM, intramuscularly; LMWH, low-molecular-weight heparin; TNF-α, tumor necrosis factor-alpha.

INDUCTION THERAPY

Antithymocyte Globulin

We administer this on day −3 (ie, 3 d before the kidney transplant [day 0]) to deplete circulating T cells. We administer a single dose of 10 mg/kg as an IV infusion in normal saline over 6 h. As it can be associated with an anaphylactic response, prophylaxis is administered before the antithymocyte globulin (ATG) in the form of diphenhydramine (5 mg/kg IV), metoclopramide (0.5 mg/kg IV), and methylprednisolone (5 mg/kg IV; Astellas, Deerfield, IL).

ATG’s efficacy in respect to reducing T-cell numbers in the blood is summarized in Table 2 and is being reported in full elsewhere.18 Recovery of T cells begins within 2 weeks but, in the presence of the maintenance IS we administer (see next), the number of T cells in the blood never reaches more than approximately 40% of the baseline (pre-ATG) number (Table 2).

TABLE 2. - Absolute numbers of T and B cells preinduction therapy (baseline, day −7), postinduction therapy (day 0, day of transplant), and posttransplantation (day 7 and at 1, 3, and 6 mo) in baboons (n = 14) receiving the immunosuppressive regimen outlined in Table 1
D/mo CD3+ (% change)a CD4+ (% change)a CD8+ (% change)a CD22+ (% change)a
−7 d 1778 977 688 454
0 d 185 (−84%) 124 (−87%) 53 (−92%) 141 (−69%)
7 d 137 (−88%) 81 (−92%) 44 (−94%) 24 (−95%)
1 mo 299 (−85%) 136 (−86%) 104 (−89%) 6 (−99%)
3 mo 224 (−67%) 109 (−89%) 96 (−86%) 41 (−90%)
6 mo 316 (−62%) 256 (−65%) 126 (−75%) 155 (−62%)
Reproduced with permission from Jagdale et al (2020).18
aPercentage reduction vs day −7.

ATG, sometimes in larger doses, has formed the basis of many IS regimens used in allotransplantation for almost 50 years.19-22 In clinical allotransplantation, some groups administer 5 mg/kg ×2 or smaller, more frequent doses before organ transplantation (if possible), and this may be safer than administering it as a single dose.23-26 In future experimental studies and in clinical trials, we will therefore plan to divide the single dose into 2 (Table 3), as is already carried out by some groups working in xenotransplantation.27,28

We have evidence from in vitro studies to indicate that ATG is rather less effective in killing baboon peripheral blood mononuclear cells than human peripheral blood mononuclear cells,29 and therefore, a larger dose may be required for in vivo studies in baboons than will be required clinically.

ATG can be associated with an anaphylactic response, despite prophylactic therapy aimed at preventing this. We have seen 2 cases of anaphylaxis in baboons during the past 20 y. Dividing the dose into 2 may help prevent this complication. However, in patients in an intensive care unit, anaphylaxis after ATG can be successfully treated.30

Anti-CD20 Monoclonal Antibody (Rituximab)

There are data in the literature suggesting that initial B-cell depletion is associated with a better outcome of pig organ xenotransplantation than if B cells remain nondepleted.4,31,32 Rituximab also forms a component of some IS regimens, especially in patients sensitized to HLA antigens, in whom desensitization is being carried out.33,34

A full course of Rituximab in a patient with a B-cell lymphoma consists of 20 mg/kg IV, weekly for 1 mo (4 doses).35 We have elected to administer just one-eighth of this dose (a single dose of 10 mg/kg IV on day −2; Table 1). In our experience (A. Jagdale, unpublished data, 2020) and that of others,36 this dose depletes all B cells in the blood (Table 2) but not in the lymph nodes or spleen. Recovery does not begin for approximately 2 mo (60 d). As with T cells, the number of B cells never recovers to more than approximately 40% of the baseline number (Table 2).

Whether a higher dose or a full course of Rituximab would be preferable remains uncertain but is probably unnecessary.37 We were influenced by a concern that a greater dosage may be associated with a higher incidence of infectious complications.5,38 In forthcoming clinical trials, we would plan to administer Rituximab at 10 mg/kg IV on day −2 (Table 3).

