By David N. Leff

Some 600 organ-graft specialists spent five days last week in the French city of Nantes, attending the Fourth International Congress of Xenotransplantation.

In a world with more and more candidates desperately waiting for replacement organs, but a critically limited supply of donor hearts, lungs, kidneys and other human spare parts, xenotransplantation aims to provide such organs from animals, primarily primates and pigs.

This prospect compounds the already dire complications of human-donor transplants — graft rejection and drastically depleted immune defenses, the latter brought about by lifelong measures to quell rejection.

"In a xenotransplant," observed Elliot Lebowitz, president and CEO of BioTransplant Inc. (BTI), "the first step is called 'hyperacute rejection.' It typically destroys the donor organ within minutes or hours. But subsequently," he added, "there are multiple additional rejection steps — acute, subacute and chronic."

BTI, located in the Charlestown district of Boston, collaborates closely with transplant researchers at neighboring Massachusetts General Hospital (MGH). Immunologist David Sachs, who heads MGH's Transplantation Biology Research Center, chairs BTI's scientific advisory board.

"Xenotransplantation," Lebowitz told BioWorld Today, "is still a futuristic concept." But closest to eventual clinical trials is joint BTI/MGH research on neutralizing an antigen that triggers hyperacute graft rejection."

In efforts to foil this immunogenic protein, known among transplanters as alpha-Gal (for alpha-galactosyl), the Boston researchers described to their Nantes audience two successful experiments.

Their basis is the known fact that *Gal is not expressed in humans or Old World monkeys, but is expressed in all other animal species. So a porcine graft put into the body of a primate animal model will trigger attack by natural antibodies, and by the cell-lysing complement cascade those antibodies turn on. Ergo, hyperacute rejection.

To set the stage for disarming *Gal, recipient primates first had their bone marrow moderately irradiated, to create a "space" in their bone marrow for a dollop of donor bone marrow. The researchers also depleted their immune systems' arsenal of mature T cells, primed to seek and destroy any and all foreign tissues.

Only then did the recipient primates get transplants of pig bone marrow, liver or kidney tissues. These "non-self" imports provoked an increase in anti-alpha-Gal antibodies in control primates, which did not get the priming regimen. Those who did, Lebowitz recounted, "had a greatly reduced antibody response."

Chimerism: Name Of Tolerance Game

"The basic concept," Lebowitz explained, "is bone-marrow chimerism, where you re-educate the immune system by mixing bone marrow from both donor and recipient in the recipient's immune system. This," he continued, "creates a new immune system, which recognizes both the specific donor tissue and the recipient's own reconstituted bone marrow as self. The strategy accomplishes this tolerance without the need for chronic immune suppression."

Lebowitz emphasized "the importance of clearing out those mature T cells, because they still remember what was self, and what was non-self. Otherwise, they would reject both the mixed bone-marrow chimerism and the transplanted organ itself."

To get rid of those menacing T cells, he observed, "BTI has developed a proprietary antibody, which has shown considerable promise in clinical studies, in terms of treating graft and bone-marrow rejection in human patients."

Another innovation the teams presented at Nantes is the use of knockout mice as a versatile alternative to primates. These rodents, developed by a scientist now on the BTI staff, lack the gene for alpha-Gal.

In the experiment reported, wild-type mice got a near-lethal dose of irradiation, which completely wiped out their own bone marrow. In its place, one cohort got back a mixture of bone marrow from alpha-Gal-minus knockouts and alpha-Gal-positive wild-types.

This regimen achieved the same pro-tolerance chimerism as with the primates. By four weeks after the reconstitution, the chimeric rodents "were alive and appeared healthy," and had reduced levels of anti-*Gal natural antibodies. A control group received the alpha-Gal-minus cells alone, and showed little or no tolerance.

"This tested the theory," Lebowitz pointed out, "that if you had sufficiently high levels of donor-plus-recipient bone marrow, you could create tolerance to the natural antibodies completely, as opposed to partially. And this panned out."

Next Up: Gene Therapy

Farther down the pike of BTI and MGH research is a gene-therapy approach to xenotransplantation.

Instead of giving donor pigs' bone marrow to primate recipients, the MGH team took several baboons' own marrow and transduced it with a key gene, which expressed MHC II (the major histocompatibility complex) from the porcine immune system. They delivered this package via a recombinant retrovirus vector.

As the researchers told their Nantes audience: "We were able to detect long-term engraftment for up to 40 weeks post bone-marrow transplantation of the genetically engineered cells." Furthermore, "autopsy samples of one primate showed expression of transgenes in various tissues at 16 weeks post bone-marrow transplantation."

They concluded: "Thus, engraftment of genetically modified cells containing miniature swine MHC genes has been achieved for the first time, by retrovirus-mediated transduction in non-myelo-ablated baboons." BTI's gene-therapy partner, Novartis A.G., of Basel, Switzerland, funded this work.

The congress conferred one of its four annual awards on another set of MGH researchers, who demonstrated that thymus tissue from mini-pigs was able to support maturation of human T cells in a live animal model.

MGH immunologist Megan Sykes, the paper's senior author, commented: "The thymus-implant research we reported represents further progress on an approach that allowed acceptance of pig skin by a mouse without using immunosuppressive drugs. This is one of several techniques," she concluded, "that may produce tolerance to, and allow long-term acceptance of, xenotransplanted tissue." (See BioWorld Today, Sept. 4, 1996, p. 1.) *