Computational protocol: Improving target cell specificity using a novel monovalent bispecific IgG design

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[…] For structural studies, the DuetMab Fab carrying the alternative interchain disulfide bond between C126 of HC and C121 of LC was produced by transient expression in HEK293F cells, purified over a cation exchange column (HiTrap SP HP [GE, CT]) and polished on a size exclusion chromatography column (Superdex 75 10/300 GL). DuetMab Fab was concentrated to ∼19 mg/ml in 10 mM Tris, pH 8.0 and 100 mM NaCl, and subjected to crystallization trials using the automated Phoenix robotic system (Art Robbins Instruments, CA). Crystals were obtained only after using seeds from an unrelated Fab. The crystal used for data collection was grown by the sitting drop vapor diffusion method with a reservoir solution (0.3 ml) containing 0.018 M of each of the following salt solutions CaCl2, CoCl2, and CdCl2, 18% PEG 3350 and 5% glycerol. Drops consisting of 2.0 μl protein + 1.0 μl precipitant mixed with 100× diluted seed stock were set up at room temperature where crystals appeared within 3-7 days. The crystals were cryoprotected by soaking in well solution supplemented with increasing concentrations of glycerol (5% per step with 5 steps) to a final concentration of 30%, then flash cooled and stored in liquid nitrogen until data collection. One diffraction data set from a single crystal was collected at a wavelength of 0.97931 Å and temperature of 100 K at the IMCA-CAT 17ID beamline of the Advanced Photon Source of the Argonne National Laboratory (University of Chicago, Chicago, IL) equipped with a PILATUS 6M detector. The 0.5° images were collected with exposure time of 0.8 s per frame. Data were processed using the XDS package. Data statistics are presented in Table S3. The best solution in molecular replacement was found when VH-VL and CH1-CL domain pairs were used as separate models. The structure was then refined with REFMAC5 using all data in the resolution range of 15 Å to 2.2 Å. Water molecules were placed in the electron density peaks within hydrogen bond distances from protein atoms. The refinement statistics are given in . All crystallographic calculations were performed with the CCP4 suite of programs. Model building was carried out using the program “O.” Figures were prepared with PyMOL (Schrodinger). The atomic coordinates and structure factors of the DuetMab Fab were deposited with the Protein Data Bank under accession number 4UB0. […]

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