Molecular engineering of a therapeutic antibody for Blo t 5–induced allergic asthma J.H. Sherlynn Chan, BSc(Hons), Yen Leong Chua, MSc, Hong Yong Peh,

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Molecular engineering of a therapeutic antibody for Blo t 5–induced allergic asthma J.H. Sherlynn Chan, BSc(Hons), Yen Leong Chua, MSc, Hong Yong Peh, BSc(Hons), Vojislav Jovanovic, PhD, Nicholas R.J. Gascoigne, PhD, W. S. Fred Wong, PhD, Fook Tim Chew, PhD, Brendon J. Hanson, PhD, David M. Kemeny, PhD, FRCPath, Paul A. MacAry, PhD Journal of Allergy and Clinical Immunology Volume 139, Issue 5, Pages 1705-1708.e6 (May 2017) DOI: 10.1016/j.jaci.2016.10.028 Copyright © 2016 The Authors Terms and Conditions

Fig 1 Molecular engineering of anti–Blo t 5 and application of chimeric variants in basophil activation. A, Inhibition of Blo t 5–specific IgE binding in patients' sera by anti–Blo t 5 murine IgG1. B, Chimeric variants generated and used in this study. C, Purified chimeric antibodies on reducing SDS-PAGE gel. D, Purified chimeric antibodies on nonreducing SDS-PAGE gel. E, Affinity ELISA of purified anti–Blo t 5 chimeric antibodies. F, Representative flow plots illustrating basophil activation test and antagonistic capacity of chimeric anti–Blo t 5 mAbs. G, Percentage inhibition of basophil activation by chimeric antibodies. *P < .05. **P < .01. ***P < .0001. Journal of Allergy and Clinical Immunology 2017 139, 1705-1708.e6DOI: (10.1016/j.jaci.2016.10.028) Copyright © 2016 The Authors Terms and Conditions

Fig 2 In vivo study of anti–Blo t 5 mAb. A, Schematic diagram depicting experimental design involving intravenous injection of Blo t 5–specific TH2 cells, intranasal challenges with Blo t 5 protein (black arrows), and antibody treatment. B, Representative images of histochemical stainings of mouse lung sections. C, Scoring of extent of inflammation and mucus. D, Total and differential immune cell counts in the bronchoalveolar lavage fluid. E, Serum total and Blo t 5–specific IgE levels. F, FEV measurements. *P < .05. **P < .01.***P < .0001. Journal of Allergy and Clinical Immunology 2017 139, 1705-1708.e6DOI: (10.1016/j.jaci.2016.10.028) Copyright © 2016 The Authors Terms and Conditions

Fig E1 Epitope analysis of anti–Blo t 5 mAb. A, Purified murine anti–Blo t 5 on reducing and nonreducing SDS-PAGE gel. B, Affinity ELISA of murine anti–Blo t 5 mAb. C, Western blot of Blo t 5 protein with anti–Blo t 5 mAb under denaturing and nondenaturing (native) conditions. D, Immuno-dot blot with Blo t 5 peptide library consisting of 15-mer peptides with 10 amino acids overlap. Peptides 1 and 2 were excluded as they represented the signal peptide, which was absent in the recombinant Blo t 5 used in this study. E, Modeling by PyMOL software to visualize the position of peptide 14 on the Blo t 5 protein. Journal of Allergy and Clinical Immunology 2017 139, 1705-1708.e6DOI: (10.1016/j.jaci.2016.10.028) Copyright © 2016 The Authors Terms and Conditions

Fig E2 Cytokine and chemokine levels in the bronchoalveolar lavage fluid. A, Eotaxin levels. B, IL-4 levels. C, IL-5 levels. D, IL-13 levels. *P < .05. Journal of Allergy and Clinical Immunology 2017 139, 1705-1708.e6DOI: (10.1016/j.jaci.2016.10.028) Copyright © 2016 The Authors Terms and Conditions

Fig E3 Pulmonary function parameters. A, Respiratory rate. B, Time of expiratory phase. C, Peak expiratory flow (PEF). D, FEV when PEF is reached. E, Dynamic compliance. *P < .05. **P < .01. Journal of Allergy and Clinical Immunology 2017 139, 1705-1708.e6DOI: (10.1016/j.jaci.2016.10.028) Copyright © 2016 The Authors Terms and Conditions

Fig E4 Basophil gating strategy. A, SSC vs FSC. B, FSC-H vs FSC-A. C, HLA-DR Pacific Blue vs CRTH2 APC. APC, Allophycocyanin; SSC, side scatter. Journal of Allergy and Clinical Immunology 2017 139, 1705-1708.e6DOI: (10.1016/j.jaci.2016.10.028) Copyright © 2016 The Authors Terms and Conditions