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Antibodies from the Rooftop of the World

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By John S. MacNeil

 

Waking up at 5 a.m. in Ghorepani, a Himalayan village in Nepal, Neil and Ella Barclay rose to catch an early morning glimpse of the Annapurna mountain range. It was cold and dark, but as the Oxford University immunologist and his wife climbed to the top of Poon Hill, the view of 25,000-foot snow-capped peaks, successively bathed in purple, reddish-brown, and then yellow light, more than compensated for the frosty temperatures. Says Barclay, “To see the sun coming up from behind all those peaks — it was quite spectacular.”

Barclay, age 52, wasn’t just in Nepal to trek around the mountains, however. Along with his wife and Nick Hutchings, a former PhD student, Barclay had traveled to Nepal in March 2001 to oversee the startup of his new company, Everest Biotech, the only such venture in Nepal, according to Barclay. While rivals established genomics and proteomics companies closer to home, Hutchings and Barclay took their seed money and plopped it down in Kathmandu.

And not necessarily because of the scenery. The idea for the company had its roots in Barclay’s declining faith in the ability of 2D gels to pick up low-abundance proteins. In discussing alternatives over coffee at Oxford in the fall of 1999, Barclay and Hutchings settled on the idea of producing antibodies against as many proteins as possible. Doing this efficiently would require employing goats to produce polyclonal antibodies, the two decided, and on the advice of Hutchings’ Nepalese classmate, Barclay turned to the goats outside Kathmandu.

“The impetus really came from the recognition that 2D gels are an imperfect drug discovery or protein discovery tool, and that if we had 10,000 antibodies against the proteins, [we could develop ways] to do high-throughput screening,” Barclay says. “The idea of goats came up because with goats you could make one big batch of antibodies. Nick Hutchings talked to his colleague Raju Adhikari, who said, ‘We’ve got to do it in Nepal. We have 5 million goats, and we actually have some quite well-trained biochemists.’” Furthermore, in theory Barclay could drastically undercut the price of competitors’ antibodies by producing them in Nepal, where overhead is relatively low.

But first he had to show that the logistics and scientific approach were viable. With the help of Hutchings, Adhikari, and another Nepalese biochemist named Ravindra Sapkota, Barclay led a small pilot project to demonstrate that they could harvest high-quality antibodies from Nepalese goats. To test the strategy, Hutchings and Adhikari traveled to Nepal in the winter of 1999, recruited a Kathmandu-based veterinarian, and met with goatherders willing to offer the use of their goats for a fee. Hutchings and Adhikari immunized five goats with five antigens for their trial, and returned 11 weeks later to extract 750 mL blood from each. Back at Oxford, Barclay devised a strategy for harvesting the antibodies to the three proteins using a method known as antigen affinity chromatography, which relies on tagged proteins to extract the antibodies of interest.

Buoyed by the success of the pilot project, Barclay raised £350,000 in early 2000 from an angel investor. With the money, Barclay’s co-founders, including Hutchings, who now serves as CEO, rented 1,000 square feet of space from the main government teaching hospital in Kathmandu, Tribhuvan University Institute of Medicine. Now, Everest employs 10 Nepalese biochemists who, under the direction of Sapkota, collect serum from the free-range goats and purify the antibodies.

“We design and synthesize peptides, immunize the goats, and then [perform] affinity purification on the peptides, so that all the antibodies are affinity purified on the peptides,” Barclay says. “Then we check for specificity by Western blot. That’s all done in Kathmandu.”

Membrane Protein Dreams

But Barclay hadn’t always dreamed of turning science into profit. He chose to study biochemistry as an undergraduate at Oxford because he enjoyed mixing biology with chemistry, and in his last year he became interested in membrane proteins while working in Nobel laureate Rodney Porter’s group on a two-month research project studying rabbit immunoglobulin proteins. Barclay then decided to pursue a PhD in Alan Williams’ group, also at Oxford, where he purified and studied Thy-1, one of the first immunoglobulin-like proteins to be sequenced. “The point was that Ig-related proteins were found in organs which were not immunological, so it was quite an exciting time,” Barclay says.

More recently, Barclay’s research has focused on studying the interactions of several well-characterized membrane proteins, including OX2 and CD2. In the mid-’90s, Barclay and his group performed a series of pull-down experiments with the two proteins, in which the protein and its interacting partners are extracted from a mixture with the aid of a tag. That work generated enough data to keep Barclay’s researchers occupied for several years trying to understand the biological mechanisms responsible for the membrane proteins’ choice of interactions.

“We wanted to know how [certain] proteins, from their interactions outside the cell, lead to signals inside, and our interest is very much in different membrane proteins,” Barclay says. “We have our friendly proteins [OX2 and CD2] that we have a particular interest in, so we set up to see what they might interact with on their cytoplasmic sides.”

In the lab, Barclay is rigorous in his dedication to proving the veracity of a protein interaction, says Marion Brown, a staff researcher at Oxford and member of Barclay’s group since 1990. “He delights in new technology,” she says, while at the same time remaining skeptical of trendy approaches. In fact, Barclay’s involvement with Everest may have rubbed off on his management style. “Some of the terminology makes me laugh,” she says. “I had a note that came around saying something about ‘action,’ and I thought, where did this come from? We laughed and said it was from the business-speak,” Brown says.

Overcoming Maoists and Skeptics

While Barclay continues to serve as scientific advisor to the company, helping decide which antibodies to produce, over time he has returned to his more academic pursuits — leaving the day-to-day management of the company to Hutchings, Adhikari, and Sapkota. Together they’re hoping to boost the company’s catalog from around 250 antibodies today to 500 by June, and eventually to 50,000. Everest uses about 360 goats at any one time, Hutchings says, and is scaling up to produce 120 new antibodies a month. The goatherders receive payment for the use of the goats, which is more than they would make if they sold the animals in the market for food, Adhikari adds.

In fact, Barclay says the goatherders are not the only party to gain from employing free-range goats. By operating in Nepal, Everest can take advantage of cheaper labor costs to produce antibodies at one-sixth the cost of producing them in Europe or the US, Hutchings estimates. In addition, using a large, dispersed herd of goats, as opposed to laboratory animals confined in smaller spaces, should reduce the prospect of contagion, Barclay says.

Not all is rosy in Nepal, however. Delivering antibodies to customers in the US and Europe from Kathmandu is not exactly a trivial task, and to complicate matters, Maoist rebels in the countryside have in the past year stepped up their attacks on police and army installations, contributing to instability. But so far Barclay and his associates have proven that the project has legs.

“Everyone laughed at it [at first],” says Hutchings. “You talk to people and you say, ‘I’m a goatkeeper in Kathmandu,’ and they say, ‘What?’ There’s been a healthy dose of skepticism, but the fact is, it’s up and running now, so we’ve proven people wrong.”

 

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