TABLE 3. - Immunosuppressive and adjunctive regimen proposed for first clinical trial of genetically engineered pig kidney transplantation
Immunosuppressive therapy
Induction
 ATG (Thromboglobulin; Genzyme, Cambridge, MA) 5 mg/kg IV on days −7 and −5
 Rituximab (Genentech; South San Francisco, CA) 10 mg/kg IV on day −3
 C1-esterase inhibitor (Berinert; CSL Behring, King of Prussia, PA) 17.5 U/kg IV on days 0, 1, 7, and 14
Maintenance
 An anti-CD154 or anti-CD40 agent that blocks this costimulatory pathway ()
 Everolimus (I.C. Laboratories, Woburn, MA) 0.75 mg/kg PO ×2 daily beginning on day –5 to maintain a blood trough level of 6–10 ng/mL
 Methylprednisolone (Astellas, Deerfield, IL) 5 mg/kg IV on day 0 (day of transplant), tapering to 0.125 mg/kg daily by day 7, followed by a tapering dose of prednisone, beginning on day 8 at 10 mg/d PO ×2 daily, reducing to 5 mg/d PO ×2 on day 14, and finally to 2.5 mg/d PO ×2 on day 21
Anti-inflammatory therapy
 Etanercept (Enbrel; Amgen, Thousand Oaks, CA) 1 mg/kg IV on day 0, and 0.5 mg/kg IV or SC, on days 3, 7, and 10
 Siltuximab (Sylvant; EUSA Pharma, United Kingdom) 11 mg/kg IV on days 0 and 21
Adjunctive
 Triiodothyronine (levothyroxine; Xgen, Big Flats, NY) a single dose of 10 µg IV before reperfusion of the graft (day 0), followed by 4 µg IV, 4 h later on day 0, and 4 µg IV ×2 daily on days 1–5
 Cefazolin (Fresenius Kabi, Lake Zurich, IL) 25 mg/kg IV ×2 on day of transplant (preoperative and postoperative)
 Valganciclovir (Genentech) 15 mg/kg/d PO, beginning on day 7
 Bactrim (sulfamethoxazole + trimethoprim; Akorn Lake Forest, IL) 75 mg/m2 PO (oral suspension) ×2/d. ×3 weekly beginning on day 7 (for 6 mo)
 Aspirin (Bayer, Deland, FL) 40 mg PO on alternate days, beginning on day 4 (for 6 mo)
 LMWH (Eisai, Woodcliff Lake, NJ) 700 IU/d SC, on days 1–28
 rhEPO (Amgen) 2000 U IV weekly, if hemoglobin <10 g/dL
ATG, antithymocyte globulin; LMWH, low-molecular-weight heparin; rhEPO, recombinant human erythropoietin.

Cobra Venom Factor

In contrast to many humans, who do not have detectable antibodies to triple-knockout (TKO) pig cells, that is, pigs in which the genes for all 3 of the known pig xenoantigens have been deleted (Table 4),39-41 all baboons have some anti-TKO pig antibodies.12,40,42 Antibody binding to the graft may activate the complement cascade and initiate hyperacute or early antibody-mediated rejection, although this may not occur if the graft expresses high levels of human complement- and coagulation-regulatory proteins (as in the pigs we plan to use in the first clinical trials12,43,44).

TABLE 4. - Known carbohydrate xenoantigens expressed on pig cells
Carbohydrate (abbreviation) Responsible enzyme Gene-knockout pig
1. Gal α1,3-galactosyltransferase GTKO
2. Neu5GC CMAH CMAH-KO
3. Sda β-1,4 N-acetylgalactosaminyltransferase β4GalNT2-KO
CMAH, cytidine monophosphate-N-acetylneuraminic acid hydroxylase; Gal, Galactose-α1,3-galactose; Neu5GC, N-glycolylneuraminic acid.

However, because of the presence of anti-pig antibodies in baboons, to be certain that we prevent hyperacute rejection, we have administered cobra venom factor (CVF; at a dose of 100 IU/kg on days −1 and 0) to deplete all complement before organ transplantation (Table 1). Our reasoning has been that, although CVF therapy may be unnecessary (because the pig kidney expresses 2 human complement-regulatory proteins), the development of hyperacute rejection would be a waste of (a) a scarce genetically engineered pig, (b) the recipient baboon, and (c) the cost of the pretransplant induction IS therapy.

CVF is not Food and Drug Administration (FDA)-approved, but until very recently, we have been unable to obtain any other agent that protects from complement-induced injury that functions in baboons. For example, eculizumab is not active in NHPs (https://www.ema.europa.eu/en/documents/scientific-discussion/soliris-epar-scientific_discussion_en.pdf). Several groups have had access to a C1-esterase inhibitor (Berinert; CSL Behring, King of Prussia, PA)28,45-48 that is FDA-approved, and has been reported to be active in NHPs (Appendix). We have recently been able to obtain it, and we now include it in our IS regimen at a dose of 17.5 U/kg IV on days 0, 1, 7, and 1428 to replace CVF.

