Journal: Cell reports

Article Title: Computational Design of Epitope-Specific Functional Antibodies.

doi: 10.1016/j.celrep.2018.10.081

Figure Lengend Snippet: Figure 1. Re-epitoping Approach for Design of an Anti-IL-17A Antibody Epitopes within the receptor-binding cavity of IL-17A were targeted for library design. (A) Depicts the PDB structure 4HSA—the IL-17A/IL-17RA complex, illustrating the large receptor-binding site and the interaction of Trp31 from the receptor in an aromatic pocket in the receptor-binding cavity. Left panel: ribbon diagram, with IL-17A dimer chains in red and blue, IL-17RA in purple, and Trp31 shown in spacefill. Right panel: surface representation of IL-17A, rotated 90 degrees about the x axis relative to left panel, with residues comprising the receptor-binding site shown in pink. IL-17RA was removed for clarity, except for Trp31, which is shown in spacefill bound in the aromatic pocket. We utilized a model of this complex as the crystal structure was not available at the time. (B) Upper panel: different epitopes within the receptor-binding cavity that were targeted based on analyses of the complementarity between a model of IL-17A and the following PDB structures: PDB: 2ZJS, 2ADG, 1GPO, 3A6C, and 3C09. The targeted epitope for each antibody is shown in pink. The region corresponding to the aromatic pocket is circled. Molecular surfaces were calculated with modeled hydrogens; interfaces/epitopes calculated as distances of 5 A˚ between heavy atoms of Ag-Ab/Receptor. Lower panel: depicts the library design for 2ZJS re-epitoping. The library contained five positions for mutation. Heavy chain is in green, light chain is in orange, and complementarity-determining regions (CDRs) (as defined by Paratome) are in blue. Positions selected for variation: heavy chain Ser55 (purple triangle) was retained or mutated to Phe or Tyr, and light chain Ser30, Asn31, Asn92, and Thr93 (2ZJS numbering, red triangle) were mutated to all potential amino acids (codon NNS). (C) Inhibition of IL-17A binding to its cell surface receptor using yeast surface display. Cells were incubated with 40 nM biotinylated IL-17A either with 120 nM IL-17RA (‘‘+IL-17+receptor’’) or without (‘‘+IL-17’’ only) or without any recombinant protein (‘‘No IL-17’’). Cells were then stained with streptavidin-allophyco- cyanin (APC) conjugate and analyzed using a flow cytometry apparatus. Results displayed as a histogram of fluorescence distribution among the population. (D) Surface representation of the crystal structure of IL-17A (red and blue) bound to two h142 FABs. The heavy and the light chains are colored green and orange, respectively. IL-17A is shown in a similar orientation to the left figure of (A). See also Figures S1, S2, and S6 and Tables S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies IL-17 Mk2 Fab This paper N/A Ant-huFab HRP Sigma-Aldrich Cat#A0293; RRID:AB_257875 Anti c-myc IgG 9E10 Santa Cruz biotechnology Cat#SC-40; RRID:AB_627268 Anti mouse IgG (Fc specific) -FITC Sigma Aldrich Cat#F4143; RRID:AB_259587 Chemicals, Peptides, and Recombinant Proteins Human IL-17A Sino Biological Cat#12047-HNAE Human IL-17A PeproTech Asia Cat#200-17-100 Human IL-17C R&D Cat#9640-IL Human IL-17F R&D Cat#1335-INS Mouse IL-17A R&D Cat#7956-ML Human IL-17RA R&D Cat#177-IR HiLoad 16/600 Superdex 75pg column GE Healthcare Cat#28989333 CaptureSelect IgG-CH1 Pre-packed Column ThermoFisher Cat#494320005 SureBlue TMB SeraCare Cat#5120-0075 Allophycocyanin-conjugated streptavidin Jackson ImmunoResearch 016-130-084 Critical Commercial Assays Streptavidin-Eu cryptate CisBio Cat#610SAKLA Anti Human IgG-XL665 CisBio Cat#61HFCXLF Human IL-6 DuoSet ELISA R&D Cat#DY206 Deposited Data Structure of the h142 Fab bound to IL-17A This paper PDB: 5N7W Experimental Models: Cell Lines CHOK1 cells Daramola et al., 2014 N/A HT1080 cells European Collection of Authenticated Cell Cultures (ECACC) Cat#85111505; RRID: CVCL_0317 Recombinant DNA Anti secY scFV library Entelechon (Eurofins Genomics) https://www.eurofinsgenomics.eu/en/ gene-synthesis-molecular-biology/genesynthesis/combinatorial-libraries/ pCTCON2 Chao et al., 2006 N/A Software and Algorithms Discovery Studio 3 Accelrys (Biovia) http://accelrys.com HEX 6.0 Ritchie and Venkatraman, 2010 http://hex.loria.fr/ GROMACS 4.54 Hess et al., 2008 http://www.gromacs.org/ Random Forest (R implementation) V 4.5-30 Liaw and Wiener, 2002 https://cran.r-project.org/src/contrib/ Archive/randomForest/ ZDOCK 2.3 (implemented in Discovery Studio) Chen et al., 2003 http://accelrys.com MODELER (implemented in Discovery Studio) Sali and Blundell, 1993 http://accelrys.com DSSP (July-95) Kabsch and Sander, 1983 https://www.embl.de/ BLAST 2.24 (tBLASTn) Altschul et al., 1997 https://blast.ncbi.nlm.nih.gov/Blast.cgi bowtie 2.2.2 Langmead and Salzberg, 2012 http://bowtie-bio.sourceforge.net/bowtie2 HTSeq 0.6.1 Anders et al., 2015 http://bowtie-bio.sourceforge.net/bowtie2 GraphPad Prism GraphPad RRID:SCR_002798 e1 Cell Reports 25, 2121–2131.e1–e5, November 20, 2018

Techniques: Binding Assay, Mutagenesis, Inhibition, Cell Surface Receptor Assay, Incubation, Recombinant, Staining, Cytometry