Novel complement inhibitors

The complement system provides natural immunity against microbes and is an effector arm of antibody-mediated humoral immunity. It promotes the inflammatory process by activating cells and facilitates microbial destruction through opsonisation and lysis. Given this tissue damaging potential, it is not surprising that nearly half of the proteins of the complement system are regulators. The complement system can mediate undesirable cellular damage in autoantibody-mediated conditions, for example myasthenia gravis, immune-complex excess syndromes, such as systemic lupus erythaematosus, ischaemia-reperfusion states, hyperacute rejection of transplants, organ failure conditions (e.g., adult respiratory distress syndrome [ARDS]), Alzheimer's disease (AD) and related neurodegenerative disorders. A complement inhibitor has been lacking in the therapeutic arsenal. However, there are now several such agents being assessed in clinical trials and others under development. Current approaches include soluble versions of membrane regulatory proteins, humanised antibodies to components, small molecule inhibitors at various stages of the pathway and transgenic animals expressing human complement regulators for xenotransplantation. These and other strategies should lead to an effective means with which to inhibit complement activation in clinical medicine.     

Pharmacotherapeutic agents in xenotransplantation

The ability to transplant pig organs into humans would resolve the current crisis in the supply of cadaveric human organs for the treatment of end stage disease. Several immunologic barriers need to be overcome if pig-to-primate transplantation is to be successful. The presence of preformed antibodies in humans, apes and Old World monkeys directed against galactose epitopes on pig vascular endothelium provides the major barrier, as binding of antibody to antigen leads to graft destruction by complement activation and other mechanisms. Hyperacute rejection can result from the action of complement. If this is prevented, delayed antibody-mediated rejection develops, which can be associated with a state of consumptive coagulopathy (disseminated intravascular coagulation, DIC). Efforts being made to overcome antibody-mediated rejection include depletion of antibody by extracorporeal immunoadsorption, prevention of an induced antibody response by co-stimulatory blockade, B-cell and/or plasma cell depletion, depletion or inhibition of complement, or the use of organs from pigs transgenic for a human complement regulatory protein, such as hDAF. The ultimate solution would be the induction of both B- and T-cell tolerance to the transplanted pig organ, which is being explored by attempting to induce haematopoietic cell chimerism. One complication of this is a thrombotic microangiopathy, similar to thrombotic thrombocytopenic purpura. The many and diverse roles in which pharmacotherapy is involved in attempts to overcome the barriers of xenotransplantation are reviewed and current progress, particularly in our own laboratory, is discussed.     

Pharmacotherapeutic agents in xenotransplantation

The ability to transplant pig organs into humans would resolve the current crisis in the supply of cadaveric human organs for the treatment of end stage disease. Several immunologic barriers need to be overcome if pig-to-primate transplantation is to be successful. The presence of preformed antibodies in humans, apes and Old World monkeys directed against galactose epitopes on pig vascular endothelium provides the major barrier, as binding of antibody to antigen leads to graft destruction by complement activation and other mechanisms. Hyperacute rejection can result from the action of complement. If this is prevented, delayed antibody-mediated rejection develops, which can be associated with a state of consumptive coagulopathy (disseminated intravascular coagulation, DIC). Efforts being made to overcome antibody-mediated rejection include depletion of antibody by extracorporeal immunoadsorption, prevention of an induced antibody response by co-stimulatory blockade, B-cell and/or plasma cell depletion, depletion or inhibition of complement, or the use of organs from pigs transgenic for a human complement regulatory protein, such as hDAF. The ultimate solution would be the induction of both B- and T-cell tolerance to the transplanted pig organ, which is being explored by attempting to induce haematopoietic cell chimerism. One complication of this is a thrombotic microangiopathy, similar to thrombotic thrombocytopenic purpura. The many and diverse roles in which pharmacotherapy is involved in attempts to overcome the barriers of xenotransplantation are reviewed and current progress, particularly in our own laboratory, is discussed.     