In the first trial in humans (who we suggest will be selected on the basis of having no pretransplant antibodies against TKO pig cells13,49), the administration of a complement inhibitor should be unnecessary, particularly if the pig organ expresses high levels of human complement-regulatory (and coagulation-regulatory) proteins. However, the administration of a C1-inhibitor or eculizumab would provide additional assurance that complement activation would not impact the initial success of the transplant (Table 3).

MAINTENANCE THERAPY

As our experience with conventional calcineurin-based IS has not been successful in preventing immune injury in pig-to-NHP organ transplantation models (even when transplanting advanced genetically engineered pig organs protected to some extent from the primate immune response),10,15-17 we have concentrated our attention on costimulation blockade, particularly of the CD40/CD154 pathway.4-6,10,15,50

Anti-CD40 Monoclonal Antibody

This monoclonal antibody (mAb; 2C10R), first introduced into xenotransplantation by Mohiuddin et al,51-53 prevents stimulation of B and T cells through the CD40/CD154 (CD40L) pathway.54 We initiate therapy with 2C10R on day −1, and it is administered throughout the course of the experiment. The dosing schedule, based on our experience4,6,10 and that of others,27,51-53 is indicated in Table 1.

With now several years’ experience with this agent, we have seen no complications directly attributable to it. We and others have measured the level in the blood, and have found a level of 1000 µg/mL to be adequate. (It is possible that a lower level might be sufficient, but to ensure satisfactory suppression of T-cell activation in the expensive pig-to-NHP experiments, we have maintained this dosage, and have not tested lower doses.) This agent is not yet FDA-approved.

Previously, we gained considerable experience with an anti-CD154 mAb in our pig-to-baboon organ transplantation model,5,15,55-61 but this was associated with platelet activation and the development of thrombotic microangiopathy, although we did not see thrombotic complications after pig islet transplantation.62,63 Some anti-CD154 agents (that are not associated with the complication of thrombotic microangiopathy) and some anti-CD40 agents are in various clinical trials, for example, in autoimmune disorders (Table 5). One of these will be required for the first clinical trial (although none is yet FDA-approved).

