A beta-lactamase with reduced immunogenicity for the targeted delivery of chemotherapeutics using antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. beta-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of beta-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. beta-Lactamase was found to contain four CD4+ T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The beta-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of beta-lactamase variant with adjuvant. The beta-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients.

An in vitro human cell-based assay to rank the relative immunogenicity of proteins.

A method to rank proteins based on their relative immunogenicity has been devised. A statistical analysis of peptide-specific responses in large human donor pools provides a structure index value metric that ranked four industrial enzymes in the order determined by both mouse and guinea pig exposure models. The ranking method also compared favorably with human sensitization rates measured in occupationally exposed workers. Structure index values for other proteins known to cause immune responses in humans were also determined and found to be higher than the value determined for human beta2-microglobulin. Using values from known immunogenic and putative nonimmunogenic proteins, a cut-off value was established. The structure index value calculation provides a comparative method to predict subsequent immunogenicity on a human population basis without the need to use animal models. Information provided by this assay can be used in the early development of protein therapies and other protein-based applications to select or create reduced immunogenicity variants.

Human population-based identification of CD4(+) T-cell peptide epitope determinants.

A human cell-based method to identify functional CD4(+) T-cell epitopes in any protein has been developed. Proteins are tested as synthetic 15-mer peptides offset by three amino acids. Percent responses within a large donor population are tabulated for each peptide in the set. Peptide epitope regions are designated by difference in response frequency from the overall background response rate for the compiled dataset. Epitope peptide responses are reproducible, with a median coefficient of variance of 21% when tested on multiple random-donor sets. The overall average response rate within the dataset increases with increasing putative human population antigenic exposure to a given protein. The background rate was high for HPV16 E6, and was low for human-derived cytokine proteins. The assay identified recall epitope regions within the donor population for the protein staphylokinase. For an industrial protease with minimal presumed population exposure, immunodominant epitope peptides were identified that were found to bind promiscuously to many HLA class II molecules in vitro. The peptide epitope regions identified in presumably unexposed donors represent a subset of the total recall epitopes. Finally, as a negative control, the assay found no peptide epitope regions in human beta2-microglobulin. This method identifies functional CD4(+) T-cell epitopes in any protein without pre-selection for HLA class II, suggests whether a donor population is pre-exposed to a protein of interest, and does not require sensitized donors for in vitro testing.