Pathological features of antibody-mediated rejection

Although cell-mediated rejection has remained the most common form of graft rejection after organ transplantation, antibody-mediated rejection has recently gained much significance in clinical transplantation. New evidence points to an antibody-mediated rejection contributing not only to hyperacute and acute but also to chronic allograft rejection. In addition, in discordant xenotransplantation, severe forms of antibody-mediated rejection, including hyperacute rejection and acute humoral xenograft rejection, represent major immunological barriers to successful xenotransplantation. Antibody-mediated rejection in both allotransplantation and xenotransplantation typically does not respond to conventional anti-rejection therapy, so it has recently been recognized as a major cause of graft loss. Histopathology remains the most definitive and reliable tool for the diagnosis of graft rejection in both allografts and xenografts. In this review, we discuss the concept that microvascular injury is a characteristic feature of antibody-mediated rejection that develops in hyperacute, acute and chronic antibody-mediated rejection in both allografts and discordant xenografts as well as in kidney and heart grafts. We also review work indicating that endothelial cell activation and endothelial cell death in the microvasculature can contribute to ultimate graft loss by triggering capillary destruction, interstitial hemorrhage, and platelet-rich microthrombi in hyperacute and acute antibody-mediated rejection as well as with the formation and progression of fibrotic scars in chronic antibody-mediated rejection.     

Xenotransplantation: application of disease resistance

1. Organ transplantation is now clinically routine for patients with end-stage organ failure. One major limitation in transplantation is chronic rejection involving the loss of the graft despite the use of immunosuppressive agents. Haematopoietic stem cell (HSC) chimerism, achieved through bone marrow transplantation (BMT), induces donor-specific tolerance to transplanted organs and prevents chronic rejection. 2. A second major limitation to organ transplantation is the donor shortage. Xenotransplantation, the transplantation of organs between different species, would have the ability to increase the availability of donor organs. 3. Current immunosuppressive therapies do not prevent the rejection of xenografts. Therefore, the only reliable method for achieving donor-specific tolerance to xenografts may require HSC chimerism. 4. In order to justify the use of BMT to induce transplantation tolerance in patients with non-life-threatening diseases, the morbidity and mortality associated with current conditioning regimens must be addressed. 5. The use of partial conditioning regimens to promote engraftment of xenogeneic HSC and the development of donor-specific tolerance may eventually make xenotransplantation in humans a clinical reality. 6. Additional advantages of xenotransplantation are the ability to genetically engineer the donor xenograft and resistance of some xenografts to infection by human viruses because of the species specificity of most viruses. 7. The clinical application of disease resistance for HIV and hepatitis B virus is the focus of the present review.     

Role of complement in xenotransplantation

1. Xenotransplantation, or transplantation across species, leads to rejection, which destroys the xenograft within hours to days of transplantation. 2. Complement is a major barrier to xenotransplantation of vascularized organs and is believed to play an important role in the rejection process. 3. The present paper reviews three aspects of complement in xenotransplantation. These include the mechanisms and regulation of complement activation as well as tissue injury mediated by complement activation.     

Human monoclonal antibodies from transgenic mice

Since the 1986 regulatory approval of muromonomab-CD3, a mouse monoclonal antibody (MAb) directed against the T cell CD3epsilon antigen, MAbs have become an increasingly important class of therapeutic compounds in a variety of disease areas ranging from cancer and autoimmune indications to infectious and cardiac diseases. However, the pathway to the present acceptance of therapeutic MAbs within the pharmaceutical industry has not been smooth. A major hurdle for antibody therapeutics has been the inherent immunogenicity of the most readily available MAbs, those derived from rodents. A variety of technologies have been successfully employed to engineer MAbs with reduced immunogenicity. Implementation of these antibody engineering technologies involves in vitro optimization of lead molecules to generate a clinical candidate. An alternative technology, involving the engineering of strains of mice to produce human instead of mouse antibodies, has been emerging and evolving for the past two decades. Now, with the 2006 US regulatory approval of panitumumab, a fully human antibody directed against the epidermal growth factor receptor, transgenic mice expressing human antibody repertoires join chimerization, CDR grafting, and phage display technologies, as a commercially validated antibody drug discovery platform. With dozens of additional transgenic mouse-derived human MAbs now in clinical development, this new drug discovery platform appears to be firmly established within the pharmaceutical industry.     