TABLE 5. - Agents that block the CD40/CD154 costimulation pathway that are currently in clinical trials
Drug name and company Class Characteristics Completed clinical trials Animal studies Adverse effects Ongoing clinical trials
Bleselumab (ASKP124/4D11)
Astellas
Anti-CD40 mAb (IgG4).64
Fully human mAb.65,66
More potent than other anti-CD40 mAbs, but equivalent to or less potent than anti-CD154 mAbs.67
No B-cell depletion.68,69
Phase Ia:
Well-tolerated in healthy humans.70
Phase Ib:
Generally, well-tolerated in de novo kidney transplant recipients, larger studies are required to assess efficacy.69
Phase II:
Kidney transplantation.68
Generally, well-tolerated in patients with moderate-to-severe plaque psoriasis. Further assessment of efficacy is still required.71
Not thrombogenic, and had no significant toxicity. in cynomolgus monkeys.65
Improved kidney, liver, and islets allograft survival in cynomolgus monkeys.64,65,72-74
Transient elevation of liver enzymes without change in bilirubin in 8.2% of patients with 1 mg/kg dose, and 4.1% with 3 mg/kg dose).69,71
Viral infections.68
Phase II
Kidney transplantation (NCT02921789)
Iscalimab (CFZ533)
Novartis
Anti-CD40 mAb, class IgG1.75
Fully human, a glycosylated mAb with Fc-silent region.76
Does not deplete peripheral blood B-cells.77 Phase I/II:
Kidney transplantation.78,79
Phase II
:Has therapeutic potential in patients with primary Sjogren’s syndrome and Graves’ disease. Further investigation is required.80,81
Good safety and tolerability profile of weekly subcutaneous doses of <150 mg/kg for 26 wk in cynomolgus monkeys.82
Improved kidney allograft survival in cynomolgus monkeys.77
Showed variable serum concentration, especially with lower doses.76 Phase II:
Kidney transplantation (NCT03663335).
Liver transplantation (NCT03781414).
Lupus nephritis (NCT03610516).
Sjogren’s syndrome (NCT03905525).
BI 655064
Boehringer Ingelheim
Anti-CD40 mAb, class IgG1.
Humanized mAb with Fc mutation.83
Phase I:
Well-tolerated in healthy humans.84
Phase II:
Favorable safety profile in patients with rheumatoid arthritis with inadequate response to methotrexate.85
In cynomolgus monkeys, BI 655064 showed potent inhibition of CD40L without alteration to platelets function.86 No demonstration of clinical efficacy yet.85 Phase II:
Lupus nephritis (NCT03385564, NCT02770170).
Dapirolizumab (CDP7657)
UCB AND BIOGEN
Anti-CD154 mAb
Fab antibody fragment conjugated to PEG87,88
No thromboembolism.89 Phase I:
Generally, well-tolerated in healthy humans and in patients with SLE.90
Preliminary evidence of safety and efficacy in SLE patients, but larger sample size is required to provide statistical significance.87
In rhesus macaques, Fc-silent anti-CD154 mAb is effective and not associated with thromboembolic complications.88 Increased rate of infection.87 Phase II:
SLE (NCT02804763).
Letolizumab (BMS-986004)
BMS
Humanized anti-CD154
dAb fused to the Fc portion of a modified IgG1.9
No thromboembolism.
Might be associated with increased Treg population.54
Phase I/II:
Immune ITP. Still under investigation (NCT02273960).
Prolonged survival of rhesus macaque renal allografts with preserved graft function, particularly when combined with conventional immunosuppressive therapy.9 Phase I/II:
GVHD; NCT03605927.
VIB4920 (MEDI4920)
VielaBio
CD154-specific nonantibody protein.91,92
Scaffold protein uses an engineered Tn3 molecule.92
No thromboembolism.93 Phase I:
Generally, well tolerated in patients with rheumatoid arthritis.(NCT02780388).
Phase II:
Has therapeutic benefit in autoimmune diseases.93
Favorable safety profile in cynomolgus monkeys. Dose-dependent inhibition of antigen-specific immune responses.94. Phase II:
Kidney transplantation (NCT04046549).
Sjogren’s syndrome (NCT04129164).
Rheumatoid arthritis (NCT04163991).
GVHD, graft-vs-host disease; ITP. immune thrombocytopenic purpura; mAb, monoclobal antibody; PEG, polyethylene glycol; SLE, systemic lupus erythematosus; TCP, thrombocytopenic purpura.

An anti-CD154 agent may have some advantages over an anti-CD40 agent,95,96 and so, if successful in our pig-to-baboon model, we might plan to use an anti-CD154 agent in the first clinical trial (Table 3). In our experience, CTLA4-Ig is effective in preventing a T-cell response in vitro, but not in vivo.10,61

Antibody-mediated rejection of pig grafts has certainly been recorded, though it is uncertain whether these have been associated with loss of control of the adaptive immune response or from continuing activity of the innate immune system10 (J.B. Foote, unpublished data, 2020). The continuing production of natural cytotoxic antibodies may be a more important factor than failure to suppress the adaptive immune response.

Rapamycin

We administer rapamycin mainly to suppress the T-cell response (as we believe costimulation blockade does not achieve this completely).97,98 In baboons, we administer a dose aimed at maintaining a trough level of approximately 6–10 ng/mL, although a level of >4 ng/mL may be sufficient (Table 1).99

We selected rapamycin in preference to other agents, for example, mycophenolate mofetil (MMF), simply because it can be administered intramuscularly (IM), whereas MMF cannot. The IM route ensures the baboon receives the desired dose, which is not always the case if an agent is administered orally (as the NHP can spit it out). However, rapamycin may have other beneficial effects in xenotransplantation.

In addition to its IS effect, rapamycin (a) is known to facilitate an increase in the number of T regulatory cells (that may help suppress the adaptive immune response and prolong graft survival), (b) has an anti-inflammatory effect, and (c) is associated with a reduced incidence of malignancy and possibly viral infection.100-113

In addition, rapamycin may be a factor in reducing the rapid growth of pig organs early after transplantation that has been reported in NHPs.4,6,114-118 Importantly, we have seen no failure of abdominal wound healing in the presence of rapamycin therapy. Data from other xenotransplantation research groups, however, indicate that MMF is equally effective.3,27,51-53

Both rapamycin and MMF have some advantages. In combination with tacrolimus, MMF therapy was reported to be associated with better renal function than rapamycin,119 whereas after simultaneous kidney-pancreas transplantation, a tacrolimus-rapamycin regimen demonstrated significantly reduced rates of acute rejection.120 As our preclinical studies in recent years have all included rapamycin (and not MMF), in the first clinical trial, everolimus or sirolimus would be administered orally, though everolimus may be preferred as it has fewer side effects.121-124

Methylprednisolone

We administer methylprednisolone beginning at a dose of 5 mg/kg/d on day 0, tapering to a maintenance dose of 0.125 mg/kg daily by day 7 (Table 1). This policy is based on years of experience of corticosteroid therapy in organ allotransplantation. In a clinical trial, we would administer methylprednisolone IV until day 7 and then convert to prednisone PO thereafter (Table 3).

