Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis.

The major obstacle to successful discordant xenotransplantation is the phenomenon of hyperacute rejection (HAR). In the pig-to-primate discordant transplant setting, HAR results from the deposition of high-titre anti-alpha-galactosyl antibodies and complement activation leading to endothelial cell destruction and rapid graft failure. To overcome HAR, we developed an enzymatic carbohydrate remodelling strategy designed to replace expression of the Gal alpha-1,3-Gal xenoepitope on the surface of porcine cells with the non-antigenic universal donor human blood group O antigen, the alpha-1,2-fucosyl lactosamine moiety (H-epitope). Xenogenic cells expressing the human alpha-1,2-fucosyltransferase expressed high levels of the H-epitope and significantly reduced Gal alpha-1,3-Gal expression. As a result, these cells were shown to be resistant to human natural antibody binding and complement-mediated cytolysis.

A novel mechanism of retrovirus inactivation in human serum mediated by anti-alpha-galactosyl natural antibody.

Type C retroviruses endogenous to various nonprimate species can infect human cells in vitro, yet the transmission of these viruses to humans is restricted. This has been attributed to direct binding of the complement component C1q to the viral envelope protein p15E, which leads to classical pathway-mediated virolysis in human serum. Here we report a novel mechanism of complement-mediated type C retrovirus inactivation that is initiated by the binding of "natural antibody" [Ab] (anti-alpha-galactosyl Ab) to the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R expressed on the retroviral envelope. Complement-mediated inactivation of amphotropic retroviral particles was found to be restricted to human and other Old World primate sera, which parallels the presence of anti-alpha-galactosyl natural Ab. Blockade or depletion of anti-alpha-galactosyl Ab in human serum prevented inactivation of both amphotropic and ecotropic murine retroviruses. Similarly, retrovirus was not killed by New World primate serum except in the presence of exogenous anti-alpha-galactosyl Ab. Enzyme-linked immunosorbent assays revealed that the alpha-galactosyl epitope was expressed on the surface of amphotropic and ecotropic retroviruses, and Western blot analysis further localized this epitope to the retroviral envelope glycoprotein gp70. Finally, down-regulation of this epitope on the surface of murine retroviral particle producer cells rendered them, as well as the particles liberated from these cells, resistant to inactivation by human serum complement. Our data suggest that anti-alpha-galactosyl Ab may provide a barrier for the horizontal transmission of retrovirus from species that express the alpha-galactosyl epitope to humans and to other Old World primates. Further, these data provide a mechanism for the generation of complement-resistant retroviral vectors for in vivo gene therapy applications where exposure to human complement is unavoidable.

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method.

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.

The receptor for ciliary neurotrophic factor.

Although neurotrophic factors were originally isolated on the basis of their ability to support the survival of neurons, these molecules are now thought to influence many aspects of the development and maintenance of the nervous system. Identifying the receptors for these neurotrophic factors should aid in identifying the cells on which these factors act and in understanding their precise mechanisms of action. A "tagged-ligand panning" procedure was used to clone a receptor for ciliary neurotrophic factor (CNTF). This receptor is expressed exclusively within the nervous system and skeletal muscle. The CNTF receptor has a structure unrelated to the receptors utilized by the nerve growth factor family of neurotrophic molecules, but instead is most homologous to the receptor for a cytokine, interleukin-6. This similarity suggestes that the CNTF receptor, like the interleukin-6 receptor, requires a second, signal-transducing component. In contrast to all known receptors, the CNTF receptor is anchored to cell membranes by a glycosyl-phosphatidylinositol linkage.

Identification of functional receptors for ciliary neurotrophic factor on neuronal cell lines and primary neurons.

The lack of reagents or molecular probes specific for the ciliary neurotrophic factor (CNTF) receptor has hindered characterization of the molecular mechanism(s) by which CNTF influences the proliferation, survival, and differentiation of cells of the vertebrate nervous system. We have developed methods for the detection and separation of cells expressing CNTF receptors by using a variety of binding assays based on a genetically engineered CNTF molecule containing an "epitope tag" at its C-terminus. These assays have allowed us to identify several neuronal cell lines, as well as embryonic and adult neurons in primary cultures, that bind CNTF and functionally respond to CNTF by rapidly activating the transcription of immediate early primary response genes.

