Advances in Engineering of Protein-Based APIs
Engineering of protein therapeutics has advanced significantly since the first biologic drugs were introduced. Today, monoclonal antibodies (mAbs) have become one of the predominant forms of protein-based biopharmaceuticals, with humanization and fully human monoclonals contributing to their increasing success. Antibody-drug conjugates are a second-generation version leveraging the attributes of both small and large molecules. Bi/multispecific and fusion proteins are emerging as promising third-generation protein drug substances. Peptides and peptidomimetic therapeutics offer the advantages of mAbs but in smaller molecules that may be formulated for oral delivery.
Advanced modeling tools are enabling the rapid engineering of more complex proteins with desirable properties. Companies are also paying attention to the developability and manufacturability of proteins and considering patient needs at the earliest engineering stages.
Fully human mAb therapeutics with fully human sequences have reduced immunogenicity potential and improved safety and efficacy, enabling chronic treatment without liabilities, according to Jennitte Stevens, director of process development at Amgen. Generation of fully human antibodies is achieved in transgenic animals. For instance, mouse cells that produce mAbs have been modified by knocking in human genes and knocking out mouse genes. Amgen’s XenoMouse technology has been used to produce the marketed mAb therapeutics denosumab, erenumab, and evolocumab.
Recently, more fully human platform technologies have been developed, such as OmniAb (Ligand), Trianni Mouse (Trianni), and huTARG (Innovative Targeting Solutions). “The latter is a fully mammalian technology that generates in-vitro antibody diversity through ex-vivo V(D)J recombination in cultured cells. These technologies enhance both the speed and success of developing antibody therapeutics,” she says.
Humanization of mAbs is another well-established method to generate therapeutic antibodies when transgenic technology is not available or feasible, according to Stevens. “Humanization techniques using complementary determining regions grafting or phage display have enabled the advancement of numerous antibody therapeutics (such as trastuzumab, pembrolizumab, and romosozumab) to the clinic and ultimately to patients. Humanized antibodies may also have improved safety and immunogenicity profiles, which is a desirable outcome for patients,” she observes.
Bispecific antibodies are an important emerging area in advancing protein engineering for pharmaceutical products, and several bispecifics based on immunoglobulin scaffolds are currently in clinical development, according to Mark Smythe, founder and currently vice-president of technology for Protagonist Therapeutics.
“Protein therapeutics traditionally were designed as single receptor/protein antagonists or agonists. But to effectively combat disease, many future drugs will require multiple proteins being agonized or antagonized to achieve efficacy, namely in the immune-oncology and inflammation space,” Stevens explains.
One example is bispecific T-cell engagers (BiTEs). Blincyto, a BiTE targeting CD3 and CD19, became the first bispecific biologic approved in the US market and demonstrates the impact bispecific molecules can have on treatment of disease, according to Stevens. “Ongoing IgG-based modular domain engineering and platform development in multispecifics present great potential to elicit synergistic activities, enhanced therapeutic index (efficacy, selectivity, novel signaling) or safety, effector cell retargeting, half-life extension and/or as a trojan horse as well as improved convenience, all of which can result in significantly improved outcomes for patients,” Stevens asserts.
Bispecifics based on non-immunoglobulin scaffolds that overcome some of the limitations of immunoglobulin scaffolds are also being pursued by multiple developers. More than 100 formats of bispecific/multispecific candidates are being engineered across the industry, many in the oncology space, according to Stevens.
Although mAbs have led to the development of meaningful biotherapeutics and provide the clearest examples of successful protein engineering technology, Smythe notes that they have significant practical limitations in therapeutic use, such as the inability to deliver biologics or peptides by oral administration. “One type of recent advance in peptide engineering has been the design of protein-based therapeutics that can bind the same targets or block the same pathways as clinically validated antibodies, while allowing oral administration. This advance can be seen in drug candidates based on constrained peptides, such as those from Protagonist Therapeutics, and in the formulation design of an oral GLP-1 agonist from Novo Nordisk,” he says.
For therapeutic applications that do not require effector function, engineering of an IgG scaffold lacking effector function can streamline development and commercialization of such a therapeutic, according Stevens. “Various strategies have been developed recently to eliminate Fc-associated effector binding and functions such as antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity activity,” she observes. For example, Amgen has developed a stable effector functionless IgG platform to eliminate off-target effects while maintaining the stability and half-life expected of a traditional antibody.
Advances have not been limited to novel protein formats. New strategies are also being applied to the development of engineered proteins that integrate developability prediction, manufacturability assessment, and quality-by-design early in the process. “This approach is critical to achieving right-first-time outcomes during the development and commercialization cycle of a biologic,” asserts Stevens.
Amgen has developed a next-generation antibody engineering strategy that enables reliable and efficient identification of pre-candidate leads from early screening lead molecules. “We translate patient needs into protein design requirements during development and engineer in attributes to meet biological performance and molecule stability, which provide improved delivery for desired patient outcomes and improved processing during manufacturing. Incorporating patient needs into molecule designs starts with a translation of the target product profile into a quality target product profile followed by application of these targets during selection and engineering of molecules. This process enables faster and more efficient advancement of novel and effective therapies to patients while improving the overall patient experience,” Stevens explains.
A combination of structure-based drug design with library approaches has become a powerful tool in protein engineering, according to Stevens. “Merging these approaches enables a focused library design that can yield desired protein characteristics rapidly by eliminating the repeated rounds of screening required in a random library approach. At the same time, it enables exploration of a much wider design space than is possible using pure rational design approaches,” she notes.
More advances can be expected, too. Continued improvements in in-silico approaches (modeling, software, and especially machine learning and artificial intelligence) for the prediction of critical molecule attributes such as aggregation, viscosity, and immunogenicity from primary sequences will significantly increase the success rate, accelerate molecule advancement, and reduce the overall resources required to advance a molecule from the discovery to the clinic, according to Stevens.
Similarly, miniaturization and high-throughput versions of protein assays will reduce analysis times and enable exploration of wider design spaces. Stevens points to nanofluidic characterization assays for antibody identification and developability assessments as one example.
Cell-free protein synthesis is also promising, but at this point is limited by the current capabilities in gene synthesis, which serves as a bottleneck for throughput. “Low cost, rapid, and precise gene synthesis will be hugely enabling in this area,” says Stevens.