Evidences that syngeneic alpha-type anti-idiotypic antibodies may non-competitively inhibit idiotype/oligomeric antigen interactions by affecting idiotype avidity

The effects of syngeneic anti-Id antibodies on the multivalent interaction between human TNF-alpha, a homotrimeric Ag, and an anti-TNF mAb (mAb₁78) have been studied. Eight anti-mAb₁78 Ig secreting hybridoma, able to inhibit TNF binding in a competitive or non-competitive mode, have been generated. Two representative clones (mAb₂1G3 and mAb₂9F1) were selected for studying the inhibition mechanism of TNF-mAb₁78 interaction. Idiotype-paratope topography studies indicated that mAb₂1G3 (IgG2a) and mAb₂9F1 (IgG1) bind two sterically distinct idiotopes on mAb₁78 (IgG1) V regions. In particular, mAb₂lG3 was found to bind an idiotope located within (or spatially close to) the Ag combining site suggesting that competitive inhibition of TNF binding to mAb₁78 by mAb₁1G3 occurs through paratope blockade. On the other hand, mAb₂9F1 recognizes an idiotope located outside the paratope, being able to bind mAb₁78 even in the presence of saturating amounts of TNF. mAb₁78-TNF molar ratio in complexes, at stoichiometric equivalence, was unchanged in the presence of a large excess of mAb₂9F1, suggesting that the functional bivalency of mAb₁78 was not impaired by this anti-Id antibody. However, bivalent mAb₂9F1 was able to partially inhibit the binding of bivalent mAb₁78 to oligomeric TNF in liquid-phase as well as in solid-phase assays, whereas no inhibition was observed with monovalent mAb₂9F1-F(ab) or mAb₁78-F(ab). This suggests that inhibition is based on a decrease of the avidity of bivalent mAb₁78 and not on allosteric effects on antigen binding sites. The effect of mAb₂9F1 on mAb₁78 arm flexibility and paratope orientation is discussed. In conclusion, the results indicate that anti-Id antibodies may inhibit Ag-antibody multivalent interactions by paratope blockade or by affecting the antibody avidity.