Configuraciones algebraicas

subclass. In contrast the dissociation rate constants ( k j showed marked differences and could be ranked in the order Ig G I > lgG2 > IgGS > lgG4. Using the calculated equilibrium affinity constants (K J it was found that antibody avidity could be ranked in the order lgG4 > IgGS > lgG2 > Ig G I. In contrast, F (a b' ) 2

fragm ents derived by pepsin digestion of the parent Ig G I, lgG2 and lgG 4 mAbs did not show any differences in rate or equilibrium constants but exhibited rate and equilibrium constants lower than their respective parent mAbs. Utilising a different set of V region identical mouse-human chimeric mAbs specific for the hapten NIP, similar differences w ere observed with lgG4 having the highest avidity and IgGS the lowest avidity. Unfortunately data w ere not available for Ig G I since the hybridoma cell line did not express antibody.

M orelock and co-workers (1994), using the less sensitive technique of competitive binding ELISA, w ere able to show functional affinity differences betw een V region-identical Ig G I, lgG2 and lgG4 antibodies specific for IC A M -1. In their study the functional affinity of the antibodies w as ranked lg G 1 > lg G 4 > lg G 2 suggesting that the functional affinity differences could be explained by flexibility in the hinge regions. T h e greater flexibility of the Ig G I hinge region m ay permit bivalent binding, in contrast to that of lgG2, the isotype thought to have the least flexible hinge region (Dangl et al. 1988; Tan et al. 1990; S chnieder et al. 1988; Ci et al. 1984). In this study, lgG4 w as found to be of highest avidity using two separate sets of mAbs directed against two different antigens. Since lgG4 is known to have a relatively inflexible hinge, it is unlikely that hinge flexibility is the sole explanation for the differences noted above.

Differences in the functional affinity of IgG subclasses have been investigated more extensively in the murine system. Fulpius et al. (1 9 9 3 ) showed that an Ig G I switch variant of an IgGS parent lacked the expected rheumatoid factor activity despite having an identical V region. Similarly, Schrieber et al. (1 9 9 3 ) dem onstrated functional affinity differences betw een an Ig G I switch variant of a V region-identical IgGS parent specific for a Pseudom onas species, with Ig G I having the lowest avidity. Cooper et al. (1 9 9 4 ) have dem onstrated differences in binding kinetics determined by BIA between m ouse Ig G I, lgG2b and IgGS directed against N-acetyl-glucosam ine (GlcNAc) of streptococcus group A carbohydrate with IgGS having the highest affinity. T h e se authors suggested that the higher functional affinity of mouse IgGS specific for GlcNAc w as due to molecular co-operativity of IgGS, whereby IgGS antibodies bound in

close proximity to antigen undergo non-covalent Fc-Fc interactions stabilising the com plex. This might explain the consistent finding of higher IgGS functional affinity in the various murine systems studied although it is unlikely to explain differences in the human IgG subclasses since they do not app ear to display co­ operative binding (G reenspan and Cooper, 1992). Furthermore, the human functional equivalent of murine IgGS is believed to be lgG2 which is thought to have the least hinge flexibility (Oi et al. 1984; Dangl et al. 1988; Schnieder et al. 1988; Tan et al. 1990) while human IgGS, in contrast to all the murine IgG subclasses has a long flexible hinge. Such differences highlight the restricted structural and functional homology betw een the m ouse and human IgG subclasses and underlines the problems in extrapolating data from the murine to the human IgG system (Callard and Turner, 1990). Most of the IgG subclasses are believed to have arisen since spéciation occurred in the m am m als and hence groups such as rodents, ungulates and primates have widely different patterns of subclass proteins.

T h e functional significance of the differences in the apparent dissociation rate constants demonstrated remains unclear. However, the ability to separately analyse association and dissociation kinetics by the BIAcore™ m ay prove crucial in our understanding of certain biological phenom ena. Foote and Milstein (1 9 9 1 ) have shown that the association constant of antibodies specific for the hapten 2- phenyl-5-oxazalone m ay be critical for B cell selection and that concurrent with affinity maturation there is also kinetic maturation. With regard to antibody function, the neutralising capacity of a panel of antibodies reactive with the V 3

loop of H IV has recently been shown to correlate directly with dissociation rate (V an C o tt et al. 1994).

S u m m a ry

It appears likely that the differences observed here in the binding kinetics of chimeric m ouse-human IgG subclasses may be due to structural differences in the human constant regions although the exact m echanisms underlying such differences are at present unclear. T h e consistent finding of high avidity lgG4 mAbs is of interest, since it has been noted that high avidity lgG 4 is produced following secondary and tertiary immunisation with the neo-antigen keyhole limpet hemocyanin (D evey et al. 1990). The exact mechanisms giving rise to such high avidity lgG4 are unclear but hinge flexibility seem s unlikely to be the sole explanation. It remains to be established w hether such avidity differences betw een the different human IgG subclasses exist in naturally occurring antibodies. The observation that engineered V region identical mAbs display avidity differences may be of importance when designing therapeutic antibodies.

The avidity of IgG subclasses specific for pneumococcal