Biological significance of carbohydrate chains on monoclonal antibodies.

Abstract

We have prepared monoclonal hapten-specific mouse IgG2b antibodies depleted of asparagine-linked carbohydrate chains by treating' the hybridoma cells with tunicamycin. 'The carbohydrate-deficient antibodies behaved in an identical manner to the normal antibodies with regard to fine antigen-binding reactivity (a Fab fragment feature) and protein A binding capacity [a, feature requiring integrity at the C2H2 and CH3 domaininteraction regions in the constant region of the heavy chain (CH)I. However, they lost 'the ability to activatevcomplement, to bind to Fc.receptors on macrophages, and to induce antibody-dependent cellular cytotoxicity. Furthermore, antigen-antibody. complexes produced from such carbohydrate-deficient antibodies failed to be eliminated rapidly from the circulation. We conclude that removal of carbohydrate chains from IgG molecules may have a profound and highly select impact on the biological activity to these antibodies.Carbohydrates have been indicated to be of significant importance in secretion of Ig molecules (1) Biosynthetic Radiolabeling of Ig and TNP Binding Assay. Hybridoma cells were put in the labeling medium (F10 medium without L-leucine containing 15% fetal calf serum pretreated with TNP-coated SRBC, 4 mM of L-glutamine, 100 pug of streptomycin per ml, 100 units of penicillin-per ml, and 10 mM Hepes) at a cell density of 1 x 106 cells per ml. In some experiments (see Fig. 1 (Cooke, UK). After incubation for 1 hr at 40C, TNP-SRBC or SRBC were washed in GVB2' dissolved in 0.2 ml of 1% NaDodSO4 solution, and transferred to 2 ml of a 2:1 (vol/vol) toluene/Triton-X-100-based scintillation solution. Ig specifically bound to TNP was determined by subtracting the radioactivity bound to SRBC, which was always <10% of that of TNP-SRBC. Furthermore, adding TNP-bovine serum albumin (TNP-albumin) in excess did inhibit >98% of the binding of Ig to TNP-SRBC (data not included). The antibody level of each sample also was measured by hemagglutination (11) of TNP-SRBC.

Endoglycosidase treatment abrogates IgG arthritogenicity: Importance of IgG glycosylation in arthritis

“…Carbohydrates present in the CH2 domain of IgG have an important role in its effector functions [19,40] and also a) Calculated affinity constants of normal and EndoS-treated (D) CII-specific mAb to four different recombinant FccR, as measured using Biacore, are shown. Affinity constants were derived from sensorgram data using simultaneous fitting to the association and dissociation phases and global fitting to all curves in the set.…”

“…Thermal stability and functionality of the CH2 domains of IgG are progressively reduced with successive removal of outer-arm sugar residues [60]. Aglycosylated IgG fails to activate complement [19], is more liable to proteolytic attack [18] and is not recognized by cells expressing FccRI and II receptors [61]. Furthermore, removal of the complete carbohydrate moiety abolished the ability to activate complement and ADCC of a human IgG1 mAb, Campath-1H, but left the antigen and protein A binding activities intact, whereas removal of terminal sialic acid residues through glycopeptidase-F digestion did not affect any of Figure 6.…”

“…The IgG heterosaccharides are known to be involved not only in the stabilization of the Fc region binding sites for Clq [19,40] but also in the structural properties of the Fc region [41]. Therefore, we tested in vitro whether the removal of carbohydrate from c-chains of IgG by EndoS reduced or abolished the ability of mAb to activate complement (Fig.…”

“…Since, there was no difference in the complement activation as well as in the ability to bind antigens between fully glycosylated and EndoS-treated Ab, we [19]. To determine whether removal of sugar moieties from the CH2 domain of the Fc part of IgG could affect the formation of stable immune complexes, single immunodiffusion assays were performed on CII-impregnated agarose gels.…”

“…Modifications in these oligosaccharides affect susceptibility to proteolytic degradation, clearance rate, Ab-dependent cellular cytotoxicity (ADCC), as well as complement activation apart from binding to FcR, monocytes, protein G and C1q/C1 [12][13][14][15][16][17]. However, Fc glycosylation is not required for either protein A binding [18] or recognition of antigen [19][20][21][22]. Recently, de-fucosylation on the N297-linked glycan in the Fc part of the Ab has been shown to increase ADCC activity, indicating the importance of glyco-engineering of Ab for improved clinical efficacy [23].…”