Like skinning a cat, there are more ways than one to ward offhyperacute rejection of pig organs transplanted into humans.Two non-competitive approaches shared the plenary podium atWednesday's third and last day of the Second InternationalCongress on Xenotransplantation at Cambridge University,England.
The conclave's organizer, David White, is a lecturer (associateprofessor) in surgery at the university medical school and a co-founder of Imutran Ltd., a biotechnology company set up toadvance research in grafting animal organs into humans.
Regulation of the human complement cascade, the theme ofWhite's plenary lecture to the congress, is the prime problemconfronting the xenotransplanters. Triggered by a transplantrecipient's natural preformed antibodies, his complementsystem releases a series of nine protein units, C1 through C9,that culminates in an attack on the alien organ's endothelialvascular cells. This totally destroys the graft in minutes orhours.
To prevent the complement onslaught from lysing the person'sown cells, the immune system provides a cascade of its own, asuccession of inhibition factors that stymie the complement'skiller proteins as fast as they are released. One of the twoprincipal ploys presented at the conference involvesconstructing transgenic pigs that carry the genes encoding theblood-borne human complement inhibitors. Three of theseblocking molecules are leading contenders to stop short thecellular mayhem.
-- DAF (decay accelerating factor, or CD46) is the earliestcomplement-blocker in the counter-cascade;
-- MCP (membrane cofactor protein, or CD55) acts further alongthe time line;
-- HRF (homologous restriction factor, or CD59) is the last-ditch,late blocker.
Current research suggests that two genes inserted in a pig'sgenome to encode two of these human inhibitors are betterthan one, and that three are best of all. Their job is to keep thecomplement's membrane-attack complex from finishing off aporcine organ graft the minute it's hooked up to a recipient'sblood supply.
White told his audience that he now has a herd of 45transgenic swine equipped with the human gene encoding DAF(CD46) early inhibitor. Next, he and his associates will generateMCP (CD55) blockers and finally a cohort of late-blocking HRF(CD59) transgenics.
Then, said the Cambridge transplant surgeon, "we'll run a kindof dating club to cross-breed carriers of each of the three genesinto one pig." He expects this plan to take "months rather thanyears."
Meanwhile, in an attempt to short-circuit the process, Whiteintends to mix the DNA for the three serial complementblockers in a single micro-injection syringe and co-inject theminto porcine embryos. But he noted that "nobody has ever co-injected three genes, and we don't know how successful thatstrategy will be."
Nor does he know how effective the entire effort of deployinginhibition factors to outwit the complement cascade will be.
"I am still unclear how much the alternative pathway ofcomplement contributes to xenograft hyperacute rejectioncompared with the classical pathway," he said. He was referringto the second string on the complement's deadly bow PP orrather, cascade. While the main string of proteins areunleashed one after the other en route to their cell-killingclimax, a second-string team, the properdine or Factor Balternative pathway, is stealing an end run around the cascade.But it does not circumvent the human inhibitory factors. Whiteproduced experimental data to demonstrate why it posespuzzling contradictions.
In one experiment, he grafted rabbit hearts into piglets, whichare born without immunoglobulin. "With no antibody at all, therabbit hearts promptly reject," he observed. "So that is clearlythe work of the alternative complement pathway."
Next, he transplanted chicken hearts into guinea pigs. "We haddiscovered that guinea pigs produce very little antibodyagainst other species, for reasons unknown," he explained."Moreover, the evolutionary divergency between chicken andguinea pig is about 200 million years. Yet the guinea pigsaccepted the chicken hearts quite happily," he said.
"So there you've got two models," White explained, "one ofwhich PP clearly the alternative complement pathway PP is all-important. The question is, what happens if you put a pig organinto a human being?" He hopes his future triple-gene swinewill supply an answer.
White's only visible rival with complement-blockingtransgenic pigs is DNX BioTherapeutics Inc. of Princeton, N.J.(see BioWorld, Sept. 28). DNX's vice president of research andpreclinical development, John Logan, attended the congress andtold BioWorld: "White demonstrated no expression in his pigs interms of endothelial cells and no activity of the gene in pigs,apart from some weak activity in lymphocytes. And he's nevershown any activity in mice. So I really think in some ways DNXis in the lead. We have multiple genes; he has a single gene.And we've shown good activity against complement; he hasn'tyet."
David Cooper, medical director of the Oklahoma TransplantationInstitute, spoke after White in the plenary session. He takes adifferent tack to thwart hyperacute rejection of heterologousorgan grafts. Instead of seeking to block the complement-mediated destruction of the pig organ with human inhibitors,he aims to take out the human recipient's T cell-mediated,natural antibodies, which trigger the complement assault in thefirst place.
In Cooper's proposed strategy, one key phenomenon isaccommodation. "If natural antibodies against histo-bloodgroups A or B can be depleted for a short period (days, notweeks), then an ABO-incompatible (i.e., heterologous) organtransplanted during this 'window' will not be hyperacutelyrejected despite the subsequent return of high levels of theantibody and normal levels of complement," he said.
Cooper pointed out that the antigenic targets against whichanti-A and anti-B antibodies are directed are carbohydratespresent on the vascular epithelium of the pig. (This is the firststructure that comes in contact with the host xenoreactiveantibodies.)
He and his group infused human plasma through pig hearts orkidneys and eluted out the pig antibodies. They found thatthese bound most strongly to oligosaccharides with an a-galactosyl terminal residue.
When the human anti-a-Gal antibodies were washed out, lysisof pig kidney cells was all but abolished.
Short of trying this tactic in a human recipient, the Oklahomaxenotransplanters turned to baboons as their surrogatepatients. These primates, Cooper explained, have the sameepitopes as humans in their vascular epithelium, as well asnatural circulating a-Gal antibodies.
While transplanting porcine hearts, Cooper and his teaminfused baboons intravenously with specific group A or Btrisaccharides against which the natural antibodies weretargeted, plus conventional post-graft immunosuppression. Theregimen held off hyperacute rejection, on average, from 19minutes to more than 28 days.
Cooper's bottom line: "The ultimate solution would be togenetically engineer a transgenic pig that does not express a-Gal epitopes on its vascular epithelium. By knocking out thesingle gene that encodes the terminal a-galactose, there wouldbe no target for the human anti-a-Gal antibodies."
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.