Membrane complement regulatory proteins: insight from animal studies and relevance to human diseases

The complement system plays an important role in host defense. However, if not properly regulated, activated complement can also cause significant damage to host tissues. To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement regulatory proteins. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59. Recent studies of membrane complement regulatory proteins from various animal species have revealed similarities as well as significant differences from the corresponding human proteins. In this review, we summarize recent advances in this area and contrast the structure, function and tissue distribution of membrane complement regulatory proteins in human and nonprimate mammalian species. We also discuss how the characterization of the animal proteins has provided important clues and might continue to show relevance to the pathogenesis and therapeutics of a number of human diseases.     

Potential use of genetically modified pigs as organ donors for transplantation into humans

1. Transplantation is currently viewed as a successful treatment for end-stage organ failure. Its more widespread use is severely limited by a shortage of human organ donors. This has stimulated the scientific and medical communities to look at the potential use of animals to solve this problem. For a number of reasons, the pig appears to be the preferred species. 2. The initial immunological problem encountered in pig-to-primate transplantation is a process of hyperacute rejection, which is mediated by the binding of antibodies to the graft endothelium followed by the activation of the complement cascade. We have sought to overcome these initial immunological challenges by focusing our attention not only on the recipient of the graft but also on the donor. Therefore, we have generated transgenic animals with specific genetic modifications designed to inhibit the activation of the complement cascade. Upon transplantation to baboons of organs derived from these transgenic pigs, we have been able to demonstrate that hyperacute rejection can be prevented. We will discuss the generation of these and other transgenic animals and their potential role in clinical xenotransplantation.     

Humanized SCID mice models of SLE

The pathological DNA-specific B cells in Systemic lupus erythematosus are a logical target for a selected therapeutic intervention. It has been recently shown that complement receptor type 1 on human B and T-lymphocytes has suppressive activity. The co-crosslinking of this receptor with the B-cell receptor (BCR) inhibits B cell activation and proliferation and it could be an attractive new target for negative signal delivery. Experimental therapy in humans is limited by many restrictions. Severe combined immunodeficiency (SCID) mice, which lack both T and B lymphocytes and accept xenogenic cells have been used for human cell transfer for evaluating the pathogenesis of human SLE. We hypothesize that it may be possible to re-establish tolerance to native DNA in humanized SCID mice with cells transferred from SLE patients by administering to them a chimeric molecule, containing a monoclonal antibody against human inhibitory complement receptor type 1 coupled to a decapeptide DWEYSVWLSN that mimics DNA antigenically. These protein-engineered molecules are able to co-crosslink selectively the antigen receptors of B-cells possessing anti-native DNA specificity with the inhibitory surface receptors, thus delivering a strong suppressive signal.     

Nude mouse xenografts as in vivo models for lung carcinomas

Eighty-eight primary and secondary lung tumor specimens were subcutaneously transplanted into athymic nude mice. One third of all carcinoma specimens yielded tumor growth. Success rates were highest if fresh tumor pieces or fresh or frozen cell suspensions were implanted or injected. More than half of squamous cell and adenocarcinoma xenografts showed a lower degree of differentiation than the original tumor. The degree of dedifferentiation led to the diagnosis of large cell carcinoma by light microscopical criteria in 4 of these cases. All small cell carcinoma xenografts showed intermediate-cell type morphology irrespective of the cell type of the original tumor, and all large cell carcinoma xenografts showed features similar to the original tumor. Tumor latent periods were approximately twice as long during the first nude mouse passage than subsequent passages, and tumor doubling times remained stable during serial passages. We conclude that the large cell lung carcinoma subtype is a mixed bag of tumors and includes highly undifferentiated squamous cell and adenocarcinomas, that the small cell carcinomas remain in that histologic subtype during xenotransplantation, and that lung carcinoma xenografts display stable morphologic and kinetic features during serial xenotransplantation. Nude mouse xenografts may serve an in vivo model to study the biologic relationship of non-small cell lung carcinomas.     

