Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. B-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 B-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. B-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 B-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 B-lactamase variant with adjuvant. The B-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. [Mol Cancer Ther 2005;4(11):1791-800]
Human CD4 þ T-cell epitopes were identified in interferon-beta (IFN-b)-1b. A prominent peptide epitope region was found that induced a proliferative response in 16% of all donors tested. Responses corresponded to the presence of the HLA-DR2 haplotype. Responsive donors expressing the HLA-DQ6 allele showed an increased level of proliferation to the epitope as compared to peptide-responsive HLA-DQ6 negative donors. A similar result was found for HLA-DR15-expressing donors. PBMC from donors expressing HLA-DR15 were more likely to proliferate in response to IFN-b in a whole-protein in vitro assay than donors who did not carry this haplotype. It is striking that the common DQ6 allele HLA-DQB1*0602 is found in linkage disequilibrium with HLA-DRB1*1501, and this combination defines the HLA genotype associated with the development of multiple sclerosis. The HLA association between a response to IFN-b and MS might explain the prevalence of neutralizing antibody development, and may underlie the etiology of the disease.
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