The Molecular Minotaur

The Molecular Minotaur

It seems to be regular calf. But in its veins flows blood full of human antibodies, which should fight diseases and protect against infections: a masterpiece of clone technology.

By Sascha Karberg, Sioux Falls

Calf No. 468 glares through the acrylic glass window of his ten square meter box. On the farm based in Iowa the young bull is isolated from the outer world. Not because he was dangerous, but because he is so precious: If confirmed by the experts, 468 could be the first bovine that produces human antibodies. For almost ten years, the clone researcher James Robl with his biotech- company Hematech has been trying to yield this breeding result. His company wants to extract the sought-after human antibodies from the blood of designer cows like 468. Antibodies are the hounds of the immune system. In medical application they can, for instance, serve to support the weakened system of aids patients, or protect soldiers and medical personnel from anthrax or other germs. There could be numerous applications, but the production can only cover the most urgent needs.

In a complicated process, the important protein molecules have to be extracted from donor's blood plasma , each of them supplying just under half a liter. A treatment with antibodies can cost up to 50,000 dollars. Contrary to humans, a cow could donate around ten liters of blood plasma twice per month. With herds of cattle like 468 Hematech could produce huge amounts of antibodies on Iowa's pastures – for the sake of patients and the company's financial balance. The vision of Robl, a professor for veterinary genetics, has now almost become reality. Back in the mid-nineties, it was merely a thought experiment that he discussed in the pub with his colleagues at Massachusetts University. After a third glass of beer it seemed absolutely plausible to use cows for the production of human antibodies. "But we lacked the appropriate technique to make the necessary adjustments", says Robl today in his posh office in Sioux Falls. Space is abundant in Iowa and South Dakota, two states that have not much more to offer than endless corn and soy fields and laid-back towns. Robl doesn't care. The world of this cool-headed researcher, who grew up on a farm, turns around small DNA-molecules and big cattle.

During the 1980s, the transfer of cell nuclei into oocytes was mainly limited to mice. Robl tried to make the cloning method applicable on productive livestock, which he managed to do in 1998 at Massachusetts University. He cloned two pieces of cattle from genetically engineered cells. And, in contrast to the already deceased clone sheep Dolly the bulls George and Charly spend their old age on a pasture between the cities of Sioux Falls and Sioux Center. Starting off from this success, Robl founded Hematech based in Sioux Falls, South Dakota. His aim was to carry out his idea of creating antibody-producing cattle. He had the necessary techniques to manipulate the bovine genome at the ready, but he didn't yet know how to transfer the huge packages of human DNA containing the construction plan for antibodies. The genes for the "light" and for the "heavy" chains of proteins that makes up the characteristic Y-shaped antibody-molecules consist of up to two million components each. This amount of genetic information is necessary since certain sections of the genes are cut out and recombined while an immune cell grows ripe. Only this arrangement serves to make sure the development of several million variants of antibodies, enough to recognize diverse viruses and bacteria, thereby serving the immune defense. Therefore, the human genes have to be implanted into the designer cows completely. But when Hematech first started off, it was only possible to transfer DNA-sections of 20,000 to 100,000 components maximum. But one day, Robl learned about a Japanese project: Researcher of the Kirin company had developed an artificial human chromosome that could be fit with any kind of genetic information before cell transfer. The Japanese on their part lacked the experience of cloning cattle, sheep or pigs that Robl could offer. So both sides decided to jointly pursue the aim of using productive

livestock for the production of antibodies. Since 2002, Hematech is a subsidiary of the Japanese pharmaceutical company.

Hematech has settled down far away from the posh biotech clusters of San Francisco and Boston: The company's headquarters lie where South Dakota borders Minnesota and Iowa - neighbouring TransOva Genetics in Sioux Center, a company that offers most modern breeding methods for livestock like artificial insemination, embryonic transfer or cloning. The ambitious Hematech- project went even beyond the capacities of TransOva, hence in 2003, both firms started their own cloning farm as a joint venture. "This is the largest cloning project ever to be endeavored worldwide," says Robl. Every weak around a hundred cloned embryos are transferred into cows ready for conception, amounting to around 4000 embryos per year. As of today, around 15,000 pieces of cloned cattle have been produced. What may sound easy – removing the genes for bovine antibodies, inserting the human antibody-genes by use of an artificial chromosome – is in fact very complicated when it comes to details, says Heiner Niemann of the Institute for Animal Breeding FAL Mariensee. A daring feat of gene technology that no-one had ever before gathered the courage to engage upon. The bovine antibody-genes have to be removed completely, because the slightest leftover of bovine protein among the various antibodies might later cause dangerous rejection reactions in the patient's blood.