COMMENT ON THE IS REGIMEN

We suggest that the IS regimen that we have used in our pig-to-baboon model to date (outlined here previously) is similar or less intensive than many regimens used in patients undergoing either an ABO-incompatible kidney allotransplant (high titer of ABO antibody)125 or allotransplantation in the presence of anti-HLA antibodies.126 In particular, the induction therapy is comparable to that used in some patients undergoing allotransplantation, and the doses of rapamycin and methylprednisolone are similar to those used in allotransplantation.

It is only the effect of the costimulation blockade agent (either an anti-CD154 or an anti-CD40 agent) that is uncertain when compared with the conventional pharmacologic IS agents commonly used, for example, tacrolimus. However, experience with these and similar agents is steadily being obtained in patients with autoimmune disorders75,80,127-130 and, to a lesser extent, in patients with organ allotransplants.68,69 The evidence to date, although limited, is that these agents are not associated with a greater incidence of posttransplant complications, for example, infection, posttransplant lymphoproliferative disease, malignant tumors, than conventional therapy.68,75,80,82,128,129

ADJUNCTIVE THERAPY

Anti-inflammatory Agents

Tumor Necrosis Factor-Alpha Blockade

We have evidence for a prolonged systemic inflammatory response after pig organ transplantation in NHPs.131-134 This is associated with an increase in the level of tumor necrosis factor-alpha (TNF-α), and so we have chosen to administer 4 doses of an anti-TNF-α agent (etanercept; Enbrel, Amgen, Thousand Oaks, CA) to try to suppress this response in the early posttransplant period (Table 1; Appendix).135 The TNF-α response may be associated with ischemia-reperfusion injury.136,137 We have no conclusive evidence of the beneficial effect of etanercept, and now that pigs expressing hemeoxygenase-1 (HO-1) are becoming available to us, it may be unnecessary to administer this agent.

However, suppression of the systemic inflammatory response by etanercept (rather than just by local protection of the graft by HO-1) may well be advantageous.138 After transplantation, inflammatory cells (eg, macrophage) produce inflammatory cytokines, including TNF-α. These cytokines activate both recipient and donor cells and recruit immune cells to the graft. As TNF-α is a potent inflammatory cytokine, in the absence of systemic TNF-α blockade, expression of HO-1 on the graft might be insufficient to suppress its effect after reperfusion.

Interleukin-6 Receptor Blockade

For some time, we included an interleukin-6 receptor-blocking mAb, tocilizumab, in our regimen as we had good evidence that it suppressed the systemic inflammatory response we had identified after pig organ transplantation in NHPs.132,133,139,140 However, subsequent studies indicated that, although tocilizumab blocked the binding of interleukin-6 (IL-6) to baboon tissues, it did not prevent binding to pig tissues.141,142 After its administration, the circulating level of IL-6 increased significantly (because it was blocked from binding to baboon IL-6 receptors), and the fact that it could only bind to the pig graft suggested to us that it may well be detrimental to the outcome of the graft. We suspected that it might be a factor in the development of histopathologic features of inflammation, associated with ureteric stenosis, seen in some grafts after transplantation6 (J.B. Foote, unpublished data, 2020). Although tocilizumab is no longer part of our regimen, siltuximab, which binds to soluble IL-6, and therefore reduces IL-6 binding to both baboon and pig tissues, warrants further investigation. In view of the association of IL-6 pathway blockade with an increase in Treg and in suppression of B-cell differentiation,143-145 we suggest that siltuximab may play a beneficial role in xenotransplantation.

Stimulation of Aerobic Metabolism Posttransplantation

There is now conclusive evidence that in deceased human potential organ donors and in patients undergoing open-heart surgery on cardiopulmonary bypass, there is a reduction in serum triiodothyronine (T3) levels, which is associated with reduced aerobic metabolism and increased anaerobic metabolism.146-149 We have documented subnormal levels of serum T3 in baboons during the first few days after pig kidney and heart xenotransplantation.150,151 It has become our practice to administer T3 to baboons immediately before reperfusion of the pig organ and for the subsequent 5 days (Table 1). We have documented excellent function of life-supporting kidney grafts throughout the early posttransplant period.