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.

Blockade of C5a and C5b-9 generation inhibits leukocyte and platelet activation during extracorporeal circulation.

Complement activation contributes to the systemic inflammatory response induced by cardiopulmonary bypass. At the cellular level, cardiopulmonary bypass activates leukocytes and platelets; however the contribution of early (3a) versus late (C5a, soluble C5b-9) complement components to this activation is unclear. We used a model of simulated extracorporeal circulation that activates complement (C3a, C5a, and C5b-9 formation), platelets (increased percentages of P-selectin-positive platelets and leukocyte-platelet conjugates), and neutrophils (upregulated CD11b expression). to specifically target complement activation in this model, we added a blocking mAb directed at the human C5 complement component and assessed its effect on complement and cellular activation. Compared with a control mAB, the anti-human C5 mAb profoundly inhibited C5a and soluble C5b-9 generation and serum complement hemolytic activity but had no effect on C3a generation. Additionally, the anti-human C5 mAb significantly inhibited neutrophil CD11b upregulation and abolished the increase in P-selectin-positive platelets and leukocyte-platelet conjugate formation compared to experiments performed with the control mAb. This suggests that the terminal components C5a and C5b-9, but not C3a, directly contribute to platelet and neutrophil activation during extracorporeal circulation. Furthermore, these data identify the C5 component as a site for therapeutic intervention in cardiopulmonary bypass.

K-252a and staurosporine selectively block autophosphorylation of neurotrophin receptors and neurotrophin-mediated responses.

The same receptor tyrosine kinase (RTK) can mediate strikingly different biological responses in a fibroblast as opposed to a neuron. We have compared the rapidly induced tyrosine phosphorylations mediated by various RTKs in both NIH3T3 fibroblasts and in the PC12 neuronal precursor cell line and found that each RTK induces a distinct pattern of protein tyrosine phosphorylations in the two cell types. These findings are consistent with a model in which various cell types present a given RTK with different menus of signal transduction components, allowing the same RTK to elicit fundamentally distinct biological responses. Although there are obvious overlaps in the tyrosine phosphorylations induced by different RTKs in the same cell, there are also clear differences. The attempt to dissect these differences revealed that the kinase inhibitors K-252a and staurosporine inhibit RTK autophosphorylation and thus the biological consequences of receptor/ligand interaction. These inhibitors displayed substantially greater specificity for a subset of RTKs (including the neurotrophin receptors) than for other RTKs and acted as remarkably selective blockers of neurotrophin action in both neuronal and nonneuronal cells. A potential therapeutic application for these inhibitors is discussed.

Xenogeneic organ transplantation.

Over the past few years, several major advances have occurred in the understanding of how the humoral and cellular immune system of humans recognizes and destroys transplanted cells, tissues and organs derived from animal sources. Consequently, armed with this new knowledge, several laboratories have now developed novel immunoprotective technologies that may allow xenotransplantation to be clinically feasible.

Epidermal growth factor induction of cellular proliferation and protooncogene expression in growth-arrested rat H4IIE hepatoma cells: role of cyclic adenosine monophosphate.

