Initial evidence that blockade of the CD40/CD154 costimulation pathway alone is sufficient as maintenance therapy in xenotransplantation

The editor

We wish to report an observation that we believe may be important to the future development of clinical pig organ xenotransplantation.

There has been considerable discussion on the strength of the primate immune response to a pig organ, even if the graft comes from a pig genetically-engineered to protect the organ from the human innate immune response (1). It has frequently been suggested that the primate adaptive immune response to a pig xenograft is stronger than to an allograft, and that the intensity of immunosuppressive therapy will need to be greater than in allotransplantation. We determined to test whether this was the case.

A baboon (B4918) that had received a kidney graft from a GTKO.hCD46.hTBM (thrombomodulin) pig (Revivicor, Blacksburg, VA) was administered our current immunosuppressive regimen (induction with anti-thymocyte globulin, anti-CD20mAb (rituximab), and a C-1 esterase inhibitor (Berinert); maintenance with an anti-CD40mAb (2C10), rapamycin, and methylprednisolone) and adjunctive therapy (Table1) (2). The aim of the study was to measure renal function, and various tests were carried out at intervals over the first 8 weeks’ post-transplantation. During this period, there were no features suggesting rejection of the graft. The serum creatinine remained <1.5mg/dl, there was no or minimal proteinuria, and good renal blood flow was demonstrated on ultrasound (carried out at least weekly).

Table 1:

Summary of immunosuppressive, anti-inflammatory, and adjunctive drug therapy administered to B4918 during the first post-transplant 8 weeks

Agent	Dose (duration)
Induction
Thymoglobulin (ATG) (Genzyme, Cambridge, MA)	5 mg/kg i.v. (days −3 and −1) (to reduce the CD3+T cell count to <500/mm3)
Anti-CD20mAb (rituximab) (Genentech, South San Francisco, CA)	10 mg/kg i.v. (day −2)
C1-esterase inhibitor (Berinert, CSL Behring, King of Prussia, PA)	17.5 U/kg i.v. (days 0, 1, 7 and 14)
Maintenance
Anti-CD40 monoclonal antibody (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 i.m. ×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 i.v. (days 3, 7, 10)
Adjunctive
Aspirin (Bayer, Deland, FL)	40 mg p.o. (alternate days), beginning on day 4.
Low molecular weight heparin (LMWH) (Eisai, Woodcliff Lake, NJ)	700 IU/d s.c., beginning of day 1.
Erythropoietin (Amgen)	500 U i.v. x2–3 weekly, beginning on day −4
Ganciclovir (Genentech)	5 mg/kg/d i.v., from day −4 to day 14 and when the baboon is sedated for blood draws (x2 weekly).
Valganciclovir (Genentech)	15 mg/kg/d p.o., beginning on day 15
Sulfamethoxazole and trimethoprim (Teva, North Wales, PA)	10 mg/kg i.v. daily, on days 4–14
Sulfamethoxazole and trimethoprim oral suspension (Akorn, Lake Forest, IL)	75 mg/m2 p.o x2/day. x3 weekly, beginning on day 15.

When the renal function studies were completed, we discontinued all drug therapy, except the anti-CD40mAb (and erythropoietin, which was inadvertently continued at weekly intervals). Throughout the subsequent two months, the baboon remained active and well in all respects, eating well, and gaining weight. The serum creatinine remained <1.5mg/dl (except on one occasion, when it increased to 1.6mg/dl, which we believe was associated with either relative dehydration (3) or was an error because, when checked 3 days later, it had normalized without any additional therapy except subcutaneous normal saline). There was no proteinuria, and renal blood flow remained unchanged. The experiment was only terminated because we were closing our laboratory and felt that histopathology was needed to confirm the absence of significant features of rejection. Necropsy revealed a macroscopically normal-looking kidney, with dilatation of the ureter secondary to a stricture at the ureteric-bladder anastomosis, and moderate hydronephrosis (that will be discussed in another report). On microscopic examination, there were minimal features of rejection (Figure1).

Figure 1: Histopathological features of pig kidney 8 weeks following discontinuation of all therapy, except anti-CD40mAb (and erythropoietin).

(A) Kidney, 20x, H+E image: Mild interstitial fibrosis and tubular dilation. (B) Kidney, 40x, H+E image: Perivascular lymphocytic infiltrate. (C) Kidney, 4x, Mason’s Tricrhrome: Mild interstitial fibrosis represented by the blue (staining collagen) Trichrome stain. (D) Kidney, 40x, Mason’s Trichrome: Mild interstitial fibrosis with lymphocytic infiltrate and mild tubular atrophy.

On one occasion previously, at 10 weeks’ post-transplantation, we discontinued only anti-CD40mAb, and the pig graft was rejected within 2 weeks (4). On the present occasion, when only anti-CD40mAb was continued (8 weeks’ post-transplant), there were no clinical or histopathological features of rejection during the following 8 weeks. We believe that this is an important observation and suggests that many of the additional post-transplant agents being administered, e.g., conventional immunosuppressive drugs, anticoagulants, anti-platelet and anti-inflammatory agents, may be unnecessary.

Whether we would have observed the same result if induction therapy had not been given or if anti-CD40mAb alone had been administered from day 1 remains uncertain, but the result does suggest that long-term maintenance therapy may not be essential, thus simplifying the regimen considerably.

As there is increasing evidence that costimulation blockade with an anti-CD154 agent is more successful than with an anti-CD40 agent (5,6), a regimen based on an anti-CD154 agent may be even more successful as maintenance therapy in the absence of additional immunosuppressive agents. We suggest that such a simplified regimen is worthy of study.