Antibody engineering holds the potential for creating antibodies with multiple specificities, greater affinity for their targets, and fewer side effects. Among these approaches, more-or-less traditional protein engineering remains a topic of intense interest.
Zymeworks whose trademarked tagline is “Building Better Biologics™,” describes its technology as “structure-guided design.” Those who remember classic medicinal chemistry will recognize the synonymous “structure-activity relationship.”
That proteins could be designed in this manner makes sense, but the idea went into hibernation after the initial hype because the tools for correlating structure to activity were primitive. “Our understanding of protein dynamics was not very advanced, not like today’s,” says Ali Tehrani, Ph.D., Zymeworks’ president and CEO.
Techniques like NMR, crystallography, and protein modeling were simply not up to the task. Now that these analytics have caught up, investigators can exploit molecular biology to its fullest.
A typical protein-engineering exercise might begin with a wild-type antibody and identification of the critical regions and amino acids responsible for binding. These regions are analogous to “functional groups” in small molecule drugs.
“We then ask which amino acids can be replaced such that desirable properties are improved, while retaining the molecule’s other critical properties,” Dr. Tehrani explains. This resembles site-directed mutagenesis, but instead of a single amino acid, Zymeworks looks for groups of building blocks that need not necessarily be contiguous. The molecular modeling engine behind this work is ZymeCAD™, which performs in silico mutagenesis to create candidate antibodies.
Zymeworks employs two molecular scaffolds: Azymetric™, an IgG1-based platform consisting of two heavy chains assembled onto one molecule, and AlbuCORE™, a multivalent antibody alternative. Being antibody-like, Azymetric can recruit T cells and bind to two antigens at the same or different sites. Azymetric is appropriate for treating cancers due to its bispecificity and effector activity.
“Rather than applying pressure to one point, you apply it to two points and create a much stronger signal for tumor destruction,” Dr. Tehrani explains.
AlbuCORE is more suited to treating cardiovascular, inflammatory, or auto-immune diseases because it does not recruit immune system cells.
In late September, Zymeworks revealed a new round of financing worth $8.1 million. Less than a month earlier, MSD/Merck signed a nonexclusive agreement with Zymeworks for developing bispecific antibodies.
Immunogenicity is known to limit dosing of certain mAbs, so eliminating it is of great interest. Antitope provides antibody humanization and protein-engineering services that focus, through in vitro and in silico tools, on screening out potential immunogenicity during preclinical development. The company also maintains a pipeline of its own molecules.
“If we start with an early-stage molecule, we can rationally design it to avoid T-cell epitopes, which are key drivers of immunogenicity,” explains chief scientist Matthew Baker, Ph.D.
Antitope concentrates on helper T-cell epitopes, not post-translational modifications. Accumulating evidence supports this approach for therapeutic proteins, including antibodies, and for drug candidates derived from plants and bacteria. The latter are more challenging than conventional proteins from an immunogenicity perspective, largely because the human body more readily recognizes these molecules as nonself.
“Depending on the bacteria, patients may have already been primed against these proteins through previous exposure,” Dr. Baker says.
Antitope’s “de-immunization” technologies amount to mapping T-cell epitopes in the sequence and removing them by point mutation, which disrupts binding to MHC. This approach, applied predominantly to antibody variable region sequences, combines elements of standard humanization with de-immunization and generates sequences that appear human in origin.
The challenge with conventional immunization technologies was incorporation of single-point mutations that disrupt a protein’s affinity for MHC without affecting stability, target binding, and efficacy. “This requires screening large numbers of sequences,” says Dr. Baker.
Tandem humanization/de-immunization combines sequence data from a large database of human variable region sequences with in silico structural analysis. Thus, Antitope can select sequence segments from unrelated, fully human antibodies and stitch them together to generate composite molecules possessing the specificity on par with nonhuman reference antibodies.
Antitope’s internal pipeline consists of seven early-stage molecules, with two that show “good efficacy” in animal models. “And obviously, we’ve addressed any immunogenicity issues as well,” Dr. Baker adds. Antitope plans to out-license its advanced drug candidates after demonstrating proof of concept.
Animals expressing genetically engineered, fully human antibodies hold tremendous promise, but access to these systems has not been straightforward. Abgenix made its name on the Xenomouse™, and Medarex with its KM Mouse™.
But Abgenix was acquired by Amgen, and Medarex by Bristol-Myers Squibb. That leaves only Regeneron’s Velocimmune mouse as a readily available—but costly—platform for generating humanized antibodies. For example, Regeneron’s two licensing deals, with Astellas and AstraZeneca, could net more than $350 million.
“It became extremely difficult and expensive to access this technology. That’s why I founded OMT [Open Monoclonal Technology; www.openmonoclonaltechnology.com],” says CEO Roland Buelow, Ph.D. His previous venture, Therapeutic Human Polyclones, which used genetically engineered rabbits to express a human antibody repertoire was acquired by Roche in 2007.
According to Dr. Buelow, OMT’s rat platform expresses fully humanized antibody as easily as rats make rat antibodies.
Rats are an ideal expression model because they exhibit very little, if any, of the strong selection pressure. By contrast, rabbits, mice, and cows endowed with human IgG loci produce very little fully humanized antibody—around 1%.
Expressing fully humanized antibodies in animals is a two-step process: first create a knockout animal by inactivating their natural antibody genes, then introduce recombinant immunoglobulin loci that encode for antibodies with fully human idiotypes. This had never been done before in rats. “It was technically difficult, but no new technology was required,” Dr. Buelow says.
At the time he founded OMT in 2008, no applicable nuclear transfer cloning protocol existed for rats. Dr. Buelow instead used zinc finger nucleases—DNA-binding proteins that generate mutations in precise locations in the genomes of higher animals.