The precise molecular events involved in growth factor-mediated cell proliferation in eukaryotes have not been entirely elucidated. Identification and characterization of the itnracellular molecular signaling systems linking growth factor function with nuclear events would provide insight into the regulatory mechanisms governing eukaryotic cell growth. In this report, we demonstrate that serum-deprived rat H4IIE hepatoma cells enter a quiescent state and remain viable in the absence of serum for up to 7 days. These cells can be stimulated to transverse the cell cycle and proliferate in response to epidermal growth factor (EGF) after a 24-h lag phase. We were able to completely mimic the mitogenic effects of EGF with 8-p-chlorophenylthio-cAMP (8-CPT-cAMP) but only partially with N6-(Bu)2-cAMP. EGF and 8-CPT-cAMP together induce a synergistic increase in H4IIE hepatoma cell proliferation. The calcium ionophore A23187 and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate had little effect on H4IIE cell proliferation. EGF treatment led to a rapid and transient increase of intracellular cAMP concentration. Both 8-CPT-cAMP and EGF were also equally effective in causing a rapid and transient induction of c-fos and c-myc protooncogene mRNA levels when added to growth-arrested H4IIE cells while A23187, N-(Bu)2-cAMP, and 4 beta-phorbol 12-myristate 13-acetate were significantly less effective. Both EGF and 8-CPT-cAMP affect protooncogene expression in growth-arrested rat H4IIE hepatoma cells primarily at the transcriptional level. Localization and semi-quantification of nuclear pp55c-fos and 63 (kilodalton)-myc protooncoproteins by immunocolloidal gold electron microscopy revealed that EGF and/or 8-CPT-cAMP treatment of quiescent H4IIE hepatoma cells led to a marked and rapid nuclear accumulation of these proteins in discrete nuclear substructures. Cummulatively, these results suggest that cAMP participates in the intracellular signaling system mediating the mitogenic and protooncogene inducing effects of EGF on growth-arrested rat H4IIE hepatoma cells.

Induction of macrophage-like differentiation of HL-60 leukemia cells by tumor necrosis factor-alpha: potential role of fos expression.

Tumor necrosis factor-alpha (TNF-alpha) is a macrophage-derived cytokine elicited during cellular responses to various microbial infections. TNF-alpha exerts direct cytotoxicity toward some tumor cells in vitro and produces hemorrhagic tumor necrosis in vivo. In human promyelocytic HL-60 leukemia cells, human recombinant TNF-alpha (rTNF-alpha) exhibits a small early proliferative effect (within 48 h), followed by marked cytostatic activity at 96 h after the addition of rTNF-alpha. Cytostasis is contiguous with an induction of cell differentiation along the monocyte/macrophage lineage. The cell proliferation effects and the induction of the differentiated phenotype are preceded by an approximate 5-fold increase in c-fos mRNA levels within 90 min after rTNF-alpha treatment of log phase HL-60 cells. Nuclear in vitro transcription assays indicate that the effect of rTNF-alpha on c-fos mRNA abundance is controlled at the transcriptional level. We have also used a postembedding immunocolloidal gold electron microscopy technique to localize and semiquantitate pp55c-fos proto-oncoprotein levels in the nucleus of both control and rTNF-alpha-treated HL-60 leukemia cells. In response to rTNF-alpha, we have observed a rapid and transient accumulation of pp55c-fos in discrete nuclear substructures within 2 h after treatment. C-fos staining appears in clusters, which are preferentially localized over semi-condensed chromatin and interchromatin granules. These results suggest that pp55c-fos is involved in the signal transduction system initiated by rTNF-alpha during the induction of HL-60 differentiation.

Humanized porcine VCAM-specific monoclonal antibodies with chimeric IgG2/G4 constant regions block human leukocyte binding to porcine endothelial cells.

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.

Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5G1.1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5G1.1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5G1.1 scFv also effectively blocked C5a generation. Finally, an intact humanized h5G1.1 antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5G1.1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases.

In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5.

Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable.

Protection of retroviral vector particles in human blood through complement inhibition.

The rapid inactivation of murine-derived retroviral vectors in human or nonhuman primate sera is largely attributed to the activity of complement mediated through the classical pathway. In this study, we have further investigated the relationship between the human complement cascade and retrovirus inactivation. Preincubation in normal human serum effectively inactivated LXSN retroviral vector particles, whereas the vector maintained the ability to transduce cells following incubation in sera deficient in either the C1, C2, C3, C5, C6, C8, or C9 human complement proteins. Preincubation of serum with monoclonal antibodies (mAbs) that functionally block specific complement components, including C5, C6, C8, and C9, successfully protected the LXSN vector from complement-mediated inactivation. Treatment of serum with cobra venom factor, which consumes terminal complement, also effectively protected the vector from inactivation. LXSN vector survival in serum corresponded inversely to the level of complement activity following treatment of serum with anti-C5 mAb as assessed in an erythrocyte hemolytic assay. Additionally, pretreatment of human whole blood with anti-C5 mAb effectively inhibited inactivation of the LXSN vector. Taken together, these data demonstrate that formation of the membrane attack complex (MAC, C5b-9) is required for the inactivation of the murine-based LXSN retroviral vector in human blood and that this process can be abrogated with the use of soluble complement inhibitors.