Therapeutic interventions in xenotransplantation

Xenotransplantation, involving the transplantation of pig organs into humans, would resolve the current shortage of organs. It involves, however, a new therapeutic approach to organ transplantation. The presence of natural antibody in primates directed against Galalpha1,3Gal epitopes on pig vascular endothelium leads to early antibody-mediated rejection. An elicited antibody response against the same target epitopes as well as against nonGal antigens intensifies the immune destruction of the organ. Even the minimal deposition of antibody appears to lead to the development of a consumptive coagulopathy that can be fatal. Approaches being investigated to overcome these barriers include depletion and inhibition of natural antibody and complement, and suppression of the elicited T cell-dependent antibody and cellular responses. In addition, however, physiologic incompatibilities between human and pig, particularly those relating to coagulation, may enhance or complicate the immune process, and may require additional therapeutic measures. Current approaches aimed at achieving successful xenotransplantation also include investigation of agents that prevent potential xenozoonotic infection of the recipient. At present, therefore, the therapeutic interventions required to attempt to overcome the barriers to xenotransplantation are multiple. Work indicating progress in the breeding of pigs that do not express the critical Galalpha1,3Gal epitopes, however, is encouraging. The introduction of these pigs may greatly reduce the therapy required, and may ultimately allow the development of methods to induce tolerance to the transplanted pig organ.     

Beyond antibody-mediated rejection: hyperacute lung rejection as a paradigm for dysregulated inflammation

The use of animal organs for transplantation in humans is seen as a potential solution to the short supply of human donor organs available for clinical transplantation. However, to develop this therapeutic option as clinical reality will require surmounting formidable obstacles. The primary immunologic barrier to pig-to-human xenotransplantation is hyperacute rejection (HAR), a phenomenon previously characterized as resulting from antibody binding and complement activation. This article will first review recent progress in the development of specific strategies to overcome hyperacute lung rejection (HALR), through production of genetically engineered pig organs, modification of the host innate immunity and control of antibody and complement. Additional therapeutic targets identified in HALR are reviewed, with particular emphasis on recent studies describing a critical role for the coagulation cascade in HAR.     

Current status of xenotransplantation

1. The transplantation of organs and tissues from animals into humans (i.e. xenotransplantation) has been a long sought objective to allow xenotransplantation to achieve its full impact in the clinical practice of medicine. 2. The main hurdles to the application of xenotransplantation are the immunological reaction of the recipient against the transplant, the functional limitations of tissues and organs in biogenetically disparate recipients and the possibility of transferring infectious organisms from the graft into the recipient. 3. Advances in a variety of fields have shed new light on these hurdles and have given rise to potential solutions and prospects for the clinical application of xenotransplant and are summarized in the report that follows.     

Complement: an inflammatory pathway fulfilling multiple roles at the interface of innate immunity and development

Complement has been long perceived as an innate immune system that plays a pivotal role in the maintenance of host defense against infectious agents and the propagation of pro-inflammatory responses in the context of human disease. Complement activation has been associated with the onset of acute inflammatory reactions leading to complications such as acute graft rejection, local tissue injury and multi-organ failure. However, recent studies have indicated that various complement activation products may exert a beneficial effect by contributing to critical developmental and regenerative processes. Appreciating this extraordinary 'versatility' of complement proteins provides a framework for revisiting the design of effective complement therapeutics. A balanced strategy will have to consider limiting the detrimental proinflammatory effects of complement while preserving those activities that promote tissue repair and regeneration, cell survival and early development.     

