The majority of protein-based biopharmaceuticals approved or in clinical trials bear some form of post-translational modification (PTM), which can profoundly affect protein properties relevant to their therapeutic application. Whereas glycosylation represents the most common modification, additional PTMs, including carboxylation, hydroxylation, sulfation and amidation, are characteristic of some products. The relationship between structure and function is understood for many PTMs but remains incomplete for others, particularly in the case of complex PTMs, such as glycosylation. A better understanding of such structural-functional relationships will facilitate the development of second-generation products displaying a PTM profile engineered to optimize therapeutic usefulness.

The adaptive immune system has the capacity to produce antibodies with a virtually infinite repertoire of specificities. Recombinant antibodies specific for human targets are established in the clinic as therapeutics and represent a major new class of drug. Therapeutic efficacy depends on the formation of complexes with target molecules and subsequent activation of downstream biologic effector mechanisms that result in elimination of the target. The activation of effector mechanisms is dependent on structural characteristics of the antibody molecule that result from posttranslational modifications, in particular, glycosylation. The production of therapeutic antibody with a consistent human glycoform profile has been and remains a considerable challenge to the biopharmaceutical industry. Recent research has shown that individual glycoforms of antibody may provide optimal efficacy for selected outcomes. Thus a further challenge will be the production of a second generation of antibody therapeutics customized for their clinical indication.

The five Ig classes have distinct biological roles. The IgG subclasses also show marked differences in their ability to mediate a variety of effector functions. A detailed comparison of the properties of the human Ig classes and subclasses is not only of interest for relating the functions of antibodies to their structures but is also of great importance for the implementation of therapy based upon immunological intervention . Indeed, this second aspect has become particularly significant as the development of techniques for the production of chimeric antibodies (1-3) should ensure that immunological intervention is now likely to make use of mAbs that have human effector functions; several cell lines have already been established that secrete chimeric antibodies directed against human cancer cells (4-6).Much of our knowledge of the properties of human Igs has been obtained from the study of myeloma proteins (reviewed in references 7-9). However, generally myeloma proteins do not bind identified antigens and, moreover, different myeloma proteins differ not only in their heavy chain class/subclass but also in their light chains and variable regions. As the initiation of antibody effector activity is usually a consequence of antigen binding and is indeed influenced by the quality of that binding, previous studies on myeloma proteins, although valuable, may not provide a sufficient picture of antibody effector function for therapeutic purposes . To carry out a detailed and controlled comparison of the effector functions of the different human C regions, we have established a panel of cell lines that secrete a matched set of human chimeric antibodies . These antibodies are directed against the hapten 4-hydroxy-3-nitrophenacetyl (NP).' This specificity for a known hapten has allowed us to compare the effector functions of the IgG subclasses not only when interacting with soluble antigen but also when interacting with cell-bound antigen. This has enabled us to determine the efficacy with which different subclasses lyse their This work was supported by grants from the Medical Research Council and Wellcome Biotechnology .' Abbreviations used in this paper. ADCC, antibody-dependent cell-mediated cytotoxicity, NP, nitrophenacetyl ; NIP, 5-iodo-4-hydroxy-3-nitrophenyl .J. Exp. MED.

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Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with FcγRII and FcγRIII, and complement. These properties render IgG4 relatively “non-inflammatory” and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology. We report here the high-resolution crystal structures, at 1.9 and 2.35 Å, respectively, of human recombinant and serum-derived IgG4-Fc. These structures reveal conformational variability at the CH3–CH3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the CH2 domain that would explain the poor FcγRII, FcγRIII and C1q binding properties of IgG4 compared with IgG1 and -3. In contrast to other IgG subclasses, this unique conformation folds the FG loop away from the CH2 domain, precluding any interaction with the lower hinge region, which may further facilitate Fab-arm exchange by destabilisation of the hinge. The crystals of IgG4-Fc also display Fc–Fc packing contacts with very extensive interaction surfaces, involving both a consensus binding site in IgG-Fc at the CH2–CH3 interface and known hydrophobic aggregation motifs. These Fc–Fc interactions are compatible with intact IgG4 molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen.

Rheumatoid factors are the characteristic autoantibodies of rheumatoid arthritis, which bind to the Fc regions of IgG molecules. Here we report the crystal structure of the Fab fragment of a patient-derived IgM rheumatoid factor (RF-AN) complexed with human IgG4 Fc, at 3.2 A resolution. This is the first structure of an autoantibody-autoantigen complex. The epitope recognised in IgG Fc includes the C gamma 2/C gamma 3 cleft region, and overlaps the binding sites of bacterial Fc-binding proteins. The antibody residues involved in autorecognition are all located at the edge of the conventional combining site surface, leaving much of the latter available, potentially, for recognition of a different antigen. Since an important contact residue is somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

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