Retroviral vector producer cell killing in human serum is mediated by natural antibody and complement: strategies for evading the humoral immune response.

The introduction of retroviral vector producer cells (VPC) into tumors as a means of increasing transduction efficiency has recently been employed in human gene therapy trials. However, the fate of these xenogeneic cells in humans is not well understood. In the present study, we used an in vitro model to examine the survival of commonly used VPC lines in serum from humans and various other species. VPC derived from the murine NIH-3T3 cell line, including PA317, Psi CRIP, and GP + E-86, were effectively killed in sera from Old World primates, including human and baboon. Conversely, the same murine cell lines survived exposure to sera from dog, rabbit, rat, and mouse. This pattern of serum killing parallels the occurrence of the anti-alpha-galactosyl natural antibody (Ab) found exclusively in Old World primates. The anti-alpha-galactosyl Ab targets the terminal glycosidic structure Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-galactosyl epitope) found on the surface of mammalian cells, excluding Old World primates. All murine-derived VPC tested expressed high levels of the alpha-galactosyl epitope as determined by FACS analysis. VPC killing was complement-mediated, because preincubation of human serum with a functionally blocking anti-C5 mAb completely abolished cell lysis. Furthermore, addition of soluble galactose(alpha 1-3)galactose (Gal alpha 1-3Gal) to human serum or down-regulation of the alpha-galactosyl epitope on the surface of VPC effectively reduced VPC killing, indicating that complement activation by these cells is primarily initiated by natural antibody recognition of the alpha-galactosyl epitope. Finally, VPC incubated with human serum for 8 hr in the presence of complement inhibition continued to produce viable retroviral particles, thus demonstrating a correlation between VPC and particle survival. Taken together, these data suggest that elimination of the alpha-galactosyl epitope or complement blockade may provide a strategy to prolong the survival of VPC and the particles that they produce in vivo.

Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.

Inhibition of complement system activation requires the development of soluble nonimmunogenic inhibitors with good tissue penetrating abilities that are themselves unable to activate complement. Chimeric mouse/human Fabs capable of blocking the activity of complement proteins are likely to fulfill these criteria. Several monoclonal antibodies that inhibit the activation of the human complement system have recently been developed. To examine the properties of chimeric Fab derived from these monoclonal antibodies, we have developed an expression system which allows the rapid production of milligram quantities of chimeric Fab. Both the chimeric light chain and the chimeric Fd were co-expressed from the same vector, pAPEX-3P. This vector contains the SV40 origin of replication, which allows the rapid production of chimeric Fab in COS cells for preliminary characterization. Additionally, pAPEX-3P contains the Epstein-Barr virus origin of replication and a puromycin selectable marker for maintenance as a stable episome in human cell lines. A production system consisting of transfected 293-EBNA cells cultured in serum free medium followed by protein G-Sepharose chromatography of the conditioned medium was found to be sufficient for the rapid production of purified chimeric Fab. Here we have utilized this expression system to demonstrate that an anti-human C5 chimeric Fab was a potent inhibitor of complement activation in both in vitro activation assays and an ex vivo model of complement-mediated tissue damage.

Complement inhibition with an anti-C5 monoclonal antibody prevents acute cardiac tissue injury in an ex vivo model of pig-to-human xenotransplantation.

