By Chester Bisbee

Special To BioWorld Today

Xenograft researchers are an optimistic lot. They face the extremely difficult challenge of getting the immune systems of organ transplant patients to accept donor tissue from pigs, the animal of choice due to the similarity in organ size and physiology to humans.

Unfortunately, due to the immunological differences, these scientists must first overcome hyperacute rejection (HAR), which causes almost immediate vascular collapse in the transplanted pig organ within minutes to only a few hours after transplantation. In this process, antibodies already present in the transplant recipient's blood combine with serum complement factors to form a complex that attaches to pig cell membranes and destroys the cells.

If HAR is prevented, delayed xenograft rejection (DXR) occurs in approximately five days. DXR results from activation of endothelial cells and the consequent up-regulation of genes that cause thrombosis and inflammation in the transplanted organ. This process also appears to involve binding of complement and the same preformed antibodies as well as infiltration of the transplant by monocytes and natural killer cells.

In addition to these two unique immune system reactions to xenografts, clinicians also must contend with the xenograft equivalent of the classical T cell-mediated response to transplanted organs. How strong this response is remains unclear as does whether it can be overcome with currently available immunosuppressive agents.

In spite of these imposing obstacles thrown up by the donor's immune system, xenografts continue to move closer to becoming a clinical reality. Exactly how these obstacles are being attacked was the subject of last week's International Business Communications conference on xenotransplantation, in Boston.

Counting On Gene Therapy

The antibodies that cause HAR react with galactose alpha 1,3-galactose (Gal alpha 1,3Gal) oligosaccharide residues found on both glycoproteins and glycolipids on the surfaces of pig cells. These antibodies are present in human serum because human tissues lack these Gal alpha 1,3Gal residues, thus treating them as foreign antigens. These antibodies can be blocked or removed from human serum using competitors of the antibody reaction to Gal alpha 1,3Gal, but they soon return as a result of being resynthesized by antibody-producing cells.

However, as described by William Fodor, director of xenotransplantation at Alexion Pharmaceuticals Inc., in New Haven, Conn., his company is genetically engineering pig cells to express the universal donor human O blood group antigen on its cells in order to prevent this antibody response. He stated, "The ultimate goal of this work is to create knock-outs that do not express the Gal alpha 1,3Gal antigen, but we haven't done that yet."

In addition, several groups are developing inhibitors of the complement cascade in order to prevent the binding of the serum complement complex that causes cell death during HAR. Una Ryan, president and CEO of T Cell Sciences, in Needham, Mass., described her company's work with a soluble inhibitor of complement receptor 1 that prevents the binding of the complement complex. She reported that "a single dose gives a survival of days as opposed to zero survival in controls. Infusion of multiple doses doubles the number of days of survival."

Using a similar strategy, Alexion is genetically engineering pig cells to express inhibitors of the C3 and C5b9 components of complement.

Nextran Inc., of Princeton, N.J., has progressed a step further and is producing transgenic animals that express complement cascade inhibitors in their cells. According to Lisa Diamond, associate director of research and development, the company "is looking for founders that express high levels of these inhibitors."

Along similar lines, Steve Stice, vice president of research at Advanced Cell Technology, in Worcester, Mass., described his company's work in cloning and producing embryonic stem cells for transgenic pigs that can be used as organ donors. Stice explained, "The key is to be able to do knock-outs so that the genetic material used in cloning can be customized."

Approaching the problem from a slightly different angle, Julia Greenstein, chief scientific officer and senior vice president of research at Biotransplant Inc., in Charlestown, Mass., described how her company is developing methods to induce recipient tolerance of pig antigens.

Biotransplant uses a therapeutic regimen that includes a radiation-induced reduction in the number of donor immune cells and a subsequent introduction of bone marrow cells from pigs in order to produce a chimeric immune system that can tolerate pig organ transplants. In addition, by using gene therapy techniques to modify the histocompatibility antigens presented on the pig cells, the company is trying to further limit the immune system response.

Greenstein said that "the stumbling block has been getting pig blood cells to grow when placed in donor animals, apparently due to differences in differentiation pathways and their control."

Additional Roadblocks Include Pig Retroviruses

The newest concern for xenotransplantation arose earlier this year when Robin Weiss and his colleagues at the Institute of Cancer Research, in London, showed that pig retroviruses can infect human cells. Weiss told the conference attendees that the potential for transmission of the viruses "remains theoretical." However, FDA concern has alerted xenotransplantation companies to the need for viral infection monitoring of transgenic herds and the testing of xenotransplant recipients for pig viruses.

Several investigators stressed that the problems presented by xenotransplantation will likely require combining several approaches. This was best stated by Ryan, who said, "Multiple technologies will have to be used to get past the problems presented by xenotransplantation."

As an example of how to act on this strategy, she pointed to the wealth of related technologies that T Cell Sciences has been able to access as a result of its partnership in this area with Novartis AG, of Basel, Switzerland.

Similarly, Fritz Bach of Harvard Medical School in Boston made this point more specifically by presenting his research showing that protecting endothelial cells from thrombosis and inflammation will require genetic engineering of several genes central to these pathways. He stated that even with these interventions "other therapeutic agents will be used." *