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.