Expression of a recombinant human anti-MUC1 scFv fragment in protease-deficient Escherichia coli mutants.

The proteolytic activity of Escherichia coli periplasmic proteases can affect the expression efficiency of many heterologous proteins such as antibody fragments that are transported to the host periplasm for folding. We investigated whether four E. coli strains that were deficient in the periplasmic proteases tsp, protease III, degP and ompT, in different combinations, affect the expression levels of an anti-MUC1 scFv fragment. The ompT protease appeared to be involved in partial degradation of the scFv since degradation products were observed in all ompT unmutated strains in Western blotting, whereas such products were absent in the ompT mutated strains. The HM120 strain that contained most mutations, expressed the scFv protein efficiently but the level of functional antibody activity was low. This was probably due to an accumulation of incorrectly folded antibody molecules in the periplasm as it was characterised by low enzyme immunoassay reactions in contrast to the intense staining of the tag in Western blots. Improved understanding of the periplasmic protease involvement in the process of the antibody expression in bacteria may allow us to design host E. coli strains that are more efficient in producing functional antibodies.

In vitro induction of somatic mutations in human peripheral B lymphocytes.

The major cellular and molecular factors that lead to the induction of somatic mutations in human B cells remain unclear. This study describes an approach which allowed us to single-cell culture antigen-specific B cells after in vitro immunization. Using single strand conformational polymorphism analysis on the descendant cultures we were able to demonstrate that the Ig gene of B cells was induced to mutate. Three clones were isolated from a single cell culture of naive B cells immunized against the semi-conserved region of V3 of HIV-1, which contained two point mutations at the CDR2 region leading to amino acid codon change. Two clones were also obtained from a single cell culture of memory B cells immunized against the recall antigen tetanus toxoid. These clones showed one point mutation at the CDR1-FR2 border region leading to amino acid codon change. Fab constructs from the mutated culture of naive B cells immunized against V3 were cloned to a procaryotic expression vector. The expression products of two clones showed anti-V3 specificity in ELISA using three different conjugates. Our results suggest that somatic mutations can be induced in human peripheral B cells through specific interaction with antigen and antigen-activated T cells.

Human single-chain Fv antibodies to MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma.

The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origins, such as breast, ovary, and colon. We isolated and describe five human single-chain (sc) Fv antibodies specific for the MUC1 variable number of tandem repeats region isolated by in vitro selection from a large naive phage antibody library containing over 6 x 10(9) different scFv antibodies. A synthetic biotinylated 100-mer peptide corresponding to five tandem repeats of the MUC1 peptide core was used for selection. Two of the antibodies were highly specific for MUC1 as judged by ELISA and flow cytometry. In immunohistochemistry, antibody clone 10A stained MUC1 in the cytoplasm and membrane of adenocarcinoma cells of breast and ovary, whereas in normal epithelium, only cytoplasmic or no staining was observed. With antibody clone 10B, staining was less pronounced and was not always membrane associated in adenocarcinoma. Determination of the fine specificity of 10A and 10B using a novel "indirect epitope fingerprinting" ELISA showed that both antibodies recognize unique epitopes that have not been described for hybridoma-derived anti-mucin antibodies of mouse origin. The selected human antibodies, like many of the murine MUC1 antibodies, recognize epitopes on the protein core of MUC1 that are abundantly present in the underglycosylated form of cell surface mucin on adenocarcinoma. The best human scFv, clone 10A, appears to distinguish normal cells from adenocarcinoma cells, which makes it an attractive candidate for use in antibody-based tumor targeting.

Retrieval of DNA sequences present at an extremely low frequency.

We describe a method for retrieving sequences with one or two point mutations of a given target sequence, which are present in a DNA population at a frequency of 1 in 466 x 10(3) and 1 in 28 x 10(3) molecules, respectively. By stringent hybridization to a stable, chemically immobilized probe, a large excess of unrelated fragments is removed, and the bound sequences are dissociated and amplified. By repeating the hybridization-amplification cycles twice, we achieved an estimated enrichment of 404,000-fold and 1612-fold, respectively, which was confirmed by cloning the resultant products and sequencing 35 clones. This procedure can be applied to retrieve mutated sequences that exist at an extremely low frequency in a DNA population.