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Epitope-specific affinity maturation improved stability of potent protease inhibitory antibodies.
Lopez, Tyler; Chuan, Chen; Ramirez, Aaron; Chen, Kuan-Hui E; Lorenson, Mary Y; Benitez, Chris; Mustafa, Zahid; Pham, Henry; Sanchez, Ramon; Walker, Ameae M; Ge, Xin.
Afiliación
  • Lopez T; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
  • Chuan C; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
  • Ramirez A; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
  • Chen KE; Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, California.
  • Lorenson MY; Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, California.
  • Benitez C; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
  • Mustafa Z; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
  • Pham H; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
  • Sanchez R; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
  • Walker AM; Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, California.
  • Ge X; Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California Riverside, Riverside, California.
Biotechnol Bioeng ; 115(11): 2673-2682, 2018 11.
Article en En | MEDLINE | ID: mdl-30102763
Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its catalytic cleft. 3A2 variants with high potency, selectivity, and improved affinity and proteolytic stability were isolated from a random mutagenesis library. Binding kinetics indicated that the affinity improvements were mainly from slower dissociation rates. In vitro degradation tests suggested the isolated variants had half lives 6-11-fold longer than the wt. Inhibition kinetics suggested they were competitive inhibitors which showed excellent selectivity toward MMP-14 over highly homologous MMP-9. Alanine scanning revealed that they bound to the vicinity of MMP-14 catalytic cleft especially residues F204 and F260, suggesting that the desired epitope was maintained during maturation. When converted to immunoglobulin G, B3 showed 5.0 nM binding affinity and 6.5 nM inhibition potency with in vivo half-life of 4.6 days in mice. In addition to protease inhibitory antibodies, the competitive FACS described here can be applied for discovery and engineering biosimilars, and in general for other circumstances where epitope-specific modulation is needed.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Evaluación Preclínica de Medicamentos / Metaloproteinasa 14 de la Matriz / Inhibidores de la Metaloproteinasa de la Matriz / Factores Inmunológicos / Anticuerpos / Afinidad de Anticuerpos / Epítopos Límite: Animals Idioma: En Revista: Biotechnol Bioeng Año: 2018 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Evaluación Preclínica de Medicamentos / Metaloproteinasa 14 de la Matriz / Inhibidores de la Metaloproteinasa de la Matriz / Factores Inmunológicos / Anticuerpos / Afinidad de Anticuerpos / Epítopos Límite: Animals Idioma: En Revista: Biotechnol Bioeng Año: 2018 Tipo del documento: Article Pais de publicación: Estados Unidos