Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft

Abstract

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

Figure 1. Expression of TBM transgene.

(a) Donor pigs with two distinct hTBM transgenes were used. ICAM-TBM and TBM-TBM. (b) Describes the hTBM expression in porcine aortic endothelial cell (PAECs) from donor pig using FACS analyses (i) and immunohistochemistry staining (ii) of pig heart tissue using human anti-porcine CD31 antibody. (c) Demonstrates the TBM molecule expression in heart tissue from the littermates of donor pigs for baboons #910 and #510 (i and ii), human heart section (iii) and wild-type pig heart section (iv). Scale bar, 50 μm.

Figure 2. Cardiac xenograft survival.

(a) Survival graph showing cardiac xenograft survival for five baboons. It also indicates the time points when the 2C10R4 dose was reduced. The long-term graft survival in two animals is plotted separately to give clear idea of the time points for antibody dose reduction. In two animals, the dose was reduced to 25 mg kg⁻¹ on day 100 (green arrow); in two long-term surviving animals dose reduction to 25 mg kg⁻¹ was made after 1 year and the antibody treatment was terminated in #910 on day 560 (blue arrow) and in #510 on day 861 (red arrow). (b) Detection of 2C10R4 levels in the plasma of the transplanted baboons. A blue triangle represents each dose. (c) Non-Gal (both IgM and IgG) antibody production in all baboons. The green and red dashed lines indicate the day αCD40 antibody treatment was stopped for baboons #910 and 510, respectively.

Figure 3. Histology of cardiac xenografts.

(a) Graft biopsy on day 182 (i) in baboon #210, demonstrating normal myocardial architecture; terminal histology of grafts from baboons #210 (ii, illustrating myocyte necrosis; #15009 (iii, showing focal myocarditis; and #110 (iv, showing myofibril loss with mineralization; × 400). (b) Baboon #910. Left panel shows normal histology of biopsy specimens obtained on days 196 (i), 350 (ii) and 567 (iii); right panel illustrates terminal histology, with multifocal fibrosis (iv), coagulative necrosis (v) and venular thrombi (vi). (c) Baboon #510. Left panel shows normal histology from biopsies on days 412 (i), 630 (ii) and 854 (iii). Middle and right panels show terminal graft histology, illustrating haemorrhage (iv), arteritis (v), coagulative necrosis (vi), neutrophilic infiltrates (vii), epicardial infiltrates (viii) and venular thrombi (ix). (d) Thrombomodulin expression in explanted grafts from baboons #510 and #910 by immunohistochemistry (IHC). Scale bar, 50 μm.

Figure 4. Laboratory blood test.

(a) Illustrates the levels of haematocrit (i), platelets (ii) and activated clotting time (iii) in each experimental animal. (b) Demonstrates the levels of troponin (i) and prothrombin time until day 600 (ii).

Figure 5. In vitro cellular assays.

(a) Mixed lymphocyte reaction of cells from baboons #910 (i) and #510 (ii). Two control specimens were used for each assay and each specimen was plated in triplicates; s.d. is also shown with bars. (b) Suppression of B-cell numbers in all five baboons for 60 days after αCD20 antibody treatment. (c)—(i) Describes the phenotypes of B lymphocytes from an untreated baboon; (ii) comparison of an untreated and experimental baboon (#510) phenotype with s.d. (d) Proliferation of B lymphocytes of naive, baboons #510 and #910 in response to human IgM antibody stimulation at one time point. (e) Antibody production from human IgM-stimulated B cells from long-term surviving baboons #510 and #910 at one time point. (f) The line graph demonstrates the rate of change in graft size in all five baboons. The time from termination of the 2C10R4 treatment to rejection is also indicated.