title: Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.
abstract: IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications.
authors: Bioprocess Development, Merck Research Laboratories, Merck & Company, Inc., Kenilworth, New Jersey 07033. Electronic address: xiaoyu.yang@merck.com.
authors: Xiaoyu
authors: X
authors: Yang
authors: Bioprocess Development, Merck Research Laboratories, Merck & Company, Inc., Kenilworth, New Jersey 07033.
authors: Fengqiang
authors: F
authors: Wang
authors: Bioprocess Development, Merck Research Laboratories, Merck & Company, Inc., Kenilworth, New Jersey 07033.
authors: Ying
authors: Y
authors: Zhang
authors: Bioprocess Development, Merck Research Laboratories, Merck & Company, Inc., Kenilworth, New Jersey 07033.
authors: Larry
authors: L
authors: Wang
authors: Discovery Operations, Merck Research Laboratories, Merck & Company, Inc., Palo Alto, California 94304.
authors: Svetlana
authors: S
authors: Antonenko
authors: Discovery Operations, Merck Research Laboratories, Merck & Company, Inc., Palo Alto, California 94304.
authors: Shuli
authors: S
authors: Zhang
authors: Discovery Operations, Merck Research Laboratories, Merck & Company, Inc., Palo Alto, California 94304.
authors: Yi Wei
authors: YW
authors: Zhang
authors: Discovery Operations, Merck Research Laboratories, Merck & Company, Inc., Palo Alto, California 94304.
authors: Mohammad
authors: M
authors: Tabrizifard
authors: Discovery Operations, Merck Research Laboratories, Merck & Company, Inc., Palo Alto, California 94304.
authors: Grigori
authors: G
authors: Ermakov
authors: Discovery Operations, Merck Research Laboratories, Merck & Company, Inc., Palo Alto, California 94304.
authors: Derek
authors: D
authors: Wiswell
authors: Discovery Operations, Merck Research Laboratories, Merck & Company, Inc., Palo Alto, California 94304.
authors: Maribel
authors: M
authors: Beaumont
authors: PPDM, Merck Research Laboratories, Merck & Company, Inc., West Point, Pennsylvania 19486.
authors: Liming
authors: L
authors: Liu
authors: Bioprocess Development, Merck Research Laboratories, Merck & Company, Inc., Kenilworth, New Jersey 07033.
authors: Daisy
authors: D
authors: Richardson
authors: Bioprocess Development, Merck Research Laboratories, Merck & Company, Inc., Kenilworth, New Jersey 07033.
authors: Mohammed
authors: M
authors: Shameem
authors: Bioprocess Development, Merck Research Laboratories, Merck & Company, Inc., Kenilworth, New Jersey 07033.
authors: Alexandre
authors: A
authors: Ambrogelly
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology*; D002845:Chromatography / Q000379:methods; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D016513:Mice, SCID; D061026:Programmed Cell Death 1 Receptor / Q000276:immunology
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D061026:Programmed Cell Death 1 Receptor; C582435:pembrolizumab
keywords: IgG4; analytical biochemistry; bioanalysis; biotechnology; bispecific antibody; cancer immunotherapy; glycoprotein; half molecule (Fab arm) exchange analysis; immunology; pembrolizumab