Cardiac xenotransplantation: from concept to clinic

Abstract

For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by the consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent 'compassionate use' transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for 2 months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Additional topics discussed include relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years.

Keywords: Heart; Non-human primate; Pig; Xenotransplantation clinical; Xenotransplantation experimental.

Graphical Abstract

Figure 1

Histopathological features of hyperacute rejection of a wild-type pig heart after transplantation into a baboon—interstitial haemorrhage, oedema, capillary occlusion; haematoxylin & eosin (HE); bar = 50 µm.

Figure 2

(A and B) Geometric mean (GM) binding and age correlation of human serum IgM (A) and IgG (B) antibodies to wild-type (WT) pig red blood cells (RBCs). There is a steady increase in IgM and IgG during the first year of life. (C and D) GM binding and age correlation of human serum IgM (C) and IgG (D) antibodies to TKO pig RBCs. There is virtually no increase in IgM or IgG antibodies during the first year of life. (Note the great difference in the scale on the Y-axis between top and bottom. The dotted lines indicate no IgM or IgG binding) (reproduced with permission from Li et al.).

Figure 3

Mechanisms of xenograft rejection and strategies to overcome them. (A) Hyperacute rejection of pig-to-primate xenografts (HAR) is triggered by the binding of recipient’s preformed natural antibodies to specific carbohydrate antigens [αGal, Neu5Gc, Sd(a)] on the surface of pig cells and subsequent activation of the complement system. In addition, bound antibodies activate natural killer (NK) cells via Fc-receptors (FcR) causing antibody-dependent cellular cytotoxicity (ADCC) by the release of lytic granules. In order to overcome HXR, donor pigs are genetically multi-modified to lack specific glycosyltransferases [α-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), β-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2), and a recently discovered B4GALNT2-like (B4GALNT2L) enzyme] and to express one or several human (h) complement-regulatory proteins [membrane cofactor protein (CD46), decay-accelerating factor (CD55), membrane inhibitor of reactive lysis (CD59)] to prevent complement-mediated cell lysis via formation of membrane attack complexes (MACs). (B) Responses of NK cells and macrophages. In addition to ADCC, NK cells exhibit direct cytotoxicity of pig cells because swine leucocyte antigens (SLAs) do not effectively bind to inhibitor receptors of human/NHP NK cells (KIRs) to prevent their activation. Additionally, activating signals, resulting from activating NK cell ligands (ALs) on pig cells with their corresponding activating receptors (ARs) on primate NK cells may be involved. NK cell activation may be prevented by expressing HLA-E/beta2-microglobulin (B2M) in transgenic pigs. HLA-E binds the inhibitory NK cell receptor CD94/NKG2. Macrophages are activated by FcRs binding the Fc portion of anti-pig antibodies. In addition, they are activated by galectin-3 binding αGal on pig cells. Porcine (p) CD47 does not activate the ‘don’t eat me’ receptor signal regulatory protein-α (SIRPα) on human macrophages. Therefore, transgenic pigs expressing hCD47 were generated to inhibit macrophage activity against xenogeneic cells. (C) Activation of T cells against xenotransplants may occur directly via porcine antigen-presenting cells (APCs) or indirectly via human/primate APCs presenting porcine peptides. In addition to the interaction of the peptide-presenting major histocompatibility complex (MHC) with the T-cell receptor (TCR), costimulatory signals are required, most importantly CD40—CD40L (CD154), which can be blocked by treatment with anti-CD40 and/or anti-CD40L antibodies to prevent T-cell activation. Another costimulatory pathway, CD80/CD86—CD28, can be blocked by treatment with CTLA4-Ig or its affinity-optimized variant LEA29Y. Another strategy is the involvement of the coinhibitory pathway PD1—PD-L1 expressing hPD-L1 in transgenic pigs. Finally, pigs lacking SLAs or expressing SLAs with reduced activating capacity have been produced to reduce T-cell activation via the direct pathway.

Figure 4

Effect of xenoantigen knockout and expression of complement inhibitors on serum cytotoxicity as measured by image-based complement-dependent cytotoxicity assay. Cytotoxicity decreased significantly from wild type with each additional knockout (columns with different superscripts, P < 0.05). Cytotoxicity was nearly eliminated when CRPs were expressed as either hCD46 alone or multi-copy hCD46, or from a single-copy bicistron composed of hCD46 and hCD55. All genotypes, except wild type, include a GGTA1-KO background (reproduced with permission from Eyestone et al.).

Figure 5

Models of pig-to-baboon cardiac xenotransplantation: (A) heterotopic abdominal, (B) heterotopic thoracic, (C) orthotopic techniques, (D) donor, and (R) recipient (modified with permission from Mohiuddin et al.).

Figure 6

Stepwise clinical translation of cardiac xenotransplantation. After the first compassionate use of a 10 × GM pig heart for a patient, ongoing pre-clinical studies need to be finalized and the donor pigs need to be transferred to a designated pathogen-free (DPF) status to get approval for a clinical pilot trial. After successful completion, stepwise expansion to routine clinical use can be envisaged.