The xenoantibody response and immunoglobulin gene expression profile of cynomolgus monkeys transplanted with hDAF-transgenic porcine hearts.

BACKGROUND: Recent work has indicated a role for anti-Gal alpha 1-3Gal (Gal) and anti-non-Gal xenoantibodies in the primate humoral rejection response against human-decay accelerating factor (hDAF) transgenic pig organs. Our laboratory has shown that anti-porcine xenograft antibodies in humans and non-human primates are encoded by a small number of germline IgV(H) progenitors. In this study, we extended our analysis to identify the IgV(H) genes encoding xenoantibodies in immunosuppressed cynomolgus monkeys (Macaca fascicularis) transplanted with hDAF-transgenic pig organs. METHODS: Three immunosuppressed monkeys underwent heterotopic heart transplantation with hDAF porcine heart xenografts. Two of three animals were given GAS914, a poly-L-lysine derivative shown to bind to anti-Gal xenoantibodies and neutralize them. One animal rejected its heart at post-operative day (POD) 39; a second animal rejected the transplanted heart at POD 78. The third monkey was euthanized on POD 36 but the heart was not rejected. Peripheral blood leukocytes (PBL) and serum were obtained from each animal before and at multiple time points after transplantation. We analyzed the immune response by enzyme-linked immunosorbent assay (ELISA) to confirm whether anti-Gal or anti-non-Gal xenoantibodies were induced after graft placement. Immunoglobulin heavy-chain gene (V(H)) cDNA libraries were then produced and screened. We generated soluble single-chain antibodies (scFv) to establish the binding specificity of the cloned immunoglobulin genes. RESULTS: Despite immunosuppression, which included the use of the polymer GAS914, the two animals that rejected their hearts showed elevated levels of cytotoxic anti-pig red blood cell (RBC) antibodies and anti-pig aortic endothelial cell (PAEC) antibodies. The monkey that did not reject its graft showed a decline in serum anti-RBC, anti-PAEC, and anti-Gal xenoantibodies when compared with pre-transplant levels. A V(H)3 family gene with a high level of sequence similarity to an allele of V(H)3-11, designated V(H)3-11(cyno), was expressed at elevated levels in the monkey that was not given GAS914 and whose graft was not rejected until POD 78. IgM but not IgG xenoantibodies directed at N-acetyl lactosamine (a precursor of the Gal epitope) were also induced in this animal. We produced soluble scFv from this new gene to determine whether this antibody could bind to the Gal carbohydrate, and demonstrated that this protein was capable of blocking the binding of human serum xenoantibody to Gal oligosaccharide, as had previously been shown with human V(H)3-11 scFv. CONCLUSIONS: DAF-transgenic organs transplanted into cynomolgus monkeys induce anti-Gal and anti-non-Gal xenoantibody responses mediated by both IgM and IgG xenoantibodies. Anti-non-Gal xenoantibodies are induced at high levels in animals treated with GAS914. Antibodies that bind to the Gal carbohydrate and to N-acetyl lactosamine are induced in the absence of GAS914 treatment. The animal whose heart remained beating for 78 days demonstrated increased usage of an antibody encoded by a germline progenitor that is structurally related, but distinct from IGHV311. This antibody binds to the Gal carbohydrate but does not induce the rapid rejection of the xenograft when expressed at high levels as early as day 8 post-transplantation.

Improved assessment of graft function by echocardiography in cynomolgus monkey recipients of hDAF-transgenic pig cardiac xenografts.

BACKGROUND: The current practice of evaluating heterotopic heart xenografts by palpation allows only detection of severe graft dysfunction, which indicates terminal graft failure. Therefore, we evaluated whether echocardiography is a better method of detecting early graft dysfunction as a marker of rejection in abdominal pig heart xenografts in cynomolgus monkeys. METHODS: Six cynomolgus monkeys received heterotopic heart transplants from pig donors transgenic for human decay-accelerating factor (hDAF). Induction therapy consisted of either cyclophosphamide or rabbit anti-thymocyte globulin. Maintenance therapy consisted of cyclosporine or tacrolimus, steroids, and sodium mycophenolate or mycophenolate mofetil, GAS914 (alphaGal oligosaccharide containing glycoconjugate), and for some animals TP10 (soluble complement receptor type 1). Echocardiography was performed immediately after transplantation and 3 times a week after surgery. We scored contractility and measured left ventricular wall thickness. Impaired contractility or increased wall thickness were considered graft dysfunction and were treated with pulse steroids. Palpation score was recorded daily. We also obtained myocardial biopsy specimens. RESULTS: Palpation score remained at 4 out of 4 in all animals until 2 to 5 days before final graft failure, whereas echocardiography detected several episodes of impaired graft function, either decreased left ventricular contractility or increased left ventricular wall thickness before graft failure. Treatment with pulse steroids improved graft function only during early episodes of graft impairment. Final graft failure was steroid resistant and caused by severe vascular rejection. CONCLUSIONS: Echocardiography is a better method of assessing graft dysfunction than is palpation. Therefore, echocardiography may detect early rejection episodes of heterotopic heart xenografts in non-human primates.