We would plan to include a modified regimen of T3 therapy in patients undergoing pig kidney transplantation (Table 3).

Prophylaxis

Prophylaxis Against Postoperative Bacterial Infection

In baboons, a 3-d perioperative course of cefazolin is administered to reduce the risk of infection, for example, systemic, pneumonia, and wound. The first dose is administered immediately before the transplant is carried out. This could be replaced by any other suitable prophylactic regimen used in the clinic. Furthermore, a 3-d course may be unnecessary, and in the first clinical trial, a shorter course may suffice (Table 3).152

Prophylaxis Against Cytomegalovirus Infection

Cytomegalovirus (CMV) is carried by a majority of humans and NHPs (unless specific pathogen-free) and, in an immunosuppressed state, can be activated and cause major infection (and exacerbate rejection153-155). After an organ transplant, CMV may be of donor or recipient origin. In view of its activation in immunosuppressed patients, prophylaxis is commonly given to patients undergoing organ allotransplantation.156 There have been several studies of CMV in relation to pig organ xenotransplantation.153-155,157

In our experimental xenotransplantation studies, therefore, although the Revivicor organ-source pigs are CMV-negative, there is a risk of activation of recipient baboon CMV, and so we have provided prophylaxis against this risk (Table 1). When IV access is available, for example, during the first 2 posttransplant weeks when an indwelling IV catheter is in situ, or whenever the baboon is sedated for a blood draw (usually ×2 weekly), ganciclovir is administered IV. On all other days, valganciclovir is administered orally (which appears to be palatable to the baboons in our studies). Using this regimen, we have not experienced a clinically obvious CMV infection.

More recently, we have been able to obtain baboons that are CMV-negative. A study of 12 baboons that underwent CMV-negative pig kidney or heart transplantation and were immunosuppressed with the aforementioned regimen (Table 1), demonstrated that there was no activation of baboon CMV over periods of up to 9 mo, either in baboons that retained a functioning pig kidney or heart or in those that rejected their graft (H. Iwase, unpublished data, 2020).

In clinical organ transplantation, if deemed necessary, prophylaxis with valganciclovir PO is usually begun within the first 2 wk, and we would follow this protocol (Table 3).

We have also monitored for Epstein-Barr virus (EBV) activation and have documented that the great majority of baboons that are negative for EBV pretransplant remained negative throughout the posttransplant course (H. Iwase, unpublished data, 2020). These data indicate that CMV-negative and EBV-negative baboons do not become positive for these viruses despite a prolonged period of IS, suggesting that the basic regimen appears to be safe, unless additional intensive therapy has to be administered for antibody-mediated rejection episodes (H. Iwase 2020, unpublished data, 2020).

Prophylaxis Against Pneumocystis

One baboon in recent years developed a pneumocystis pneumonia, and so we now administer Bactrim (sulfamethoxazole and trimethoprim) throughout the posttransplant period of follow-up (Table 1). In immunosuppressed patients with organ allografts, this policy is followed for at least the first 6–12 mo posttransplant (until the intensity of IS has been reduced).158,159 This would be our policy in the first clinical trial of pig kidney transplantation (Table 3).

Prophylaxis Against Thrombotic Complications

The development of a thrombotic microangiopathy in the graft, sometimes associated with a consumptive coagulopathy in the recipient, was a major problem in experimental xenotransplantation until transgenes for human coagulation-regulatory proteins were introduced into the organ-source pig.5,6,10,51,53,160 Our recent data indicate that thrombotic microangiopathy and consumptive coagulopathy are no longer problematic (J.B. Foote, unpublished data, 2020). However, there is still perceived to be a risk. With the organ-source pigs currently in use by our group (that express both human thrombomodulin and endothelial protein C receptor), this complication is now rare. Nevertheless, we still administer low-dose aspirin (for 6 mo) and low molecular weight heparin (for 1 mo) as prophylaxis (Table 1).

Whether this is now necessary or beneficial remains uncertain. However, as the therapy is relatively safe and may help resist the development of thrombotic microangiopathy, in the first clinical trial we will continue to include both of these agents in the regimen (Table 3).

Prophylaxis Against Anemia

It has been reported that pig erythropoietin, produced by the kidney, does not function in humans (and thus possibly not in NHPs).161 However, we have been unable to find any conclusive evidence for this in the literature, and we are currently investigating this point ourselves in our pig-to-baboon kidney transplantation studies.

