title: A beta-lactamase with reduced immunogenicity for the targeted delivery of chemotherapeutics using antibody-directed enzyme prodrug therapy.
abstract: 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.
authors.affiliation: Genencor International, 925 Page Mill Road, Palo Alto, California 94304, USA.
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Harding
authors.forename: Amy D
authors.initials: AD
authors.lastname: Liu
authors.forename: Marcia
authors.initials: M
authors.lastname: Stickler
authors.forename: O Jennifer
authors.initials: OJ
authors.lastname: Razo
authors.forename: Regina
authors.initials: R
authors.lastname: Chin
authors.forename: Nargol
authors.initials: N
authors.lastname: Faravashi
authors.forename: Wendy
authors.initials: W
authors.lastname: Viola
authors.forename: Tom
authors.initials: T
authors.lastname: Graycar
authors.forename: V Pete
authors.initials: VP
authors.lastname: Yeung
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Aehle
authors.forename: Daan
authors.initials: D
authors.lastname: Meijer
authors.forename: Stephanie
authors.initials: S
authors.lastname: Wong
authors.forename: M Harunur
authors.initials: MH
authors.lastname: Rashid
authors.forename: Ana M
authors.initials: AM
authors.lastname: Valdes
authors.forename: Volker
authors.initials: V
authors.lastname: Schellenberger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002511:Cephalosporins / Q000494:pharmacology; D002846:Chromatography, Affinity; D002986:Clinical Trials as Topic; D004305:Dose-Response Relationship, Drug; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D007769:Lactams / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011355:Prodrugs / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012306:Risk; D013601:T-Lymphocytes / Q000276:immunology; D013997:Time Factors; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D002511:Cephalosporins; D000939:Epitopes; D007074:Immunoglobulin G; D007769:Lactams; D010455:Peptides; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases; C021720:nitrocefin