title: The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody.
abstract: High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition.
authors.affiliation: Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Claudio
authors.initials: C
authors.lastname: Ciferri
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Lipari
authors.affiliation: Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Wei-Ching
authors.initials: WC
authors.lastname: Liang
authors.affiliation: Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Alberto
authors.initials: A
authors.lastname: Estevez
authors.affiliation: Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Julie
authors.initials: J
authors.lastname: Hang
authors.affiliation: Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Scott
authors.initials: S
authors.lastname: Stawicki
authors.affiliation: Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Paul
authors.initials: P
authors.lastname: Moran
authors.affiliation: Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Mike
authors.initials: M
authors.lastname: Elliott
authors.affiliation: Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.affiliation: Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A.
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Katschke
authors.affiliation: Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A. vanlookeren.menno@gene.com dak@gene.com.
authors.forename: Menno
authors.initials: M
authors.lastname: van Lookeren Campagne
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A. vanlookeren.menno@gene.com dak@gene.com.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kirchhofer
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001666:Binding Sites, Antibody; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D018604:Epitope Mapping; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D008545:Melanoma / Q000188:drug therapy* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D057134:Antibodies, Neutralizing; D000936:Antigen-Antibody Complex; D000970:Antineoplastic Agents; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; C492073:HtrA1 protein, mouse; D012697:Serine Endopeptidases
keywords: HtrA1; age-related macular degeneration; antibody; phage display; serine protease