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.
Harding
Fiona A
FA
Genencor International, 925 Page Mill Road, Palo Alto, California 94304, USA.
Liu
Amy D
AD
Stickler
Marcia
M
Razo
O Jennifer
OJ
Chin
Regina
R
Faravashi
Nargol
N
Viola
Wendy
W
Graycar
Tom
T
Yeung
V Pete
VP
Aehle
Wolfgang
W
Meijer
Daan
D
Wong
Stephanie
S
Rashid
M Harunur
MH
Valdes
Ana M
AM
Schellenberger
Volker
V
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*
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