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts.

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. RESULTS: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: influence of pre-existing anti-pig antibodies and prevention by the alpha GAL glycoconjugate GAS914.

BACKGROUND: Our introductory pig-to-cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay-accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti-pig antibodies. We evaluated the efficacy of GAS914, a Gal alpha 1-3Gal trisaccharide linked to a poly-l-lysine backbone, in inhibition of HAR. METHODS: hDAF transgenic heterotopic heart (n = 15) or life-supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti-thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti-pig antibody levels were determined in various assays. RESULTS: Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre-treatment, did not show HAR (chi-square, P < 0.05). Two of four kidney transplants without GAS914 treatment ended with HAR. Four subsequent transplants with GAS914 did not show HAR. Animals with HAR showed extremely high antibody levels. Samples just before transplantation showed significantly higher antibody levels in recipients presenting with HAR. In all assays antibody levels were significantly lowered by GAS914 pre-treatment. CONCLUSIONS: HAR of hDAF solid organs could be ascribed to high levels of anti-pig antibodies. It is hypothesized that the hDAF transgene shows a threshold in efficacy, above which an overwhelming attack by antibodies and complement activation cannot be modulated to prevent HAR. HAR does not occur when animals with lower levels are used, or when antibodies are effectively depleted from the circulation by GAS914 treatment.

Anti-non-Gal porcine endothelial cell antibodies in acute humoral xenograft rejection of hDAF-transgenic porcine hearts in cynomolgus monkeys.

BACKGROUND: Anti-Gal alpha 1-3Gal (Gal) antibodies play a major role in hyperacute rejection and acute humoral xenograft rejection (AHXR) in porcine-to-nonhuman primate transplantation. The role of anti-non-Gal antibodies in AHXR is less well defined. METHODS: Eleven cynomolgus monkeys received a heterotopic heart transplant from a human decay-accelerating factor transgenic pig, and maintenance immunosuppression with cyclosporin A or tacrolimus, steroids, mycophenolate sodium or mycophenolate mofetil, and in 10 animals the Gal-containing soluble glycoconjugate GAS914. Six ended with AHXR (6 to 78 day survival) and five did not show AHXR (9 to 36 day survival). Anti-Gal antibodies were depleted in vivo with GAS914, or in vitro with Gal-coated Sepharose beads. IgM- and IgG-class anti-non-Gal antibodies in serum depleted of anti-Gal antibodies were measured by flow cytometry using porcine endothelial target cells. RESULTS: Compared with pre-transplant values, all six recipients with AHXR showed a substantially higher level of anti-non-Gal IgM antibodies at rejection; in five animals there was also an increase in IgG-class antibodies. There was no relevant change in recipients without AHXR. AHXR at time of cessation of heart contraction could be preceeded by a steady increase in antibody level starting 2 to 3 weeks earlier. CONCLUSIONS: AHXR is invariably associated with increased circulating anti-non-Gal antibodies. These antibodies are not observed in recipients without AHXR, and five of six recipients with AHXR were adequately depleted of anti-Gal antibodies by maintenance GAS914. This indicates that anti-non-Gal antibodies play a significant role in the pathogenesis of AHXR. Also, the assessment of these antibodies could be used as an early monitor of AHXR.

Anti-pig antibody levels in non-human primates of various origin.

BACKGROUND: Natural anti-porcine antibodies play a major role in hyperacute solid organ xenograft rejection in the pig-to-non-human primate model. Work from other groups and our experience in transplantation experiments has shown that antibody levels are highly variable between non-human primate species, and that extremely high levels can mediate hyperacute rejection even if organs from animals transgenic for human decay-accelerating factor are used. METHODS: Sera were obtained from cynomolgus monkeys wild-caught in Mauritius, captive-bred in the Philippines, captive-bred in Indonesia (Indonesia-Ind), and originating from Indonesia but colony-bred in USA (Indonesia-USA), from baboons wild-caught in Kenya, and from rhesus monkeys originating from India but colony-bred in USA (10 animals in each group). Antibody levels were determined using assays for haemolytic antibody (APA), IgM and IgG class anti-Galalpha1-3Gal antibody, and IgM and IgG class anti-endothelial cell antibody. RESULTS: Cynomolgus monkeys from the Philippines and Indonesia-USA and rhesus monkeys showed median APA and IgM antibody levels in the same range as a pooled human serum standard, and median IgG levels well below the level in this standard. Cynomolgus monkeys from Mauritius and Indonesia-Ind showed extremely high APA levels (median seven to 10 times the human serum standard): IgM class antibodies were also higher, while IgG class antibodies were in the range of the level in the human serum standard. Antibody levels in baboons were in between these two categories. The results of the APA assay showed a highly statistically significant correlation with the assays of IgM antibody, and this was also the case for the IgM antibody assays, indicative of the assessment of the same antibodies in these assays. The same was observed for the assays for IgG antibody. Taking body weight as an indicator for age, there was no relationship between body weight and levels of antibodies. CONCLUSIONS: Natural antibody levels show a significant variation between various groups of non-human primates, with levels in some groups well above those in a human serum standard.