As well as interacting with the target antigen, antibody derived therapeutics have the potential to be able to interact with components of the immune system. Interactions with the innate immune system trigger effector functions such as antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody dependent cellular phagocytosis (ADCP), which result in target cell death. The ability of monoclonal antibodies, antibody drug conjugates and Fc-fusion proteins to interact with the immune system is dependent on the IgG subclass, primary sequence, structure and glycosylation profile. Due to this potential to induce an immunological response, there is an expectation that these Fc mediated effector functions will be assessed as part of the protein characterization package to determine the impact on product safety and efficacy (EMA, Guideline on Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related Products).
Fc receptors and C1q
Interactions with the innate immune system are mediated by receptors present on effector cells known as Fc gamma receptors (FcγR). These receptors are FcγRI (CD64), FcγRIIa (CD32a) FcγRIIb (CD32b), FcγRIIIa (CD16a) and FcγRIIIb (CD16b). All of the Fcγ receptors activate effector cells on binding, with the exception of FcγRIIb which down-regulates the immune response. Fcγ receptors bind below the hinge, at the site of glycosylation between the two heavy chains of the Fc region of antibodies. Fc glycosylation impacts the conformation of the antibody and results in a more open structure compared to a non-glycosylated form, facilitating binding of the Fcγ receptor. The glycan structure can have a significant impact on the affinity of the interaction with the Fcγ receptors. This is best understood in the interaction with FcyIIIa. The majority of antibody glycans contain fucose. However, antibodies show increased binding affinity to FcyRIIIa in the absence of fucose, which increases ADCC.
C1q is a large multi-subunit protein, which can initiate CDC on binding to the Fc region of antibodies. Binding of C1q is the first step in the complement cascade, which induces a series of protein hydrolysis events, resulting in the formation of the membrane attack complex on the surface of the target cell. While C1q binds to the external surface of the Fc heavy chains, the affinity is impacted by the Fc glycan structure and in particular, galactose is known to increase C1q binding and therefore C1q activity. The structure of the hinge region can also inhibit binding by C1q and Fcγ receptors. Both IgG2 and IgG4 have more rigid and inaccessible hinge regions, which inhibit binding. This leads to lower Fc mediated effector functionality in IgG2 and IgG4 molecules, compared to IgG1.
FcRn and antibody recycling
FcRn is another protein which can bind to the Fc region of antibodies. While it does not induce an immune response, it plays an important role in extending the half-life of antibody based therapeutics within the body. On average, serum proteins have a half-life of approximately 5 days. However, antibodies are known to have a half-life of 21 days or more. Upon entering an epithelial cell from the blood, the Fc region of antibodies is bound by FcRn within the acidic endosome. This prevents the antibody entering the lysosome and being degraded. Instead FcRn transports the antibody to the cell surface and releases it back into the circulation. Unlike the Fcγ receptors, FcRn does not display significant differences in binding affinity for the different IgG subclasses. However, it is impacted by post-translational modifications such as oxidation.
A risk based approach for Fc mediated effector function
Depending on the mode of action, IgG subtype, and structure, therapeutic antibodies can be assigned a high, medium or low potential for Fc mediated effector function. This can be used to generate a risk based strategy for assessing effector function. For example, an IgG4 therapeutic targeting a soluble antigen could be classified having as a low potential for Fc effector function, while an IgG1 binding to a cell surface antigen would be classified as medium to high risk.
Initially, all new antibody therapeutics and biosimilars require evaluation of the Fc binding responses and glycosylation profile as part of the characterisation package. As we gain more knowledge of the structure and function of the antibody, the potential for Fc mediated effector function and therefore the analytical testing programme can be refined. If Fc mediated effector function is confirmed, this can be defined as a critical quality attribute and added to the product specification. This would require the generation of validated assays to monitor the glycan profile and effector functions.
In addition, if combined with simple formed degradation studies, the impact of other factors can also be established, characterised and risk assessed too aid drug development.
Characterisation of Fc binding interactions
Covance has extensive experience in characterising the binding of Fcγ receptors and C1q protein to antibody-based therapeutics, as well as developing cell based assays to determine Fc effector functions. We have developed platform surface plasmon resonance methods to enable the screening of the binding affinities of Fcγ receptors and C1q, to enable a rapid assessment of products for potential Fc mediated effector functions. These assays can then be customised for continual monitoring of the appropriate receptors. The screening assay also helps to support subsequent decisions for monitoring of the effector function using cell based assays.