Nevertheless, because the recipient baboons in our studies are receiving IS (thus suppressing the production of cells in the bone marrow), and blood is drawn for various tests and assays at frequent intervals, it has been our policy to administer recombinant human erythropoietin (rhEPO) ×2–3 weekly to the recipient baboon (Table 1). Clinically significant anemia has not developed. In 1 recent baboon, we discontinued rhEPO therapy after 6 mo, and did not observe any decrease in the hematocrit or hemoglobin during the next 3 mo (H. Iwase 2020, unpublished data, 2020.)

In the first clinical trial, if the hematocrit falls to <20%, it would be our policy to administer rhEPO to the patients for at least 3 mo (Table 3) and longer if there is continuing evidence of a low hematocrit or hemoglobin.

Prophylaxis Against Gastritis/Peptic Ulceration

An NHP wearing the jacket and tether that is used to facilitate blood draw and fluid/drug administration in the perioperative period is believed to be under stress (although the jacket/tether does not place any limitation on activity). In the opinion of many veterinarians, these baboons are susceptible to peptic ulceration. During this period, famotidine is administered to reduce this risk (Table 1). We have not seen peptic ulceration in >20 y in NHPs receiving famotidine, but we have no control group with which this experience can be compared.

In the first clinical trial, the need for prophylaxis for stress would possibly be reduced. The policy in patients undergoing allotransplantation would be followed (Table 3). In general, patients usually receive either an H2 blocker or a proton pump inhibitor.

Comment on the Adjunctive Therapy

The adjunctive therapy we have been employing in our preclinical model is similar to that used in patients with kidney allografts today. There should be no greater risk of complications from it than seen in patients with allografts.

DISCUSSION

The innate immune response (which was the major cause of graft failure in the early days of xenotransplantation research) has now largely been resolved by the introduction of genetically engineered pigs in which the known xenoantigens have been deleted and human “protective” transgenes expressed.43,44 However, the adaptive immune response needs to be controlled to prevent a T-cell response and de novo elicited antibody production. In the future, this will almost certainly be achieved by further genetic modification of the organ-source pig, but at present, it needs to be controlled by IS. The regimen outlined in Table 1 appears to have been largely successful in this respect.

The role of anti-inflammatory agents remains unproven, but, in view of the considerable evidence of a systemic inflammatory response to the presence of a xenograft,131,134,139,140 it seems important to suppress this response, at least during the early posttransplant period. It also seems necessary to provide prophylaxis against the several other complications of xenotransplantation and/or IS that may develop, for example, infection, thrombosis, and anemia.

As discussed briefly previously, the major difference in the IS regimen planned for the first clinical trials of pig organ xenotransplantation from those used today in clinical allotransplantation is the replacement of a calcineurin inhibitor by a costimulation blockade agent. However, this does not appear to increase the intensity of the regimen or its complications. Furthermore, a regimen based on inhibition of the CD40/CD154 costimulation pathway may be less nephrotoxic than a conventional IS regimen that includes tacrolimus or cyclosporine.65,69,71,72

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        APPENDIX: MECHANISMS OF ACTION AND POTENTIAL SIDE EFFECTS OF THE IMMUNOSUPPRESSIVE AND ANTI-INFLAMMATORY AGENTS INCLUDED IN OUR EXPERIMENTAL PIG-TO-BABOON STUDIES AND/OR PROPOSED FOR A CLINICAL TRIAL OF PIG KIDNEY OR HEART XENOTRANSPLANTATION

        INDUCTION THERAPY

        ATG (Genzyme, Cambridge, MA)

        ATG acts mainly on depletion of peripheral circulating T-lymphocytes through complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and apoptosis (depletion of T cells in lymphoid tissue),19 hence preventing cellular rejection. The main action of ATG is to deplete peripheral T-lymphocytes.20 However, data suggest that ATG to some extent affects other key cells involved in the immune reaction (eg, B cells, natural killer-T cells, and dendritic cells), via modulation of surface adhesion molecules or chemokine receptor expression.162 ATG can cause some adverse events, such as cytokine release syndrome (fever, chills, and nausea/vomiting) and thrombocytopenia.21,163,164

        Anti-CD20 mAb (Rituximab, Genentech, South San Francisco, CA)

