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Feature Articles : Jun 15, 2009 ( )
Protein Engineering Reaches New Frontiers
More Detailed Knowledge of Structure and Function Drives Field Forward Quickly
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.”
Enhancing Anticancer Antibodies
Antibody conjugates have been the main focus of research at ImmunoGen since its origin in 1981. “The goal, even then, was to arm an antibody with a cytotoxic payload in order to make an effective anticancer product,” explained John Lambert, Ph.D., evp and CSO.
The company’s Tumor-Activated Pro-drug technology (TAP) includes a monoclonal antibody attached to a cell-killing agent via a linker. After trying several molecules, maytansinoids proved most amendable to manipulation. As derivatives of maytansine, DM1 and DM4 are a thousand- to ten-thousand-fold more potent than chemotherapeutics and target tubulin and microtublins, so they only kill dividing cells, Dr. Lamber said. “This contributes to the excellent toxicity profile shown in the clinic.”
The company’s approach is to use linker chemistry that reacts with the lysine amino group of the antibody. The linker reaction is controlled by an average of four lysine residues, which maintains the molecule when in circulation, yet enables the cell-killing agent to exert full potency once inside the cancer cell.
New linkers are being developed that can control what is released inside the cell. “We know maytansine can bind to the tubulin no matter what the linker is. Our new polar linkers resist the efflux mechanisms associated with multidrug resistance much more readily.”
This efflux process pumps out hydrophobic molecules, so the more hydrophillic the compound, the more it stays inside the cell. “We think our new linkers will enable use to target diseases where multidrug resistance becomes a clinical problem such as colon cancer.”
Although Dr. Lambert explained that the company is currently focused on applying this technology to cancer, it could be expanded to other diseases such as rheumatoid arthritis, where killing a specific population of cells would have a beneficial effect.
New Drug Platform
There is a long history of bi-specific antibodies attempting to engage T cells, but none of these early attempts worked well. Micromet has developed novel proteins using its BiTE® technology. This involves using recombinant DNA, whereby the two binding domains of a T-cell binding antibody are connected by a linker sequence to become one polypeptide—a single-chain antibody. This process is repeated with a second antibody binding a cancer cell. The two different single-chain antibodies are finally connected with a third linker.
“The advantage of the novel protein is that it is small (only 55 Kd) compared to a regular antibody (150 Kd),” explained Patrick Baeuerle, Ph.D., senior vp, R&D and CSO. Another advantage is that it is produced as a single polypeptide encoded only by one gene. The linkers are of low immunogenicity because they lack hydrophobic amino acids.
“There have been no immune responses to BiTE antibodies in clinical trials to date,” added Dr. Baeuerle. In addition, the linker sequence is flexible, allowing the antibody to grab two epitopes on two opposing cell surfaces.
A key factor is the way these antibodies bind to T cells—only if the BiTE is presented to T cells on the surface of a target cell will it activate the T-cell receptor CD3. If it binds to the T cell with just one arm, the T cells are not activated. This safeguard mechanism is a core feature of the technology and is referred to as conditional T-cell activation.
The company currently has two BiTE antibodies in clinical studies. MT103 (blinatumomab) binds CD19, present on lymphoma cells and MT110 binds to an epithelial cell adhesion molecule (EpCAM) present on most solid tumors.
The company recently showed data (in vitro and mouse assays) confirming that MT110 eradicates colorectal cancer stem cells expressing EpCAM, Dr. Baeuerle noted. Additional data demonstrated that a BiTE antibody developed from the binding domains of Erbitux can engage T cells and overcome the limitation of this mAb. Human colorectal cancer cells mutated in KRAS and BRAF genes were efficiently killed in a mouse model by the Erbitux-based BiTE antibody, while Erbitux was ineffective, he added.
Many researchers have investigated the drug-targeting abilities of transferrin and have conjugated bioactive agents to it in attempts to exploit its natural properties. In addition, the transferrin receptor is upregulated on highly proliferating cancer cells and at inflammation sites. Other beneficial properties include a long circulating half-life of eight days, and since it’s the second most abundant protein in blood, it’s nonimmunogenic. There is also the possibility of oral uptake as reported in the literature.
Novozymes Biopharma has engineered transferrin to contain an unpaired cysteine that enables use of a free thiol group to attach a bioactive compound, forming an active therapeutic bioconjugate called Thio-transferrin. It is being developed for use with customers’ preferred bioactive, which could be a protein, peptide, drug, nucleic acid, or nanoparticle. It is produced in the same yeast expression system used to manufacture animal-free recombinant transferrin for cell culture applications. It allows site-specific conjugation, overcoming the problems of plasma-derived transferrin and nonspecific conjugation.
“One of the main challenges in developing thio-transferrin was identifying stable amino acid sites for modification,” commented Joanna Hay, Ph.D., senior research scientist. “We wanted to engineer transferrin to introduce an unpaired cysteine, but at the same time retain transferrin’s physiological properties namely iron and receptor binding.”
The company used its computer-modeling and protein-engineering experience to identify suitable amino acids of transferrin for modification to a cysteine residue. Engineering a reactive cysteine into proteins with several disulfide bonds is challenging since it could interfere with structural and functional properties of the protein. A year-long study showed the thiotransferrin variants had the necessary characteristics—a free thiol group able to bind iron and the transferrin receptor.
Llama-Derived Protein Therapeutics
Ablynx has developed a method using llama proteins to create their Nanobodies. These have a high homology with humans, and immunogenicity is not an issue. They also require no engineering—the company’s process removes the VHH domain. In addition, they are about one-tenth the size of monoclonal antibodies and can access targets such as viral canyons, GPCRs, and chemokines that are usually difficult for mAbs and other fragments, reports the company.
“Our formatting feature allows us to link multiple Nanobodies together to increase potency and to obtain bi-specific Nanobodies that target two different disease targets,” explained Eva-Lotta Allen, CBO. This also enables tailoring the half-life depending on whether it’s an acute or chronic indication.
These molecules can be delivered by alternative routes such as pulmonary, oral, and skin patch because they are stable to pH, enzyme, and temperature changes. Since highly potent Nanobodies can be generated, a lower dose can be administered, which aids the alternate delivery route, she added.
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