Protein engineering has come a long way since the first monoclonal antibody was humanized in 1988. Novel techniques are improving safety and efficacy, as well as providing alternative delivery routes. In addition, new methodologies are improving the characteristics of current products. Several companies presented their latest efforts at CHI’s “PEGS” meeting held recently in Boston.
Researchers at the University of Texas, Austin are engineering human therapeutic enzymes for amino-acid depletion therapy in cancer. “We use rational design with high throughput to isolate enzymes or other proteins that have the requisite antibodies for therapeutic applications,” explained George Georgiou, Ph.D., professor, department of biomedical engineering.
One of the challenges, he said, is finding the mutations that confer a particular function. In addition, it’s a matter of fine-tuning the science because it requires developing a protein that fulfills its ability to function in a particular biochemical activity, remains stable in physiological fluids, doesn’t aggregate, and can be administered to humans without causing an immune response.
“You have to plan and execute the development program to fulfill all these goals, because not having one of these considerations would render the whole exercise a moot point.”
Certain cancers require amino acids to grow. Amino-acid depletion therapy has been validated and is effective in acute lymphoblastic leukemia where asparaginase derived from bacteria is used to deplete asparagine. This enzyme is used in conjunction with chemotherapy. The bacterial origin, however, is immunogenic.
Dr. Georgiou’s lab is developing methods to humanize this enzyme while retaining its properties to deplete asparagine, as well as making a human enzyme to deplete arginine, which has therapeutic effects for liver, melanoma, and prostate cancers.
Dr. Georgiou’s group uses two approaches to extend the short half-life of enzymes. One is to fuse the protein to the Fc domain in bacteria (faster and less expensive than a mammalian-cell process), and the second method involves conjugation of polymers (polyethylene glycol) onto a protein of interest. “We’re developing more refined ways to do it so the protein stays in circulation for a longer period of time.”
Prion diseases are well-characterized protein-folding diseases (e.g., Alzheimer’s and Parkinson’s), with early research identifying the conversion of a normal protein, PrP cellular (PrPc), into the pathogenic form, PrP scrapie (PrPsc), mostly in the brain and lymphoreticular systems, as the critical event. This pathologic protein causes loss of neutrophils creating spongiosis and leading to astrocytosis.
“We started thinking a few years ago that if we could engineer a way to deliver and express an antibody that could block the conversion to PrPsc, that might be a therapeutic strategy to extend life in individuals with prionoses,” stated Howard Federoff, M.D., Ph.D., executive vp for health sciences and executive dean, Georgetown University Medical Center.
His group engineered a series of single-chain antibodies with one in particular, D18, being the most effective. A mouse model demonstrated that when delivered via a recombinant adeno-associated virus vector, D18 extended the life of mice given the PrPsc antigen by approximately 33%. “This is a proof-of-concept model that showed that this antibody interfered with the conversion process of PrPc to PrPsc. It didn’t prevent death, but it did extend life.”
Single-chain antibodies are small and can be expressed at high levels and easily engineered because they don’t involve multiple polypeptides interacting with one another. In addition, they can diffuse away from a point source much more readily than a larger antibody. Since they lack the Fc region effector function, they are unlikely to produce Fc-related processes, which translates into fewer adverse events. Dr. Federoff believes that the only potential disadvantage to single-chain antibodies is they have a shorter half-life, requiring higher expression levels.
Additional studies being conducted by Dr. Federoff’s lab included another set of novel single-chain antibodies his group has identified. One involves antibodies against a protein involved in Parkinson’s disease. Another uses intrabodies (single-chain antibodies that are expressed and retained intercellularly), one of which has recently shown to protect against the production of extracellular amyloid implicated in Alzheimer’s disease. “We are interested in whether these antibodies have the capacity to be translated further and have potential clinical applications.”