Review
Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action

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More than twenty recombinant antibody molecules are now licensed for the treatment of a variety of cancers and chronic diseases. Initially, the attraction of antibodies was their specificity for target antigens; however, it is now appreciated that the downstream consequences of engaging antigen, after the formation of immune complexes, is crucial to clinical outcomes in vivo. This review introduces the structural and functional activities of the IgG class of recombinant antibodies, in vitro, and criteria that determine choice between the four subclasses. Importantly, we demonstrate that, although accounting for only 2–3% of antibody mass, glycosylation of the IgG-Fc is essential to the activation of downstream biologic mechanisms (effector functions). Additionally the precise structure of the attached oligosaccharide can influence biologic efficacy. These findings have led to cellular engineering to enable the production of selected glycoforms of antibody that are considered to be optimal for the disease indication to be treated.

Introduction

Recombinant antibody therapeutics (rMAbs) are exemplars of translational medicine. The rMAbs currently licensed represent a substantial success in terms of clinical benefit delivered and revenue (profit) generated within the biopharmaceutical industry. Additionally, it is estimated that ∼30% of new drugs likely to be licensed during the next decade will be based on antibody products 1, 2, 3. The efficacy of rMAbs results from their specificity for the target antigen and biological activities (effector functions) that might be activated by the immune complexes formed. Recombinant monoclonal antibody therapy might be applied to achieve one, or more, of several outcomes: (i) killing of cells or organisms, for example cancer cells, bacteria; (ii) neutralisation of soluble molecules, for example cytokines in chronic disease or toxins in infection; (iii) as agonists or antagonists of cellular activity; (iv) induction of apoptosis; (v) delivery of a cytotoxic payload such as a chemotherapy drug, catalytic toxin, radioisotope, or enzyme. Importantly, multiple antibodies binding the same antigen but recognizing different surface structures (epitopes) might be generated that can differ in their functional activity and might be used, to advantage, in selected combinations.

Five classes of human antibody (immunoglobulin, Ig) are defined: IgM, IgG, IgA, IgD and IgE; within IgG and IgA four and two subclasses (IgG1, IgG2, IgG3, IgG4 and IgA1, IgA2) are designated, respectively, according to their relative concentrations in serum. Each antibody class and subclass expresses a unique profile of effector functions 4, 5, 6. The IgG antibody class predominates, quantitatively, in blood and extra vascular space and is relatively easy to purify from whole serum. Consequently it has been the subject of structural and functional studies over decades. The initial difficulty in obtaining meaningful sequence data was overcome by analysis of monoclonal IgG proteins present in the sera of patients having multiple myeloma, a cancer resulting from clonal proliferation of a single antibody secreting plasma cell. Because the IgG1 subclass predominates in blood and in the incidence of myeloma, it was the first to be comprehensively investigated. Similarly, the IgG antibody class predominates in the blood of mice and the hybridoma technique for generating monoclonal mouse antibodies leads to the production, predominantly, of mouse IgG antibodies. The considerable understanding of the mode of action of the IgG antibody class resulted in it being the isotype of choice when recombinant techniques allowed for the generation of mouse/human chimeric antibodies. All currently licensed rMAb are of the IgG class and predominantly of the IgG1 subclass. They are manufactured in Chinese hamster ovary (CHO), mouse NSO or mouse Sp2/0 cell lines.

Section snippets

Choice of IgG subclass

The choice of IgG subclass is a crucial decision when developing rMAb therapeutics 5, 6. In oncology the IgG1 subclass has been the isotype of choice as it has maximal potential to eliminate targeted cancer cells by inducing antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) and apoptosis. However, in chronic diseases neutralisation of a soluble target, for example a cytokine, might be the central objective and excessive effector activity could be

Mechanisms of therapeutic action

When an antibody binds to a target antigen the latter is said to be opsonised given that the immune complex formed might activate downstream processes that result in the removal and elimination of the complex. This principle has been known for more than a century and laboratory-based in vitro research has elucidated many parameters that influence activation and outcomes of these processes. There remains a substantial challenge to elucidate, understand and anticipate mechanisms of action and

Pharmacokinetics and pharmacodynamics of IgG

Normal human IgG equilibrates between the vascular and extra-vascular compartments, ∼60% being extra-vascular. After intra-vascular injection of IgG there is a rapid fall in blood (serum) concentration as equilibration with the extra-vascular space takes place; this is referred to as the α phase. After equilibration, serial sampling enables the β phase of turnover to be determined and establishes half-life values of ∼20–25 days for IgG1, IgG2 and IgG4 subclasses and ∼7 days for IgG3. The long

Adverse events

It is common for short-term adverse reactions to be experienced on first exposure to rMAb therapy; however, treatment protocols have generally been refined to minimise these effects. When large cohorts of patients receive such therapies a minority might experience more serious adverse reactions, including immediate hypersensitivity reaction, for example patients receiving the rMAb cetuximab. This rMAb has an additional glycosylation site within the variable region of the heavy chain and

Conclusions

The presence of ‘core’ oligosaccharide is essential for the expression of IgG-Fc effector functions, and the addition of outer arm sugar residues has a variable influence on the efficacy of specific functions. Thus, an informed choice of IgG subclass and glycoform is a step towards optimisation of an rMAb for a given disease indication. It seems that the intimate association of the oligosaccharide with the protein structure limits access to galactosyl and sialyl transferases expressed in CHO,

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