Such complex gene manipulations have been examined step by step in trials on mice. For each manipulation one mouse would be bred who then would be cross-bred with otherwise manipulated mice until one of the resulting offspring eventually carries the requested changes in its genome. But since cows reach sexual maturity at the age of eight to ten months, the same procedure would have taken up to 20 years' time. Robl therefore devised a new strategy: All genetic manipulations happen in the cell culture. But then again: those virtually immortal lines of embryonic stem cells that might simplify the procedure, don't exist for bovines. Robl had to resort to cellular connective tissue, the so-called fibroblasts. "They die after a few dozen cell divisions, and cannot be deep-frozen", Niemann explains. But Robl made use of a trick: He "rejuvenated" the cells by fusing the newly manipulated cells with oocytes containing no genome on their own – in other words: he cloned them. From the resulting embryos new fibroblasts can be yielded. The rejuvenated cells can be deep-frozen or used for the next manipulation stage. Some of the cloned embryos may grow to become cows so that the researchers of Hematech can study the effects of a certain manipulation step. There is Black Angus cows, and Holstein stallions, all of them clones representing different stages of genetic manipulation.

Todd Stahl is proud of the cloned bovines. Driving around in his small cross-country vehicle, the director of the cloning farm passes the grazing cattle. Then he stops in front of a small area where a single calf is kept. Its legs are twitching, its fur is shaggy, it is coughing and breathing heavily. Calf No. 454 lacks the genes that a bovine needs to produce antibodies, and it doesn't possess any human antibodies either. Being fed with colostrum, the foremilk saturated with antibodies, more or less protects it from grave illnesses. Like all the other clones, 454 was born in the "Separation Unit", a construction of bars where the surrogate mother cows give birth. "This morning we already had four cesareans", Stahl says. The method was more hygienic and easier to determine, he stresses.

The newborn calves are examined and put under a warming red lamp in one of the nursery chambers. When the clone calves grow older, each gets his own stable consisting of a plastic hut and around nine square meters of additional space. Finally Stahl shows the box where clone calf No. 468 is kept. The first calf with only human antibodies in its blood – presumably. Robl doesn't want to definitely confirm that, until the referees of the specialized journal have made their judgment. "We are in one year's distance from a production line, that's suitable for commercial products, approximately", he dares to admit. Cows producing human antibodies – that is

tantamount to "a money printing factory", as the cell biologist Christian Erck of Helmholtz Center for Infection Research in Braunschweig puts it. "Whoever has such animals on his pasture can produce antibodies for numerous applications.” But even if the genetic manipulations work out as desired, there are still some hidden traps to the Hematech-project: "There still seem to be problems with the concentration of human antibodies in bovine blood", says Niemann. If the level is too low, the yield of blood plasma isn't profitable either. And despite genetic similarities between human and bovine the immune systems of both species' differ considerably. While the antibody-producing immune cells of humans stem almost exclusively from the bone marrow, in bovines the milt takes this function. Apart from that, antibodies like most protein molecules are combined with saccharine residues or otherwise chemically modified. If these alterations proceed not after a human but a bovine pattern, the antibodies could behave abnormally when entered into a human system.

And then there’s still the risk that the artificial chromosome might get lost with the passing of generations. However, Robl does not worry. Even after years, 80 percent of bovine cells still carry the chromosome, he says. "And those immune cells that lost the chromosome are sorted out anyway." Only the debate on the mad cow disease BSE disturbed the scientist a while. Ever since BSE came up, bovine products have to be defended against the general suspicion of being infected with infectious prions. Robl therefore not only removed the antibody-genes but also the prion- gene from the genome. "Prions can be filtered today, so I suppose we don't really need animals without prions", he sighs. However, being so close to his goal he doesn't let himself be disconcerted. He looks ahead “enthusiastically”. 

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