Computational protocol: Efficient Generation of Bispecific Murine Antibodies for Pre-Clinical Investigations in Syngeneic Rodent Models

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[…] Mouse IgG2a-T370K Fc mutant was recombinantly expressed in HEK293 cells and purified by Protein A affinity chromatography followed by size exclusion chromatography at Sino Biological, Inc. (China). Crystallization experiments employing the sitting drop vapor diffusion method were set up as 300 nL drops in Corning 3550 96-well plates. Crystals were grown at 20 °C. Initial crystals obtained in 23–25% (w/v) PEG 1,000, 0.1 M HEPES, pH 7.5, 5% (w/v) PEG 400 were used as seeds in subsequent refinement of crystallization conditions. Final crystals were obtained with a reservoir solution comprising 40% (w/v) PEG 200, 0.1 M MES, pH 6.5.Crystals were cryoprotected in reservoir solution supplemented with 20% (v/v) glycerol, and snap frozen in LN2. X-ray diffraction data were collected using a 1.000 Å wavelength at the Advanced Photon Source (APS) beamline 17-ID (IMCA-CAT) at Argonne National Laboratory equipped with a DECTRIS Pilatus 6 M pixel array detector and were processed with the program XDS.Initial phases were determined by the method of molecular replacement using as a search model isolated CH2 and CH3 domains from previously refined internal structure of similar composition. The initial model underwent rounds of rebuilding and refinement using the programs Coot and PHENIX, respectively. Toward the end of refinement the structure was processed with PDB_REDO, and final refinement was performed using the program Refmac5. The final model had good stereochemical quality with 98.4% percent of residues in the favorable region of the Ramachandran map and without Ramachandran outliers. Data processing and refinement statistics are presented in Supplementary Table . [...] Explicit solvent molecular dynamics (MD) simulations of the CH3 dimer were performed for human IgG1 wild-type (PDB ID: 3AVE), mmIgG2a wild-type (PDB ID: 3ZO0), and the crystal structure of mmIgG2a-T370K determined here. For each system, the crystal structures of the CH3 dimer were used as the starting conformation. The simulations were performed under NPT conditions with a fixed number of atoms, constant pressure of 1 bar, and constant temperature of 300 K. The simulations were set up in Maestro and run using the GPU version of Desmond, both part of the Schrodinger 2016-3 suite (Schrodinger, LLC, New York, NY). In the initial setup, the crystal structures were solvated in a cubic box of size 70 Å. Due to the same simulation box size and similar size and shape of the different CH3 domains, all simulations had roughly the same number of water molecules (~10,000). The OPLS3 force field was used for the protein and the SPC model was used for the water molecules. Initial equilibration was performed using standard protocol (see, for example, ref. ) in Desmond. The production run was 250 ns long using a time step of 2 fs and conformations were saved every 100 ps generating a trajectory with 2500 frames. The frames were aligned on the respective crystal structures based on backbone atoms of residues 370, 371, 399 and 405 from one CH3 domain and residues 364′ and 409′ from the opposite CH3 domain. In order to inspect the water structure in the interface, a three dimensional normalized histogram of the water oxygen atoms was computed after discretizing the simulation box into cubic cells of side 0.5 Å. The water occupancy analysis was performed using the grid utility in the AmberTools 16 and visualized in VMD. [...] Data sets were compared using one-way ANOVA analysis (Dunnett’s multiple comparison) (GraphPad Prism for Windows, version 5.01; GraphPad). Log-rank Mantel-Cox analysis was applied to tumor progression curves (SPSS statistics 20; IBM). Statistical significance was accepted when P < 0.05. […]

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