Preclinical efficacy of Virulizin in human breast,ovarian and prostate tumor models

Virulizin is a novel biological response modifier (BRM) approved for the treatment of melanoma and is currently in a phase III clinical trial against advanced pancreatic cancer. The purpose of this study was to define the anti-cancer activity of Virulizin against a number of solid human tumors. The therapeutic effect of Virulizin was evaluated in mouse xenograft models, and the results demonstrate that Virulizin has high efficacy against breast, ovarian and prostate tumor xenografts. Seventy-seven percent inhibition, with an optimal T/C value of 24.8%, was observed in human beast MDA-MB-231 xenografts treated with Virulizin as compared to saline-treated controls (p=0.0004). In human ovarian SK-OV-3 tumor xenografts, administration of Virulizin inhibited tumor growth by 77.6% compared to saline controls (p=0.0439). Furthermore, high anti-tumor activity was also demonstrated in DU145 and PC-3 prostate tumor xenografts, as indicated by 72.6 and 49.1% suppression of tumor growth (versus saline controls, p=0.0007 or p=0.0049), respectively. Direct comparisons with the anti-tumor activities of conventional drugs demonstrated that Virulizin has higher or equal efficacy against all four tumors tested. Finally, addition of Virulizin into co-cultures of tumor cells and macrophages stimulated the cytolytic activity of the macrophages against the tumor cells in a dose-dependent manner. This result suggests that stimulation of immune cells is at least part of the anti-tumor mechanism of action of Virulizin. These results clearly demonstrate that Virulizin inhibits the growth of human breast, ovarian and prostate tumors, indicating great potential for expansion of the clinical indications for this novel BRM.     

Herbal formula enhances 5-fluorouracil activity through suppression of thymidylate synthase

Traditional Chinese herbal medicine(TCM) has been shown to enhance the efficacy of standard anticancer agents. However, there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM. OBJECTIVE To identify biologically active formulas that were effective against colorectal cancer(CRC)by screening TCM formulas in in vitro and in vivo animal models. METHODS Cell growth assays, cell cycle analysis,immunoblot analysis and q RT-PCR were performed to investigate the mechanism(s) of action of the formulason human CRC cells. In vivo animal models were used to evaluate the antitumor activity of formulasalone and in combination with5-FU. RESULTS We identified Huangqin Gegen Tang(HQGGT) which suppressed the in vivo growth of human CRC HT-29 xenografts. HQGGT significantly inhibited the growth of CRC cell lines. HQGGT enhanced the cytotoxicity of5-FU against human 5-FU-resistant cells(H630 R1) and mouse colon cancer cells(MC38). This synergy was the result of suppression of thymidylate synthase expression by HQGGT. HQGGT significantly enhanced the antitumor effect of5-FU in mice bearing MC38 xenografts. Ongoing studies have identified Huangqin as the herb responsible for TS inhibition. CONCLUSION These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrimidine chemotherapy for CRC treatment.     

Complement and complement regulatory proteins as potential molecular targets for vascular diseases

By-products of complement activation and complement regulatory proteins are increasingly recognized to play an important pathogenic role in a variety of vascular diseases including atherosclerosis, ischemia and reperfusion injury, hyperacute graft rejection, vasculitis, and the vascular complications of human diabetes. "Self" damage by autologous complement is mediated by activation products of the complement cascades or by direct insertion of the membrane attack complex (MAC) into cell membranes. Specifically, insertion of MAC complexes into endothelial cells results in the release of an array of growth factors and cytokines that induces proliferation, inflammation and thrombosis in the vascular wall. This paper reviews complement and complement regulatory proteins with specific focus on the vasculature and vascular diseases; it highlights complement and its regulators as potential targets for the rational design of mechanism-specific drugs for the treatment of some of the most prevalent human diseases.