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A Cornell-led collaboration has devised a potentially low-cost method for producing antibodies for therapeutic treatments. They've bioengineered bacteria with an overlooked enzyme that can attach complex sugars, known as glycans, to monoclonal antibodies (mAbs) to boost their immune defenses.

The research is in Nature Communications. The lead author is Belen Sotomayor, M.S. '23, Ph.D. '25.

"We think this is a really crucial step toward democratizing access of therapeutic antibody drugs by engineering their biosynthesis in low-cost production technologies like recombinant bacteria," said Matt DeLisa, the William L. Lewis Professor of Engineering in Cornell Engineering and the paper's senior author.

"These engineered bacteria are amenable to large-scale biomanufacturing schemes that afford dramatically accelerated production speeds. Another advantage is that the cost of goods associated with making protein products in bacterial cells is significantly lower versus with conventional technology."

The current leading method for manufacturing antibody drugs relies upon the use of Chinese hamster ovary (CHO) cells—a very slow and expensive process that isn't practical when a rapid response is needed, for example, during an emergency outbreak of disease. Bacteria cells are an exciting alternative because they grow, divide and produce proteins much faster than CHO cells. For more than a decade, the DeLisa Research Group has been working to bioengineer bacteria for this purpose.

One reason bacterial systems have not been widely adopted for producing protein therapeutics is that most bacteria don't naturally perform protein glycosylation—an essential life process whereby a complex carbohydrate is attached to a protein. Glycosylation impacts more than 50% of all proteins produced in the human body, making this modification critical for human protein drug development, DeLisa said.

"Glycosylation is super important for the structure and function of the antibody drug itself. Without their attached glycans, the antibodies lack important immune functions," said DeLisa, who is director of the Cornell Institute of Biotechnology. "So if you're not able to install these glycans, then you really don't have access to a therapeutically relevant product."

Working with his longtime collaborator, Michael Jewett of Stanford University, DeLisa's team explored the machinery that makes this modification by mining the genomes of more than 50 different bacterial species. They identified a complicated enzyme known as an oligosaccharyltransferase (OST) in the bacterial species Desulfovibrio marinus, or D. marinus, that has a unique ability to install glycans at the specific location on mAbs of the immunoglobulin G (IgG) subtype that requires glycosylation.

"That's a very unique property. Most enzymes from this family are incapable of catalyzing this specific reaction. In fact, none of them that we've tested do, except for this one special enzyme," DeLisa said. "It was quite a surprise. The genome of D. marinus is the last place we would have expected to find an enzyme that possesses this functionality."

There is only a limited collection of bacterial species that even have OST enzymes, DeLisa noted, and most of them, like D. marinus, have not been exhaustively studied.

After they identified the enzyme, the researchers took the gene from D. marinus and installed it in E. coli bacteria—"a major workhorse of the biotechnology enterprise," according to DeLisa—essentially giving it the requisite machinery for executing the desired protein glycosylation reactions.

After producing glycosylated IgG-type antibodies in the engineered bacteria, the researchers used a chemoenzymatic remodeling technique, pioneered by co-author Lai-Xi Wang of University of Maryland, to remodel their glycan into a structure that would be more compatible in humans. Then a team led by Parastoo Azadi at the University of Georgia's Complex Carbohydrate Research Center (CCRC) performed mass spectrometry analysis to confirm the results.

Because all IgG antibodies are structurally similar, the researchers' creation of glycosylated antibodies in E. coli should enable the biosynthesis of virtually any antibody drug product. These products could be used to treat everything from cancer to autoimmune and infectious diseases. But first, the team will need to explore producing them in higher concentrations.

"CHO cells are slow and expensive, but they're really good at making antibody drugs at incredibly high production titers," DeLisa said. "So although we now have the machinery for executing these reactions, next steps would certainly have to focus on optimizing the production of these glycosylated antibody proteins in order to become competitive with existing CHO cell technology."