Although overlooked by some as simply a structural backbone, the Fc domain is critical to the functioning of an antibody and has been the focus of many engineering efforts. Broadly speaking these approaches can be broken down into three main areas:
• Increasing effector functions
• Decreasing effector functions
• Extending serum half-life
Increasing effector functions through Fc engineering
One of the most significant therapy areas for therapeutic antibodies is oncology, with more than 200 antibodies having passed through clinical testing (1). One of the key mechanisms of action for such antibodies is the targeted killing of tumour cells through recruitment of the immune system, which is achieved through interaction of the Fc domain with the complement component C1q or Fcγ receptors. However, many of these antibodies have failed in clinical trials due to insufficient efficacy (2). This has, therefore, lead to efforts to increase the potency of antibodies through enhancement of their ability to mediate cellular cytotoxicity functions such as antibody dependent cell mediated cytotoxicity (ADCC) and antibody dependent cell mediated phagocytosis (ADCP).
In particular efforts have focussed on increasing the affinity of the Fc domain for the low affinity receptor FcγIIIa. A number of mutations within the Fc domain have been identified that either directly or indirectly enhance binding of Fc receptors and through this significantly enhance cellular cytotoxicity (2-5). Researchers at Genentech identified the mutations S239D/A330L/I332E (dubbed 3M) (2, 3), MedImmune identified the mutation F243L (4) and Xencor identified G236A (5).
An alternative approach has focussed on glycosylation of the Fc domain. It is known that FcγRs interact with the carbohydrates on the CH2 domain and that the composition of these glycans has a substantial effect on effector function activity (6). Perhaps the best example of this is afucosylated (non-fucosylated) antibodies, which exhibit greatly enhanced ADCC activity through increased binding to FcγRIIIa (7-10).
Decreasing effector functions through Fc engineering
Activation of ADCC and CDC is often desirable for therapeutic antibodies but there are circumstances in which an antibody unable to activate effector functions is preferential. For these purposes IgG4 has commonly been used but this has fallen out of favour in recent years due the unique ability of this sub-class to undergo Fab-arm exchange, where heavy chains can be swapped between IgG4 in vivo (11, 12).
Fc engineering approaches have been used to determine the key interaction sites for the Fc domain with Fcγ receptors and C1q and then mutate these positions to reduce or abolish binding. Through alanine scanning Duncan and Winter first isolated the binding site of C1q to a region covering the hinge and upper CH2 of the Fc domain (13, 14). Researchers at Genmab identified mutants K322A, L234A and L235A, which in combination are sufficient to almost completely abolish FcγR and C1q binding (15). In a similar manner MedImmune later identified a set of three mutations, L234F/L235E/P331S (dubbed TM), which have a very similar effect (16).
An alternative approach is modification of the glycosylation on asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction. A loss of binding to FcRs has been observed in N297 point mutations (3, 17), enzymatically degylcosylated Fc domains (18), recombinantly expressed antibodies in the presence of a glycosylation inhibitor (19) and the expression of Fc domains in bacteria (20, 21).
At Absolute Antibody, we’ve developed Fc Silent™ antibodies for research and assay development use, which have a genetically engineered Fc domain with key point mutations that abrogate binding of Fc receptors and abolish antibody directed cytotoxicity (ADCC) effector function.
Enhancing serum half-life of IgG through Fc engineering
IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Despite this there have been a number of efforts to engineer the pH dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. Researchers at PDL BioPharma identified the mutations T250Q/M428L, which resulted in an approximate 2-fold increase in IgG half-life in rhesus monkeys (22), and researchers at MedImmune have identified mutations M252Y/S254T/T256E (dubbed YTE), which resulted in an approximate 4-fold increase in IgG half-life in cynomolgus monkeys (23, 24). Whilst these enhancements are yet to be shown in humans it is hoped that significant increases to half-life will bring about the possibility of decreasing administration frequency whilst maintaining or improving efficacy.
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