Science Editor

News from a telephone press briefing yesterday emanated from Sioux Falls, S.D., the new headquarters of Hematech LLC. The company's CEO, James Barton, and its president and chief scientific officer, developmental biologist James Robl, reported the research leading to successful cloning of calves with human antibody genes.

"Along with our collaborators, Kirin Brewery Co., of Tokyo," Barton led off, "Hematech is announcing today the development of cattle that will produce fully human antibodies in their blood. In recent months," he continued, "Hematech and Kirin scientists have successfully introduced into a bovine fetal fibroblast cell line a human artificial chromosome vector - HAC for short - which contains the full repertoire of genes required for human antibody production. From this modified cell line, Hematech has cloned four healthy calves.

"Each of these animals has the capacity to produce human antibodies in its blood. We are now well on our way to knocking out the bovine genes that control antibody production. The result of these efforts will be cows that can be immunized against a wide variety of pathogens, and about three months thereafter these animals will produce polyclonal antibodies targeted against the immunized pathogens."

"At the present time," Robl said, picking up the story, "this type of immunoglobulin can be secured - if at all - from human blood donors. Some 60,000 blood donors a year contribute intravenous immunoglobulins - human antibodies - to counterattack infectious pathogens. The 16 million grams (18 metric tons) are sold each year for $700 million to treat 50,000 patients with immunodeficiency or autoimmune ills or infections," Robl recounted.

Immunoglobulins Avoid Bacterial Drug Resistance

"In hospitals, as a result of hospitalization," Robl told his press conference listeners, "many patients get infections, some of which are due to antibiotic-resistant organisms. So it would make sense instead of just using an antibiotic to protect them against infection, to use a therapeutic antibody, which does not allow an organism to develop drug resistance. Also," he added, "antibiotics retain their effectiveness in the blood stream for about a month. So they'd make a good kind of prophylactic treatment while the patient is recovering from whatever trauma or surgery they might have gone through. In the case of bacterial infections - like Staphylococcus aureus, which is well known for its ability to become resistant to antibiotics - a polyclonal antibody treatment could be beneficial. Also for pediatric ear infections, in which kids have Staph recurrence even when treated with antibiotics. And of course in biodefense," Robl pointed out, "any of the bioweapon agents we're concerned about could be treated prophylactically with antibodies."

Robl was the first person to clone a cow three or four years ago. He is senior author of an article in Nature Biotechnology, released online today and scheduled to appear in the journal's September 2002 issue. His paper is titled: "Cloned transchromosomic calves producing human immunoglobulin."

"Our overall finding," Robl told BioWorld Today, "was that we could put the entire immunoglobulin [Ig] loci into a calf, and show appropriate processing and expressing. What's novel is that in comparison to previous approaches for making large transgenic animals in particular, the size of the genetic insert is typically about 20,000 base pairs. In this particular case we were not able to use those approaches because the two immunoglobulin genes were 1 million to 1.5 million base pairs in length. So we constructed an artificial chromosome in which the DNA fragment we put in was about 20,000 bps long, compared to 20 million, which allowed us to insert these very large sequences. And we found that the HAC was stable, retained at a high rate, and functioned appropriately with a humanized immune system.

"The Ig genes," Robl explained, "contain many different DNA segments. During normal development an enzyme comes in and clips the two genes expressing the heavy and light Ig chains, allowing them to rearrange. So random mixing and matching takes place, permitting each cell to produce a unique immunoglobulin molecule. The important thing here is that we're not just producing a single protein: The HAC system can generate literally millions of different polyclonal antibody proteins, allowing it to bind a wide array of target epitopes on various pathogens or molecules. This contrasts with monoclonal antibodies, which are basically made up of two single genes.

"Our next approach," he recounted, "was to transfer the light-chain gene into the chromatin fragment that carried the heavy-chain gene. This chromosome-cloning approach got recombination to produce a single 10-million-bp fragment that contains all of the necessary components for replication of the chromosome and the two Igs. What we did then was introduce the fragment into a cow's skin cell, which we used to clone the calf. We've now got about 20 of these transchromosomic calves that carry the chromosome fragment, with a fully diversified expression of the antibody gene.

Robl made the point, "The potential clinical application is for production of human polyclonals. Currently these are used for treating a wide variety of immune-deficiency and autoimmune diseases, which consumes the 16 million grams of the stuff sold each year."

Polyclonals Out-Do Monoclonals In Many Ills

"Right now we have cows that are producing low levels of human antibody," Robl said. "Our next step - among other hurdles - is to inactivate the bovine antibody genes, so that we have a cow producing only human antibody - hence at high levels. Human trials are not on the immediate horizon," he observed, "but we hope that we can start producing at least some products in the next two to three years, to begin moving into human clinicals. Probably the first place that we'll be looking at may be a biodefense type of product - at first, smallpox, anthrax and botulinum toxin.

"The thing we'd like to emphasize," Robl summed up, "is that from monoclonal antibodies, which everybody has heard about, to something new and different from traditional transgenic approaches in that we make a single animal we can use for production of multiple therapeutics. From a biotech industry standpoint, the important thing is that once you've got the animal, the time frame until you've got product is about two months.

"So you immunize the cow," Robl concluded, "and within two months you're collecting therapeutic polyclonal antibodies."