Enhancing Biotherapeutic Properties with Cell-Line Engineering

Neha Mishra, Ph.D, Senior Scientist Bioproduction, Revvity.

Monoclonal antibodies (mAb) have established themselves as one of the leading classes of biotherapeutics, making up over 50% of first-time regulatory approvals in recent years. With total mAb sales expected to surpass US$200 billion in 2024, many companies and academic researchers are investing significantly into methods to enhance the therapeutic properties of these proteins.

Choosing the right cell line

Chinese Hamster Ovary (CHO) cell lines have long been established as the most popular cell line for the production of biotherapeutics. With short doubling times (16-22 hours), CHO cells effectively grow in suspension culture as well as chemically-defined media. These key attributes allow biotherapeutic developers to establish controlled manufacturing pipelines that support scale-up.. Perhaps most importantly, CHO cells have been heavily involved in biotherapeutic production since they were used in the first mammalian-expressed recombinant therapeutic—human tissue plasminogen activator, in 1987. Hence, CHO cells have a long history of safety data that helps to ensure that the resulting biotherapeutics can navigate a stringent modern regulatory landscape.

In addition, CHO cells possess the ability to produce biotherapeutics with human-like post- translational modifications (PTMs). They are also significantly less susceptible to viral infection than alternative cell lines. These factors combined highlight CHO cells as an extremely robust cell line able to produce biotherapeutics with reduced potential immunogenic responses, alongside minimising risk of potentially adventitous agents that may cause safety concerns.

Glycosylation

Post-translational modifications, such as glycosylation, play a key role in several aspects of monoclonal antibodies, including efficacy and half-life. As such, the presence and type of PTMs on therapeutic antibodies is something that must be closely measured and controlled. The process of glycosylation involves the addition of oligosaccharide units to individual amino acid residues within a protein structure. Oligosaccharide addition occurs within the endoplasmic reticulum (ER) and golgi apparatus, and most commonly happens via two distinct pathways denoted by where the linkage is made:

i) Nitrogen (N)-linked glycosylation: when oligosaccharides are attached to the nitrogen of an asparagine residue.

ii) Oxygen (O)-linked glycosylation: when sugars are attached to the oxygen of a serine or threonine residue.

The majority of the mammalian proteins are glycoproteins with N-linked glycans, which often confer specific properties to the polypeptide chain. Variation of glycosylation patterns within N-linked glycans is particularly pertinent to biotherapeutics since they can have an effect on protein folding, stability, pharmacokinetics and immunogenicity, among others. Glycosylation variations and their impacts on biotherapeutics are also highly dependent on the region of the protein.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important immune response to foreign antigens, through which FcγRIIIa receptor-bearing natural killer (NK) cells are recruited to attack targets that express antigens derived from, for example, tumours or pathogens. ADCC response is mediated by the binding of antibodies to specific ligands present on the surface of a target cell. The Fc region of the antibody then binds to the FcγRIIIa receptor expressed by NK cells, which are then activated to release cytotoxic granules that ultimately eliminate the target cells. The ADCC response is extremely variable and dependent on the binding affinity of a given antibody to the receptor. Given the importance of the ADCC response on the efficacy of mAb therapeutics, it is important to ensure this response is controlled and consistent. Consequently, structural studies conducted by Ferrera et. al suggest that the presence of fucose within the core glycan structure of the Fc region of an antibody can significantly decrease receptor:antibody binding affinity.

The absence of fucose from a given molecule, called afucosylation, is an important consideration when designing more potent therapeutic antibodies. Increased binding affinity and cytotoxic effects offers several advantages when compared to fucosylated counterparts. Stronger binding affinity to receptors leads to less competition with circulating antibodies present in serum, which allows for lower dose requirements and a minimised risk of undesirable side effects. Moreover, afucosylation can support the development of anti-cancer biopharmaceuticals for tumours that express low levels of surface antigens. This factor significantly increases the range of cancer types that can be considered, bringing with it more therapeutic opportunities. 

Genome editing to alter glycan composition

Monoclonal antibodies generated using CHO cells are typically characterized by a glycan core structure that is fucosylated. This attribute can have implications for the biological activity of the therapeutic antibodies, as well as for their effector function in the case of ADCC reponse. As a result, modifying the glycosylation pathways in CHO cells could lead to improved therapeutic properties. For many years, there has been a growing interest in developing methodologies able to control the glycan composition in therapeutic proteins. Several strategies have been explored to enrich the proportion of afucosylated antibodies in the final product such as; controlling cell host metabolism, using fucosylation enzyme inhibitors, expressing enzymes to reduce available fucose within the cells, and using RNAi to reduce the expression of key fucosylation enzymes. Nevertheless, glycan composition is highly sensitive to process and media conditions, product and overall behaviour of cells in culture, and the use of most of these technologies make it virtually impossible to generate therapeutic preparations with 0% or 100% of their molecules containing a specific glycan composition. Additionally, batch-to-batch glycosylation variability that is inherent to the nature of the cell culture control systems, may have significant effects on controlling drug potency and safety, posing additional pressure during the manufacturing and quality control in bioproduction.

Comments restricted to single page For this purpose, next-generation genome editing tools provide an effective means for engineering expression host cells capable of producing therapeutics with specific characteristics. CHO host cells can be engeneered to lack entirely the ability to incorporate a fucose molecule in the glycan structure. This can be achieved by generating a complete functional knockout (KO) of the fucosyltransferase gene in CHO cells using gene editing tools such as recombinant Adeno-Associated Virus (rAAV) or CRISPR-based technologies to generate enzymatically inactive fucosyltransferase proteins. CHO cell lines that lack the fucosyltransferase gene show similar growth and productivity performance to the parental cell line and are capable of consistently producing fully afucosylated mAbs (0% fucose) without any batch-to-batch variation. In addition, monoclonal antibodies produced in CHO hosts with fucosyltransferase gene KO exhibit a markedly higher efficacy in eliciting an ADCC response, when compared to those expressed in the parental cell line.

Genetically modified CHO cells can enable production of afucosylated antibodies with enhanced ADCC activity, which can make possible the development of more effective treatments in oncology, infectious diseases or autoimmune disorders, enabling better control over product quality and potency of new therapeutics.