Molecular determinants of polyreactive antibody binding: HCDR3 and cyclic peptides

Abstract

Human monoclonal antibody 63 (mAb63) is an IgM/lambda polyreactive antibody that binds to multiple self and non-self antigens. The molecular basis of polyreactivity is still unclear. The present study was initiated to prepare a recombinant Fab of mAb63 and use it to study the determinants involved in polyreactivity. The baculovirus system was employed to express large amounts of mAb63 Fab in Sf9 cells. Our experiments showed that infected Sf9 cells secreted a soluble 50-kD Fab heterodimer that bound to multiple self and non-self antigens. The antigen-binding activity of mAb63 Fab was inhibited by both homologous and heterologous antigens. To study in more detail the molecular determinants involved in polyreactivity, the heavy chain complementarity-determining region 3 (HCDR3), which is known to play a key role in the binding of monoreactive antibodies to antigens, was subjected to site-directed mutagenesis. A single substitution, alanine for arginine, at position 100A resulted in complete loss of antigen-binding activity. The 19 amino acids comprising the HCDR3 of mAb63 were then synthesized and a cyclic peptide prepared. The cyclic peptide showed the same antigen-binding pattern as the parental mAb63 and the recombinant mAb63 Fab. A five amino acid motif (RFLEW), present in the HCDR3 of mAb63, was found by searching the GenBank in three of 50 other human polyreactive antibodies, but in none of nearly 2500 human antibodies thought to be monoreactive. It is concluded that HCDR3 plays a major role in polyreactivity and that in some cases cyclic peptides comprising the HCDR3, by themselves, may be polyreactive.

Fig. 1

Baculovirus transfer vectors used to express recombinant human polyreactive IgM mAb63 Fab. The pBac-mAb63 μFd_his vector was used to express the mAb63 μFd chain (a) and the pBac-mAb63 λ vector was used to express the mAb63 λ-chain (b). Heterodimers of these chains produced mAb63 Fab. MCS, Multiple cloning site; HBM signal, honeybee melittin secretion signal.

Fig. 2

Expression and characterization of recombinant mAb63. Recombinant baculoviruses containing cDNA encoding mAb63 Fd and/or λ were used to infect Sf9 cells. Uninfected Sf9 cells and Sf9 cells infected with wild-type baculovirus (AcNPV) served as negative controls. Cell lysates and culture supernatants were subjected to 12% SDS–PAGE. Proteins were transferred to nitrocellulose membranes and immunoblotting was carried out using goat anti-human μ- or λ-chain. (a,b) Cells were infected for 72 h with either mAb63 Fd or λ or (c,d) co-infected with mAb63 Fd and λ recombinant baculovirus for 24–96 h. (e) Secreted recombinant mAb63 Fab (2 μg) was subjected under reducing (R) and non-reducing (NR) conditions to 12% SDS–PAGE, transferred to nitrocellulose and immunoblotted with goat antibodies to human μ- and λ-chain.

Fig. 3

Dose-dependent binding of (a) parental mAb63 (IgM), (b) recombinant mAb63 Fab, (c) recombinant mAb63 (97 S/R) Fab, (d) recombinant mAb63 (100A R/A) Fab, (e) cyclic peptide 63, and (f) cyclic peptide 6l to solid-phase ssDNA, insulin (Ins), IgG Fc, thyroglobulin (Tg), and tetanus toxoid (TT). The molecular weights of mAb63 (IgM), mAb63 Fabs, cyclic peptide 61, and cyclic peptide 63 are 900 000, 50 000, 2130 and 2680, respectively.

Fig. 4

Site-directed mutagenesis of residues within heavy chain complementarity-determining region 3 (HCDR3). The substituted residues are shown within boxes.

Fig. 5

Dose-dependent inhibition of binding of recombinant mAb63 Fab (a) and recombinant mAb63 (97 S/R) Fab (b) to solid-phase insulin (Ins) in the presence of different concentrations of soluble antigen (i.e. Ins, IgG Fc, thyroglobulin (Tg), ssDNA, or tetanus toxoid (TT)). Dose-dependent inhibition of binding of mAb63 Fab to solid-phase antigens (i.e. Ins, IgG Fc, ssDNA, or TT) in the presence of different concentrations of soluble cyclic peptide 63 (c) and cyclic peptide 61 (d). Dose-dependent inhibition of binding of cyclic peptide 63 to solid-phase insulin in the presence of different concentrations of soluble antigens (i.e. Ins, IgG Fc, ssDNA or TT) (e). The molecular weights of ssDNA, Ins, IgG Fc, Tg, TT, cyclic peptide 61, and cyclic peptide 63 are 500 000, 6000, 25 000, 640 000, 110 000, 2130 and 2680, respectively.

Fig. 6

Schematic drawing of cyclized and biotinylated peptides derived from the heavy chain complementarity-determining region 3 (HCDR3) of mAb63 and mAb61.