Testing therapeutic potential

TSRI chemists design ‘miniecosystems’ to test drug function

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LA JOLLA, Calif.—Scientists at The Scripps Research Institute are using droplet-sized “miniecosystems” to determine whether a molecule can function as a potential drug. The research was supported by the JPB Foundation.
As the researchers reported in the journal Proceedings of the National Academy of Sciences, in an article entitled, “Antibody selection using clonal cocultivation of Escherichia coli and eukaryotic cells in miniecosystems,” the new method will enable researchers save critical time and funding by simultaneously testing how drug candidates bind to their cellular targets and alter cell function. The Scripps team used the technique to assess the therapeutic potential of antibodies, the focus of much drug discovery research.
According to Dr. Tianqing Zheng, a postdoctoral associate on Scripps Research’s California campus and first author of the new study, “This could save a lot of time in drug discovery by reducing the steps needed to assess drug candidates.” Other authors were Jia Xiea, Zhuo Yangb, Pingdong Taob, Bingbing Shi, Lacey Douthita, Peng Wue and Dr. Richard A. Lerner.
Building on 30 years of research led by study senior author Lerner, who is the Lita Annenberg Hazen Professor of Immunochemistry at Scripps Research, the study takes advantage of antibody phage display, a technology that scientists can use to label and test antibodies for their ability to bind to a biological target. This technology has boosted the development of pharmaceuticals, but there is still a bottleneck: in the vast group of antibodies with a binding affinity for the disease target, there may be only a few antibodies with the right biological functions. Testing these antibodies for function adds time and expense to the drug discovery process.
In the article, the researchers described a method for the rapid selection of functional antibodies, using the co-cultivation of Escherichia coli that produce phage with target eukaryotic cells in very small volumes.
As the article explained, “The antibodies on phage induce selectable phenotypes in the target cells, and the nature of the antibody is determined by gene sequencing of the phage genome. To select functional antibodies from the diverse antibody repertoire, we devised a selection platform that contains millions of picoliter-sized droplet ecosystems. In each miniecosystem, the bacteria produce phage displaying unique members of the antibody repertoire. These phage interact only with eukaryotic cells in the same miniecosystem, making phage available directly for activity-based antibody selection in biological systems.”
Testing for affinity and function at the same time, the miniecosystems are held in the droplets, which contain a mammalian cell and E. coli bacteria. The bacteria produce phage, which stand as carriers for antibody drug candidates, and the antibodies on the surface of the phage are capable of interacting with the mammalian cell in the miniecosystem.
“Co-cultivation of mammalian and bacteria cells in mini-ecosystems makes it possible to select functional antibodies directly with phage display,” Zheng explained.
The mammalian cell in the droplet was engineered to express a fluorescent protein if properly targeted by an antibody, meaning that scientists can test antibody affinity and function in a single step. To confirm the system's function, the researchers whipped up millions of miniecosystems with mammalian cells and bacteria that produce phage-tethered antibodies, which were tested against a receptor on brain cells, called TrkB.
Not only did the system work, but the researchers were surprised to see that the antibodies did a better job at targeting TrkB when attached to phage, rather than the antibody alone. Zheng said that moving forward, they will apply this method to select functional antibodies against other targets of interest.
Describing the significance of the research, the article summarized, “We constructed a library of miniecosystems that can translate the information from antibody phage display directly into signals of biological function, thereby allowing for rapid selection of antibodies with the function of interest. Compared with the conventional phage display platform that can only isolate antibodies based on their binding affinity toward antigens, our new method bypasses the step of affinity-based selection, and the selection is based purely on the activity of antibodies in a biological system without concern for their relative affinity for antigens. This new method bridges the gap, which has existed for almost three decades, between affinity-and activity-based antibody selection for phage display of combinatorial antibody libraries, thus advancing antibody drug discovery.”

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