Clonal analysis of a human antimouse antibody (HAMA) response.

Circulating human antimouse antibodies (HAMAs) directed to mouse immunoglobulin G (IgG) are clinically significant, compromising mouse antibody therapy and imaging, and interfering in immunological assays. To investigate the HAMA response, 20 stable cell lines secreting human monoclonal antibodies reactive with mouse IgG were established from a donor with a history of exposure to mice. Their subclass and domain specificities were established by solid-phase binding, indirect haemagglutination assays and immunoblotting, using Igs of known subclass and Ig fragments. The heavy-chain variable region gene usage was determined for 12 HAMAs. Eight HAMAs were IgM, 11 HAMAs were IgG4 and one HAMA was IgG1, indicating an IgG4-dominated response. All of the IgG HAMAs reacted with epitopes present on the Fc portion; one was subclass-specific, nine were subclass-restricted and two were pan-IgG-reactive. Measurement of their affinities gave dissociation constants typically in the nanomolar range. Seven and five HAMAs were derived from variable heavy-chain 3 (VH3) and VH1 gene segments, respectively. The IgG HAMAs used different VH segments to the IgM HAMAs. JH regions were coded by JH4 in eight HAMAs. DH segment usage appeared to be restricted in the IgM HAMAs. Two IgG HAMAs were clonally related. These monoclonal HAMAs are potentially useful as reagents for detecting mouse IgG and as reference reagents for the investigation of the HAMA response in patients undergoing mouse monoclonal antibody therapy and for the investigation of the influence of HAMAs on immunodiagnostic tests.

Characterization and V(H) sequences of human monoclonal anti-F(ab')(2) autoantibodies from normals and Sjögren's syndrome patients.

To investigate the genetic background of anti-F(ab')(2) autoantibodies and the mechanism behind their production we have analyzed 10 human monoclonal antibodies directed against IgG F(ab')(2) and IgG Fab. They were all derived from peripheral blood by the EBV/hybridoma technique. Eight were from three healthy individuals and two from two patients with primary Sjögren's syndrome (pSS). They react with epitopes on distinct regions of IgG, including epitopes present on or near the hinge of IgG, epitopes on the Fd gamma, and an antigenic determinant(s) present on lambda light chains. These determinants are either exposed on the intact IgG molecule or revealed following pepsin or papain digestion. The V(H) germline gene repertoire used is diverse and with considerable overlap with that used by rheumatoid factors (RF). The two IgG antibodies from normals are extensively mutated (13 and 24 mutations/V(H)), but with a replacement to silent mutation ratio in the CDR(H)1 + 2 of only 3.7. The IgM antibodies from normals are also heavily mutated (mean 10 mutations/V(H)). This suggests that anti-F(ab')(2) from normals are generated by an antigen-driven somatic hypermutation mechanism. In contrast, the two IgM antibodies from pSS are virtually unmutated in both V(H) and V(L). Together with published data of pSS RF and anti-Ro 52-kDa sequences (1-3), this suggests that there is an expanded population of naïve B cells with autoantibody specificities in the peripheral blood of pSS patients.