title: Crystallization of a challenging antigen-antibody complex: TLR3 ECD with three noncompeting Fabs.
abstract: The mechanism of action of therapeutic antibodies can be elucidated from the three-dimensional crystal structures of their complexes with antigens, but crystallization remains the primary bottleneck to structure determination. Methods that resulted in the successful crystallization of TLR3 ECD in complex with Fab fragments from three noncompeting, neutralizing anti-TLR3 antibodies are presented. The crystallization of this 238 kDa complex was achieved through fine purification of the quaternary complex of TLR3 with the three Fab fragments combined with microseed matrix screening and additive screening. Fine purification entailed the application of a very shallow gradient in anion-exchange chromatography, resulting in the resolution of two separate complex peaks which had different crystallizabilities. Subsequent structure determination defined the epitopes of the respective antibodies and revealed a mechanistic hypothesis that is currently under investigation. The results also showed that cocrystallization with multiple noncompeting Fab fragments can be a viable path when an antigen complex with a single Fab proves to be recalcitrant to crystallization.
authors.affiliation: Centocor R&D, 145 King of Prussia Road, Radnor, PA 19087, USA. tmalia@its.jnj.com
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Malia
authors.forename: Galina
authors.initials: G
authors.lastname: Obmolova
authors.forename: Jinquan
authors.initials: J
authors.lastname: Luo
authors.forename: Alexey
authors.initials: A
authors.lastname: Teplyakov
authors.forename: Raymond
authors.initials: R
authors.lastname: Sweet
authors.forename: Gary L
authors.initials: GL
authors.lastname: Gilliland
mesh_terms: D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D003460:Crystallization; D005110:Extracellular Space / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D051196:Toll-Like Receptor 3 / Q000737:chemistry* / Q000276:immunology
chemical_list: D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3
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