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Cell Signaling Technology Using Proteomics and NGS to Isolate Monoclonal Antibodies in Human Plasma

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Biotech firm Cell Signaling Technology has begun applying its proteomics-based antibody discovery platform to human donors, isolating circulating human monoclonal antibodies from plasma.

The technology, which combines mass spec-based proteomics and next-generation sequencing to identify antigen-specific antibody sequences, could prove useful for vaccine research and developing human monoclonal antibodies for therapeutic use, among other applications, CST chief scientific officer Roberto Polakiewicz told ProteoMonitor.

The company presented an initial version of the platform applied to rabbits and mice in a paper published in Nature Biotechnology in March 2012 (PM 3/30/2012). It presented a paper detailing the method in human samples in a November paper in the same journal.

The mammalian immune response works by generating a range of different antibodies via changes in the genome of B-cells, each of which contributes a specific monoclonal antibody to the broad polyclonal response attacking the targeted infection.

Researchers and technicians have used these polyclonal antibodies for more than a century, but without having precise knowledge of their composition.

With the advent of monoclonal antibody technology, researchers could use methods like B-cell immortalization, single-cell sorting, and phage display to generate monoclonal antibodies to specific antigens.

However, these approaches also have their downsides. For instance, said Polakiewicz, B-cell immortalization causes many B-cells to die, limiting researchers' view of the original B-cell repertoire.

Additionally, he said, with single-cell methods, the cells a researcher manages to culture aren't necessarily the B-cells that generated the antibodies in circulation. "Essentially you are making a leap," he said. "The cultured cells can produce an antibody, but that doesn't mean it was really the antibody that was doing the job in circulation."

The CST team addressed these challenges via a proteomic method in which they used LC-MS/MS analysis to identify the specific monoclonal components of the polyclonal antibodies circulating in infected subjects. These monoclonal sequences could be synthesized and cloned and the antibodies expressed and screened for specificity and activity.

Because an individual subject's B-cell antibody repertoires are constantly changing in response to foreign antigens, the CST scientists had to generate custom proteomics reference databases for these proteins, which they did using next-generation sequencing to sequence RNA from B-cells taken from the study subjects.

In the original paper, Polakiewicz and his colleagues used the method in rabbits and mice to develop antibodies against five proteins – PR A/B, pMET, Lin28A, Sox1, and phospho-p44/42. In their more recent work in humans, they used it to identify and clone high-affinity, antigen-specific monoclonal antibodies from the plasma of a subject vaccinated against hepatitis B virus and to clone neutralizing human monoclonal antibodies against human cytomegalovirus from a healthy, naturally infected individual.

Although fundamentally similar, adopting the technique for use in humans presented certain new challenges, Polakiewicz said. In particular, he noted, the researchers had to overcome the limitations in sampling that came with using human subjects.

"The key with a human versus animal model is that in an animal model you have access to B-cell sources like the spleen," Polakiewicz said. "You can tap into bone marrows, lymph nodes."

On the other hand, "with humans you really only have blood," he said. "So we wanted to demonstrate that you could do this from simple blood draws from donors. And the challenge there was whether the B-cell repertoire you see from blood donors is sufficient to identify the antibodies in circulation by doing mass spec."

The process is made additionally challenging by the fact that, while animals used in antibody development are often hyperimmunized – receiving multiple boosts of target antigen – this isn't typically done in human subjects.

In the case of the hepatitis B vaccine work presented in the Nature Biotechnology paper, the subjects received two boosts, considerably less than the four, five, or six boosts used in animal work, Polakiewicz said. In the case of the HCMV work, the researchers used healthy donors with only environmental exposure to the virus.

Both versions of the technique rely on recent advances in mass spectrometry and improvements in the instruments' mass accuracy, Polakiewicz said. In the animal work the researchers used a Thermo Fisher Scientific LTQ Orbitrap Velos. For the human work they used an Orbitrap Elite, the latest and highest performance instrument in the Orbitrap line.

The ability to isolate monoclonal antibodies from human plasma makes the technique applicable to "any situation where there are people walking around with interesting antibodies – mostly therapeutically interesting antibodies," Polakiewicz said. "This could be a very efficient way of obtaining those antibodies, and therefore applicable to different types of clinical areas."

For instance, it could prove useful for isolating monoclonal antibodies for therapeutic purposes, as in the HCMV work. Or, Polakiewicz said, it could allow researchers to better monitor vaccine response.

"If you're developing a vaccine, you can use this method to monitor at the individual molecular level the response to the vaccine through the whole process," he said. "You can start comparing vaccines through the different boosts and seeing what kinds of antibodies the vaccine generates."

Polakiewicz noted that other researchers, both in academia and industry, are exploring alternative techniques for isolating circulating human antibodies such as cell sorting followed by functional testing via cell-based methods, but that CST believes its approach could prove faster and more efficient.

"The cell-based methods are quite labor intensive and complex and suffer from attrition because you can only keep the cells alive for a [short] while and you can only screen a relatively small number of cells unless you have very powerful robotics," he said.

The CST approach is also equipment intensive in that it requires high-end mass spectrometry, Polakiewicz said, but, he added, "it is very efficient, very focused, and much faster."

Additional studies will be required to prove its value, however, he said.

CST has not begun offering the service commercially or offering commercial antibodies produced using the technique, but, Polakiewicz said, "we are in the process of planning to do so."

The company is in discussions with several outside research groups and several companies about work involving the technique, he added, but said that he could not provide further details.

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