Kinetic and genetic bases for the heteroclitic recognition of mouse cytochrome c by mouse anti-pigeon cytochrome c monoclonal antibodies

The B lymphocyte response to pigeon cytochrome c (CYT) in BALB/c mice was p reviously shown to initiate as a heteroclitic response specific for the sel f antigen mouse CYT. As the immune response progressed, the mAb that were p roduced became less heteroclitic and often bound pigeon CYT with higher aff inity than they bound mouse CYT [Minnerath, J, et al., 1995. Proc. Natl. Ac ad. Sci. USA 92, 12379-12383]. To study the basis for heterocliticity and i ts loss in this system, the H and L chain amino acid sequences of anti-pige on CYT mAb obtained from the primary and secondary Ab responses were first compared. The most frequent somatic mutations and Ig gene joints were then introduced into an engineered single-chain Fv (scFv) that expressed the ger mline-encoded V-H and V-L amino acid sequences. The effects of those change s on the on-rate, off-rate, and affinity constants in binding both mouse an d pigeon CYT were determined by surface plasmon resonance. In this system, the heterocliticity of primary mAb was largely due to a decreased on-rate c onstant for binding pigeon CYT relative to mouse CYT, Various combinations of the three frequently occurring H chain somatic mutations (H31, H56, and H58) increased the on-rate constant to maximal levels. Only one of the muta tions (H58) decreased the off-rate constant when in combination with the ot her mutations and the effect of H58 was greater for scFv binding mouse CYT than pigeon CYT. Consequently, the mutated scFv and many secondary mAb rema ined heteroclitic, although their affinities for pigeon CYT increased. Seco ndary mAb that were no longer heteroclitic expressed non-canonical amino ac id sequences in the V-H-D-J(H) joint in the context of the canonical V gene s or expressed different V genes. In addition to providing insight into the molecular basis for heterocliticity, our findings confirm that both faster on-rate and slower off-rate constants are favored during affinity maturati on of the Ab response. (C) 2001 Elsevier Science Ltd. All rights reserved.