Computational protocol: Complex Interplay between Epitope Specificity and Isotype Dictates the Biological Activity of Anti-human CD40 Antibodies

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Protocol publication

[…] ChiLob 7/4-h1 was digested with pepsin (10 mg/ml at room temperature). Cleavage of the heavy chain occurs below the cysteine rich sequence of the hinge region. The cleaved product was subjected to size exclusion chromatography (SEC) using two coupled S200 columns (GE Healthcare; 70ml volume in total) to remove the Fc fragment. The resulting F(ab’)2 fragments were reduced with DTT to produce Fab’ and quenched by Iodoacetamide. The final Fab’ product was verified by HPLC and CE-SDS.The Fab’ fragment was incubated with CD40 ECD (CRD 1-4) in PBS at room temperature for 30 minutes at a molar ratio of 1:4 (CD40:Fab’), followed by SEC using an S200 10/300 column (GE Healthcare) using HEPES buffer (150 mM HEPES, 50 mM KCl pH 7.0). Complex formation was confirmed by HPLC. The complex was concentrated to 13.6 mg/ml for crystallisation in sitting drop plates, set up using a TOPS () screen at 20°C using a Gryphon (Art Robbins Instruments). Crystals grew over 3 weeks in condition A11 (20% w/v PEG8000, 0.2 M Ammonium Sulphate, 0.1 M Sodium Cacodylate, pH 6.0) and were harvested in a cryo-protectant (crystallisation buffer containing 20% glycerol), flash frozen and stored under liquid nitrogen.Diffraction experiments were carried out at beamline ID23-1 (ESRF, France) at a wavelength of 0.9763 (12.7KeV); images were collected using a Pilatus 6M detector. The final dataset in space group P3121 was processed using XDS to a resolution of 3Å ()All data manipulation was carried out using the CCP4 suite (). Data reduction and scaling was performed using AIMLESS (). Molecular replacement was carried out with PHASER using pdb models 1U6A for the Fab’ and 3QD6 for CD40 (). Iterative model building and reciprocal space refinement were carried out using COOT () and REFMAC5 (). Refinement statistics are given in . The final model and structure factors have been deposited in the PDB. […]

Pipeline specifications

Software tools XDS, CCP4, Coot, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Autoimmune Diseases, Neoplasms
Chemicals Cysteine