American Association for Clinical Chemistry
Better health through laboratory medicine
NACB - Scientific Shorts
NACB - Scientific Shorts (formerly NACB Blog)
By Gregg Wallis, PhD, CMgr.

The classic paradigm of antibody structure is that a single mature plasma cell produces one type of immunoglobulin heavy chain and one type of immunoglobulin light chain. These are combined within the cell to produce a tetrameric molecule composed of two identical heavy chains and two identical light chains. Immunoglobulin light chains exist as two types, kappa (κ) and lambda (λ), but each plasma cell can only produce one of these; thus secreted immunoglobulins have only κ or only λ light chains.

Within the last 10 years it has been shown that human IgG4 molecules are dynamic and can exchange half molecules in vivo to become bi-specific (monovalent) antibodies (1-3). This process involves the swapping of a heavy-light chain pair (half antibody) between different IgG4 molecules, reforming new disulphides in the hinge region but without disruption of the heavy-light inter-chain disulphide bond. Thus, an immunoglobulin with 2 different antigen binding domains can be produced. Elegant biochemical studies have identified the amino acid residues implicated in the control of this mechanism (4-5). This process is presumably a dynamic equilibrium and the localised concentrations of different IgG4 molecules will govern the extent of bi-specific antibody formation.  The in vivo location of the exchange process has not been determined, but it is likely to be related to local redox conditions in the blood or cell surfaces. The process has somewhat controversially been termed “Fab-arm” rather than “half-molecule” exchange.

We were intrigued to discover if the half-molecule exchange process can result in IgG4 with both kappa and lambda light chains on the same immunoglobulin molecule. Polyclonal IgG4 purified from pooled or single donors of normal serum contained a substantial portion that could not be fractionated by light chain specificity. Size exclusion chromatography showed that this material was principally composed of immunoglobulin monomers (150 kDa) and not aggregates. Analysis of these monomers by ELISA and western blotting suggested the presence of both kappa and lambda light chains on the same IgG4 molecule.  MALDI-Mass spectrometry showed that polyclonal light chain hybrid IgG4κ/λ had an average molecular weight approximately half-way between that of IgG4κ and IgG4λ. Reduction with DTT released both kappa and lambda light chains from only the hybrid.  The analytical techniques clearly indicated that IgG4 proteins containing different light chains on the same molecule were present. Based on the molecular weight these molecules were formed of 2 IgG4 heavy chains plus 1 kappa and 1 lambda light chain. Compositional analysis indicated that about 30% of purified polyclonal IgG4 was of the hybrid κ/λ form. 

These results have demonstrated that hybrid asymmetrical IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal (non-immunized) human serum. This is a logical outcome from the process of half-molecule exchange in vivo. IgG4 has been considered an odd antibody since it cannot crosslink antigen and poorly activates complement (6-7). Now these recent studies have shown that structurally it is also odd, undergoing dynamic half-molecule exchange to produce asymmetrical antibodies with different immunoglobulin light chains on the same molecule. These structural anomalies most likely contribute to the incongruous functionality; however does the occurrence of IgG4 hybrids play a role in the pathology of IgG4-related diseases?

The presence of light chain hybrid IgG4 molecules could pose problems for accurate diagnosis and monitoring of IgG4 monoclonal gammopathy patients with low serum paraprotein concentrations since the likelihood of exchange with polyclonal molecules is high. At higher concentrations a monoclonal IgG4 molecule is presumably more likely to exchange with another identical monoclonal molecule.  Any diagnostic strategies that rely on subtle evaluation of paraproteins such as MGUS progression, definitions of complete remission and early relapse (clonal evolution) could be compromised by IgG4 half-molecule exchange.

1) van der Neut Kolfschoten M, Schuurman J, Losen M, Bleeker WK, Martínez-Martínez P, Vermeulen E, et al. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science 2007;317:1554-7.
2) Labrijn AF, Buijsse AO, van den Bremer ET, Verwilligen AY, Bleeker WK, Thorpe SJ, et al. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo. Nat Biotechnol 2009;27:767-71
3) Lewis KB, Meengs B, Bondensgaard K, Chin L, Hughes SD, Kjaer B, et al. Comparison of the ability of wild type and stabilized human IgG(4) to undergo Fab arm exchange with endogenous IgG(4)in vitro and in vivo. Mol Immunol 2009;46:3488-94.
4)Labrijn, AF, Rispens, T,  Meesters, J, Rose, RJ,  den Bleker, TH,  Loverix, S,et al. Species-Specific Determinants in the IgG CH3 Domain Enable Fab-Arm Exchange by Affecting the Noncovalent CH3-CH3 Interaction Strength.  J. Immunol. 2011;187: 3238-3246  
5) Rispens, T, Ooijevaar-de Heer,P, Bende, O and Aalberse, R C. Mechanism of immunoglobulin G4 Fab-arm exchange. J. Am. Chem. Soc. 2011;133:10302-10311.
6) Aalberse RC, Stapel SO, Schuurman J, Rispens T. Immunoglobulin G4: an odd antibody. Clin Exp Allergy 2009;39:469-477.
7) Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med 2012;366:539-51


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