        Rituximab is a mAb, which targets CD20, a protein expressed on the surface of B cells. It acts by depleting CD20+ B cells mainly through 3 different mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and apoptosis).33 When used in high dose, Rituximab can be associated with infection resulting from hypogammaglobulinemia.165 However, the reduced dose used in patients undergoing ABO-incompatible kidney transplantation has less risk of serious infection.166 Rituximab therapy can be associated with neutropenia and thrombocytopenia.167,168

        CVF (Complement Technology, Tyler, TX)

        CVF is a complement-activating protein isolated from cobra venom. It is an analog of complement C3.169 Both CVF and C3b can bind to factor B and subsequently form the C3/C5 convertases (CVF, Bb) or (C3b, Bb), respectively,170 activating the alternative complement pathway.171 However, (CVF, Bb) is more stable than (C3b, Bb)172 and is also resistant to inactivation by the regulatory proteins (factor H and factor I).173 Because of that, (CVF, Bb) will continuously consume C3 and C5, eventually leading to complement depletion.174 Possible adverse events appear to be related to either the sudden release of C3a and C5a during complement activation, that might cause mild transient lung inflammation,175,176 or from a reduced serum complement activity.177 Complement depletion by CVF will never completely eliminate C3, allowing for residual, but important, protection against infections.178

        C1-esterase Inhibitor (C1-INH, Berinert; CSL Behring) (if Required)

        C1-esterase inhibitor is a potent complement-blocking agent (a serum protease inhibitor [serpin]) that binds covalently and inactivates C1r, C1s, and mannan-binding protein-associated proteases,46 thus inhibiting the first enzymes in the classical and lectin pathways, leaving the alternative pathway relatively unaffected.48 C1-INH is associated with some adverse effects, for example, headache, nausea/vomiting, and rash.47 Studies in a larger group of patients with longer follow-up are needed to fully establish its safety in transplantation.45

        MAINTENANCE THERAPY

        Anti-CD40 mAb (2C10R4)

        The anti-CD40mAb (2C10R4; NIH NHP Resource Center, Boston, MA) acts by binding to and interrupting the CD40/CD40L costimulation pathway, which has been shown to be a potent stimulator of B and T-cell activation through conventional antigen-presenting cell interactions.50 Anti-CD40 mAb has been widely used in pig organ xenotransplantation experiments in NHPs without serious adverse events.4,6 In clinical trials, for example, in autoimmune disorders, the most common adverse effects have been headache and cough.68-70

        Rapamycin (Sirolimus)

        Rapamycin (Sirolimus; LC Laboratories, Woburn, MA) is a macrolide isolated from Streptomyces hygroscopicus. It modulates the activity of the mammalian target of rapamycin, which in turn inhibits IL-2 activity, resulting in cell-cycle arrest in the G1-S phase, thus inhibiting T-cell proliferation.179 Because the mammalian target of rapamycin is essential for cell metabolism, its modulation can result in undesired adverse effects, for example, (a) impaired wound healing,180 (b) anemia, which is generally mild and dose-dependent,181 and (c) thrombocytopenia.182

        Methylprednisolone

        Methylprednisolone binds to the intracellular glucocorticoid receptor and translocates into the nucleus, where it interacts directly with specific DNA sequences known as glucocorticoid-responsive elements, finally upregulating anti-inflammatory genes (eg, IL-1 receptor-II and IL-10) and downregulating proinflammatory genes (eg, IL-1-α and IL-1-β).183,184 It also competes for or blocks the function of transcription factors, such as nuclear factor-kappa-B and activator protein-1, which are required for transcription of proinflammatory mediators.185 Methylprednisolone affects monocytes and macrophages by decreasing the production of monocyte/macrophage inflammatory cytokines (IL-1 and TNF-α) and phagocytic function.186 Antigen presentation and expression are also downregulated.187 Collectively, these account for the significant impact of methylprednisolone on acquired immunity. Among the adverse effects of long-term methylprednisolone administration are hypertension, hyperlipidemia, hyperglycemia, and osteoporosis.188 Because of its numerous side effects, efforts are being made to minimize or even avoid methylprednisolone therapy.189

        ANTI-INFLAMMATORY THERAPY

        TNF-α Inhibitor (Etanercept)

        The TNF-α inhibitor, etanercept, is composed of 2 extracellular ligand-binding domains of the TNF p75 receptor linked to the Fc portion of human IgG1.190 It functions as an anti-inflammatory agent through TNF-α inhibition by competitively binding to TNF-α and preventing its activation of the inflammatory cascade.135,191 Adverse events associated with etanercept are neutropenia and thrombocytopenia.192

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