Prevention of hyperacute xenograft rejection in the pig-to-primate combination has been accomplished by removal of natural antibodies, complement depletion with cobra venom factor, or prevention of C3 activation with the soluble complement inhibitor sCR1. Although these strategies effectively prevent hyperacute rejection, they do not address the relative contribution of early (C3a, C3b) versus late (C5a, C5b-9) activated complement components to xenogeneic organ damage. To better understand the role of the terminal complement components (C5a, C5b-9) in hyperacute rejection, an anti-human C5 mAb was developed and tested in an ex vivo model of cardiac xenograft rejection. In vitro studies demonstrated that the anti-C5 mAb effectively blocked C5 cleavage in a dose-dependent manner that resulted in complete inhibition of both C5a and C5b-9 generation. Addition of anti-C5 mAb to human blood used to perfuse a porcine heart prolonged normal sinus cardiac rhythm from a mean time of 25.2 min in hearts perfused with unmodified blood to 79,296, or > 360 min when anti-C5 mAb was added to the blood at 50 micrograms/ml, 100 micrograms/ml, or 200 micrograms/ml, respectively. In these experiments, activation of the classical complement pathway was completely inhibited. Hearts perfused with blood containing the highest concentration of anti-C5 mAb had no histologic evidence of hyperacute rejection and no deposition of C5b-9. These experiments suggest that the activated terminal complement components C5a and C5b-9, but not C3a or C3b, play a major role in tissue damage in this porcine-to-human model of hyperacute rejection. They also suggest that targeted inhibition of terminal complement activation by anti-C5 mAbs may be useful in clinical xenotransplantation.

Protection of porcine aortic endothelial cells from complement-mediated cell lysis and activation by recombinant human CD59.

Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC). Human CD59 was constitutively expressed in nPAECs at levels similar to that of native CD59 in human umbilical vein endothelial cells. The protein was tethered to the cell surface by a glycosyl-phosphatidylinositol anchor, as demonstrated by its removal following treatment with phosphatidylinositol-specific phospholipase C. In a model of antibody-dependent complement activation, nPAECs expressing human CD59 were protected from membrane pore formation and cell lysis by complement derived from either human or baboon sera. Conversely, nPAECs expressing CD59 were not protected from lysis by rabbit or dog complement, indicating that recombinant CD59 retained its species-restricted inhibitory activity. Additionally, CD59 expressed on nPAECs inhibited the C5b-9-dependent generation of membrane prothrombinase activity. Collectively, these data establish that stable expression of human CD59 on xenotypic (porcine) endothelial cells renders these cells resistant to both the cytolytic and procoagulant effects of human complement. We propose that expression of recombinant human CD59 on porcine donor organs may prevent complement-mediated lysis and activation of endothelial cells that leads to hyperacute rejection.

Expression of human CD59 in transgenic pig organs enhances organ survival in an ex vivo xenogeneic perfusion model.

The serious shortage of available donor organs for patients with end stage organ failure who are in need of solid organ transplantation has led to a heightened interest in xenotransplantation. The major barrier to successful discordant xenotransplantation is hyperacute rejection. Hyperacute rejection results from the deposition of preformed antibodies that activate complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement -- however, these molecules are species-restricted and therefore are ineffective at inhibiting activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in the pig-to-human combination, F1 offspring were generated from a transgenic founder animal that was engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was detected in the hearts and kidneys of these transgenic F1 animals, similar to expression levels in human kidney tissue. The hCD59 was expressed on both large vessel and capillary endothelium. Ex vivo perfusion experiments, using human blood as the perfusate, were performed with transgenic porcine hearts and kidneys to evaluate the ability of hCD59 to inhibit hyperacute rejection. These experiments demonstrated that transgenic organs expressing hCD69 resisted hyperacute rejection, as measured by increased organ function for both the hearts and the kidneys, as compared with control pig organs. Hearts from hCD59-expressing animals demonstrated a five-fold prolongation in function compared with controls, 109.8 +/- 20.7 min versus 21.2 +/- 2.9 min (P = 0.164). The hCD59-expressing kidneys also demonstrated significantly prolonged function at 157.8 +/- 27.0 min compared with 60.0 +/- 6.1 min for controls (P = 0.0174). Deposition of C9 neoantigen In the vasculature of porcine organs perfused with human blood was markedly reduced in organs expressing hCD59. These studies demonstrate that C5b-9 plays an important role in hyperacute rejection of a porcine organ perfused with human blood and suggest that donor pigs transgenic for hCD59 may be an integral component of successful clinical xenotransplantation.