April 6, 2020
More Potent and Safer Products: The Importance of Effector Function.
Monoclonal antibodies (mAbs) have high specificity and low toxicity, which when combined with their versatility, makes them very attractive for disease treatment, whether it’s a cancer or an infection. Cancer cells are targeted by mAbs through specific recognition of tumour-associated antigens. The binding of a mAb with its cognate target results in recruitment of immune effector functions. This occurs through the Fc region binding to receptors on a range of immune cells and on components of the complement pathway. In an infectious disease, binding of the mAb to target can also trigger responses through cell and complement based pathways.
For many years use of recombinant mAbs was focussed on anti-cancer and anti-immune therapies. Recombinant mAbs against pathogens were first approved in 1998 (Synagis®), with another 3 approved 2016. The increasing frequency of infections caused by multidrug resistant bacteria or viruses and the recent viral disease epidemics feeds the growing need for new prophylatic or therapeutic approaches that include anti-infective mAbs. A hurdle to successful use is the need for a better understanding of how the Fc receptor, isotype, and other structural regions mediate protection.
Modes of effector function (recruitment of immune cells, complement dependent cytoxicity, and phagocytosis) for mAbs in these three disease areas involve binding of the Fc region to Fcγ receptors. Efficient interaction of a mAb with relevant FcγRs is central to effector function efficacy. Modulating effector function can improve safety by reducing off-target cytotoxicity, unwanted cytokine secretion, and (possibly) reduce the frequency of escape variant appearance.
There are three general approaches to modulating effector function:
- Protein engineering of the Fc region
- Manipulation of Fc glycans
- Manipulation of manufacturing process conditions.
Protein engineering of the Fc region can enhance effector function. For example, margetuximab was mutated to reduce FcγRIIb binding resulting in enhanced ADCC activity. Protein engineering can create new combinations of effector function. IgG1 is the most potent ADCC activator whilst IgG3 is most potent for CDC. Antibodies with chimeric CH regions from IgG1 and IgG3 show 25-60% increase in ADCC and CDC activities. In addition to enhancing activity, effector function can also be reduced. IgG2 and IgG4 have very limited ability to elicit ADCC activity, alongside lower CDC activity, compared to IgG1 and IgG3. Soliris® combines sequences from IgG2 and IgG4 to eliminate effector function. Mutations that enhance low pH binding to FcRn translate to increased half-life in humans.
With regard to manipulation of Fc glycans, evidence is strongest for the involvement of fucose with possibly a supportive role for other sugars. The absence of fucose leads to stronger binding between Fc glycans and FcγRIII receptors, as stearic hindrance by fucose inhibits interactions between the mAb and the FcγRIII receptor, which enhances ADCC activity. A number of approaches to reducing the fucose content have been developed ranging from: knockout of FUT8 (such as in Potelligent® CHOK1SV® and other CHO host cell lines); insertion of a bisecting GlcNAc that stops the oligosaccharide being a substrate for α-1,6-fucosyltransferase; redirecting fucose biosynthesis through use of a heterologous enzyme; use of small chemical inhibitors of glycosyltransferases e.g. 2-deoxy-2-fluoro-L-fucose. These approaches result in a varying reduction in fucose content, enabling tuning of ADCC function to help meet the needs of an indication.
Increasing the terminal galactose potentially enhances both CDC and ADCC, but the benefit may be antibody specific. Terminal galactose introduces conformational changes in the CH2 domain enhancing FcγR binding. Some highly sialylated mAbs have reduced ADCC effector function as bulky Neu5Ac groups may reduce flexibility in the mAb’s hinge region, thereby reducing affinity for RcγRIIIa.
Due to the potential immunogenicity of some glycan structures and their role in clinical efficacy, glycosylation is now considered a critical quality attribute that must be within an appropriate range to ensure the desired product quality, safety and efficacy is achieved reproducibly. There is an intricate relationship between mAb glycosylation and culture conditions used to express the protein. This complexity creates many opportunities to affect glycan structures – for good or bad! The importance of process in modulating effector function will be explored in a future blog article.
Antibodies with engineered effector function are not some future idyll; they have been here for a while – Soliris® was approved in 2007 and by 2018 at least 3 glycoengineered mAbs were approved. Historically, the primary use of mAbs was in anti-cancer and anti-immune therapies. In such therapies, there is a strong link between effector function and clinical efficacy, and a good understanding of how effector function can be manipulated is needed. With the appearance of multidrug resistant pathogens, and the need for rapid responses to the appearance of novel pathogens (e.g. the current coronavirus outbreak), there is the need for new prophylactic and therapeutic approaches - mAbs will play a role. If mAbs are to be successful in such circumstances, a better understanding of how effector function mediates protection is needed. However, there is a large body of knowledge available that will enhance the rapid exploitation of that new understanding.