In VitroAffinity Maturation of Human IgM Antibodies Reactive with Tumor-Associated Antigens

Abstract

Human lymphocytes secreting tumor cell-specific IgM antibodies were enriched in vitro following the stimulation of allogeneic human splenocytes from nontumor-bearing donors with cytostatic tumor cells or tumor cell plasma membrane fractions. The antibodies were generally of the IgM class and displayed low intrinsic affinity (K(d) > 100 nM). Nonetheless, the avidity arising from multivalent binding sites permitted the identification of multiple monoclonal antibodies (MAbs) displaying specificity for cultured tumor cells. Five antibodies were cloned from the B cells and two of these were expressed as human Fabs with IgG(1) constant regions. Although the avidity of the human IgM antibodies was sufficient to permit detection in the original screening, the monovalent Fabs displayed low binding activities, consistent with their low intrinsic affinity. Thus, in vitro affinity maturation was used to rapidly generate multiple variants of both antibodies displaying greater than 100-fold higher affinity. Two of the antibodies were characterized further and shown to have distinct specificities. One of the targets, LH11238, is associated both with the plasma membrane and with lysosomes and is rapidly internalized following incubation of the antibody with intact live cell monolayers. The second antigen, designated LH13, is a secreted antigen that has been enriched 200-fold from conditioned media and consists of two reactive bands at 42 and 45 kDa on denaturing Western blots. The stimulation and enrichment of human lymphocytes in culture coupled with rapid in vitro affinity maturation of low affinity antibodies potentially enables the discovery of human antibodies to a broader range of epitopes, including those that might be of greater therapeutic relevance.

Generation and Activity of a Humanized Monoclonal Antibody That Selectively Neutralizes the Epidermal Growth Factor Receptor Ligands Transforming Growth Factor-αand Epiregulin

“…Fabs were expressed and confirmed to bind to mouse/rat and human Tgfa. Affinity maturation was done as described in Pancook et al (2001) and yielded an affinity optimized Fab that was converted into monoclonal antibody (mAb)41. The mouse mAb41 antibody was chosen for humanization based on its performance in the in vitro myofibroblast proliferation assay and demonstrated in vivo efficacy in mice.…”

Identification and validation of cell surface antigens for antibody targeting in oncology

“…Moreover such MAbs have a low risk for immunogenicity in man, as they are human, and often have few or no somatic mutations (Bra¨ndlein et al 2003b). An intriguing complication is that most, if not all, tumor reactive antibodies isolated from cancer patients are IgMs (Vollmers & Bra¨ndlein 2002), necessitating either isotype switching or molecular reconfiguration to an IgG (Pancook et al 2001) to facilitate preclinical and clinical development. Several tumor-selective IgMs from cancer patients have been used to identify their cognate antigens including SC-1 (Hensel et al 1999) and PAM-1 (Bra¨ndlein et al 2003a).…”

A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

“…Even if IgM antibodies have low intrinsic affinity for tumor-specific epitopes, the high avidity of multivalent binding sites could increase the specificity for tumor cells. However, IgM is not appropriate to target intracellular proteins in live cells due to its large size[29,30]. We combined IgG or IgM antibodies directed against hTERT with Tat peptides and compared both of their activities in hTERT-negative and hTERT-positive cells(Fig.…”