Because IgG1 predominates in human serum and has been readily available as a monoclonal protein isolated from the serum of patients with plasma cell cancers (multiple myeloma), early structural and functional studies were focused on this IgG subclass. It transpired that IgG1 defined all of the IgG-Fc-mediated ligand binding and effector activation functionsparative studies demonstrated that IgG3 mediates comparable functional activity, while IgG2 and IgGcuatro exhibit restrictions in one or more activity. 29 Few studies have addressed the impact of IgG-Fc polymorphisms on structure and function; however, IgG3 exhibits a very extensive polymorphism with allotypic forms that differ in their ability to bind SpA. 32 By contrast, some studies have focused on possible influences of IgG polymorphisms on clinical responses to infective agents. 33,34
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Following the initial scan of mutant IgG1-Fc for binding and activation of Fc?RI, 17,18 Shields et al. 35 published a comprehensive study that employed full-length chimeric IgG1, IgG2, IgG3, and IgG4 variants of an anti-IgE antibody. All solvent-exposed residues of the IgG1 isotype were sequentially substituted by alanine. The IgG proteins and His-tagged extracellular domains of Fc?R and FcRn were expressed in HEK293 cells, now shown to generate atypical Fc?RIIIa glycoforms. The IgG protein products were classified according to their binding to the subtypes of Fc?R and FcRn in an ELISA assay: Class 1 exhibited reduced binding to all Fc?Rs; Class 2, reduced binding to Fc?RII and Fc?RIIIa; Class 3, improved binding to Fc?RII and Fc?RIIIa; etc. 234–L–G–G237– sequence, were shown to influence binding to all Fc?R subtypes tested but not FcRn. These data were counterintuitive at the time, as the –L234–L–G–G237– sequence did not yield defined conformation in the Deisenhofer IgG-Fc structure 18 and was assumed to be mobile; this conundrum was resolved with publication of an x-ray serwisy randkowe dla meksykanГіw crystal structure for IgG1-Fc in complex with an aglycosylated form of Fc?RIIIb that showed binding to be in the lower hinge region and embracing the –L234–L–G–G237– sequence that assumed defined structure within the complex. 37,38 Flexibility within the hinge region of the intact IgG molecule allows each Fab region to engage its specific antigen with the generation of an equilibrium of IgG-Fc conformers that may, separately, accommodate binding to an individual Fc?R. The subtlety of IgG-Fc conformation and Fc?R recognition is underlined by the finding that substitution of certain residues within the CH3 domain also influenced Fc?R binding affinity. In a subsequent publication, Shields et al. 38 demonstrated a 50-fold increase in the binding affinity of a non-fucosylated glycoform of IgG-Fc for Fc?RIIIa and a consequent increase in NK-cell-mediated ADCC. In the decade following these landmark publications, research has been directed towards differentially maximizing or minimizing binding to and activation of selected Fc?R and FcRn, with the aim of generating antibody therapeutics with an effector function profile that might be optimal for a given disease indication in vivo. A particular goal has been to differentially up- or downregulate interaction with activating and inhibitory Fc?R.
Initial evaluation of the mechanism of action of rituximab indicated ADCC, mediated through engagement of Fc?RIIIa expressed on NK cells. This conclusion was supported by the finding that disease remission in non-Hodgkin lymphoma was linked to polymorphic variants of the Fc?RIIIa receptor that differ in their affinity for IgG1-Fc. 39,40 The allelic products differ in the presence of valine (V) or phenylalanine (F) at position 158 (Fc?RIIIa-V158 and Fc?RIIIa-F158) in the extracellular domain. These residues were later shown to be located within the IgG-Fc/Fc?RIII interaction site. 36,37 Consequently, research has been directed toward generating antibodies with IgG-Fc mutations that result in increased binding affinity for products of each Fc?RIIIa allele, with the expectation of a consequent increase in ADCC irrespective of a patient’s Fc?RIIIa haplotype. The earlier demonstration of increased affinity for afucosylated IgG1-Fc was consolidated with the development of stable production cell lines generating non-fucosylated antibodies. 38,41,42 The improved efficacy for killing cancer cells demonstrated in vitro for these antibodies encouraged other research groups and companies to attempt to improve Fc?RIIIa-mediated ADCC through protein engineering. In addition to the intellectual challenge, success could generate significant intellectual property (IP) and opportunities for patent acquisition that might be exploited for the generation of biobetter therapeutics.