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The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells.A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA.Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K(a)) range between 8 x 10(7) and 10(9) M(-1). Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23).In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.

Immunotherapeutic approaches to cancer offer an attractive adjunct to conventional modalities, although human antiglobulin responses can be an obstacle to repeated treatment. This study of a large number of patients with B-cell malignancies, over an extended period of time, characterized their human antimouse antibody (HAMA) seroconversion.A total of 617 samples from 112 subjects were analyzed for HAMA titers. Eighty-five patients with B-cell malignancies; 12 breast cancer patients, and 15 volunteers were titered for comparison. Fifty-six B-cell malignancy patients were titered for HAMA throughout Lym-1 radioimmunotherapy (RIT); 29 were titered after a single imaging dose of Lym-1 antibody.Baseline titers did not correlate with subsequent HAMA seroconversion against Lym-1. Only 1 of 29 (3%) of the patients developed HAMA after an imaging dose of Lym-1. Of the RIT trial group, 37 of 56 (66%) never developed HAMA above baseline despite multiple doses. Of those who did (19 of 56; or 34%), the HAMA responses fell into two categories. Thirteen responded rapidly (median of 31 days) and were termed "early responders," whereas 6, termed "late responders," had a median response time of 111 days. Early responders developed higher peak HAMA titers with fewer exposures to Lym-1 and took longer to return to baseline than did the late responders. The frequency of new antiglobulin seroconversion decreased as the number of exposures increased.Seventy-seven percent of B-cell malignancy patients developed no response or a weak response after multiple doses of mouse Lym-1 antibody. Positive responders occurred in all histology types and fell into two categories differing in seroconversion time and titer, possibly indicative of the initial state of the immune system.