The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody

• Published in: Biochemical journal Vol. 472; no. 2; p. 169
• Main Authors: Ciferri, Claudio, Lipari, Michael T, Liang, Wei-Ching, Estevez, Alberto, Hang, Julie, Stawicki, Scott, Wu, Yan, Moran, Paul, Elliott, Mike, Eigenbrot, Charles, Katschke, Kenneth J, van Lookeren Campagne, Menno, Kirchhofer, Daniel
• Format: Journal Article
• Language: English
• Published: England 01.12.2015
• Subjects: Allosteric Regulation; Amino Acid Substitution; Animals; Antibodies, Neutralizing - chemistry; Antibodies, Neutralizing - genetics; Antibodies, Neutralizing - metabolism; Antibodies, Neutralizing - pharmacology; Antibody Specificity; Antigen-Antibody Complex - chemistry; Antineoplastic Agents - chemistry; Antineoplastic Agents - metabolism; Antineoplastic Agents - pharmacology; Binding Sites, Antibody; Catalytic Domain; Cell Line, Tumor; Epitope Mapping; High-Temperature Requirement A Serine Peptidase 1; Humans; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - metabolism; Immunoglobulin Fab Fragments - pharmacology; Immunoglobulin G - chemistry; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Immunoglobulin G - pharmacology; Melanoma - drug therapy; Melanoma - enzymology; Melanoma - metabolism; Mice; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutant Proteins - pharmacology; Neoplasm Proteins - antagonists & inhibitors; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Protease Inhibitors - chemistry; Protease Inhibitors - metabolism; Protease Inhibitors - pharmacology; Protein Subunits - antagonists & inhibitors; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Serine Endopeptidases - chemistry; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; antibody; HtrA1; serine protease; phage display; age-related macular degeneration
• ISSN: 1470-8728
• DOI: 10.1042/BJ20150601

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.