Analysis of BR96 binding sites for antigen and anti-idiotype by codon-based scanning mutagenesis.

We performed a scanning mutagenesis study of heavy chain complementarity-determining region (CDR) residues to identify how mutations affected binding of the anti-carcinoma mAb BR96 to Ag, Lewis Y, and to an anti-Id Ab (anti-Id). By ELISA, we demonstrated that the anti-Id bound close to the Ag binding site of BR96, but the anti-Id and Ag sites were not identical. Immunoblot analysis and screening of light and heavy chain CDR libraries with multiple mutations in each CDR suggested that the heavy chain had greater involvement in anti-Id binding.

A combinatorial library strategy for the rapid humanization of anticarcinoma BR96 Fab.

We have used a combinatorial mutagenesis strategy to humanize BR96, a monoclonal antibody that binds to the Lewis Y class of tumor antigens. This approach allows simultaneous assessment of hundreds of humanized variable regions to identify the molecules that best preserve affinity, thus overcoming the major drawback of current humanization procedures, the requirement to construct and analyze each humanized antibody separately. Murine residues of BR96 were mutated to human if they were solvent-exposed residues that did not participate in the formation of the antigen binding site and were not at the interface of the light and heavy chain.

Affinity maturation of the BR96 anti-carcinoma antibody by codon-based mutagenesis.

We have increased up to 65-fold the avidity of BR96, a mAb recognizing Lewis Y (Le(y))-related Ags expressed on the surface of many human carcinomas. Libraries of mutations in the complementarity-determining regions (CDRs) of BR96 were constructed in an M13 phage Fab expression vector by codon-based mutagenesis, a method that efficiently introduces large numbers and potentially all combinations of amino acid substitutions. Two mutants that improved the affinity of BR96 to tumor Ag were identified by screening the libraries on carcinoma cell lines.

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF.

The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membrane protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence.

Analysis of the Pseudomonas aeruginosa major outer membrane protein OprF by use of truncated OprF derivatives and monoclonal antibodies.

TnphoA mutagenesis of the cloned oprF gene was utilized to generate 16 classes of fusions encoding differing lengths of the amino terminus of OprF fused to either alkaline phosphatase or to peptide tags of 1 to 20 amino acids, depending on the orientation and reading frame into which TnphoA was inserted. Representatives of each of the 16 classes were sequenced to determine the precise fusion joint. Four of these 16 representatives which produced in-frame fusions to alkaline phosphatase and another 8 with fusion joints in the amino-terminal half of OprF failed to react with a panel of 10 specific monoclonal antibodies.