title: Combining somatic mutations present in different in vivo affinity-matured antibodies isolated from immunized Lama glama yields ultra-potent antibody therapeutics.
abstract: Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.
authors.affiliation: arGEN-X BVBA, Technologiepark 30, Zwijnaarde 9052, Belgium Department of Cell Biology, Science Faculty, Utrecht University, Padualaan 8, Utrecht CH 3584, The Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Klarenbeek
authors.affiliation: arGEN-X BVBA, Technologiepark 30, Zwijnaarde 9052, Belgium.
authors.forename: Christophe
authors.initials: C
authors.lastname: Blanchetot
authors.affiliation: Department of Cell Biology, Science Faculty, Utrecht University, Padualaan 8, Utrecht CH 3584, The Netherlands.
authors.forename: Georg
authors.initials: G
authors.lastname: Schragel
authors.affiliation: Department of Cell Biology, Science Faculty, Utrecht University, Padualaan 8, Utrecht CH 3584, The Netherlands.
authors.forename: Ava S
authors.initials: AS
authors.lastname: Sadi
authors.affiliation: arGEN-X BVBA, Technologiepark 30, Zwijnaarde 9052, Belgium.
authors.forename: Nico
authors.initials: N
authors.lastname: Ongenae
authors.affiliation: U-Protein Express BV, Padualaan 8, Utrecht CH 3584, The Netherlands.
authors.forename: Wieger
authors.initials: W
authors.lastname: Hemrika
authors.affiliation: INSERM, Unité 1098, University of Franche-Comté, 1 bd A. Fleming, Besançon 25020, France.
authors.forename: John
authors.initials: J
authors.lastname: Wijdenes
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Unité Mixte de Recherche 7257 Centre National de la Recherche Scientifique and Aix-Marseille University, Marseille Cedex 09 13288, France.
authors.forename: Silvia
authors.initials: S
authors.lastname: Spinelli
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Unité Mixte de Recherche 7257 Centre National de la Recherche Scientifique and Aix-Marseille University, Marseille Cedex 09 13288, France.
authors.forename: Aline
authors.initials: A
authors.lastname: Desmyter
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Unité Mixte de Recherche 7257 Centre National de la Recherche Scientifique and Aix-Marseille University, Marseille Cedex 09 13288, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.affiliation: arGEN-X BVBA, Technologiepark 30, Zwijnaarde 9052, Belgium.
authors.forename: Anna
authors.initials: A
authors.lastname: Hultberg
authors.affiliation: RuiYi Inc, 505 Coast Blvd S, Suite 300, La Jolla, CA 92037, USA.
authors.forename: Anke
authors.initials: A
authors.lastname: Kretz-Rommel
authors.affiliation: arGEN-X BVBA, Technologiepark 30, Zwijnaarde 9052, Belgium.
authors.forename: Torsten
authors.initials: T
authors.lastname: Dreier
authors.affiliation: arGEN-X BVBA, Technologiepark 30, Zwijnaarde 9052, Belgium Department of Cell Biology, Science Faculty, Utrecht University, Padualaan 8, Utrecht CH 3584, The Netherlands hdehaard@argen-x.com r.c.roovers@gmail.com.
authors.forename: Hans J W
authors.initials: HJ
authors.lastname: De Haard
authors.affiliation: Department of Cell Biology, Science Faculty, Utrecht University, Padualaan 8, Utrecht CH 3584, The Netherlands hdehaard@argen-x.com r.c.roovers@gmail.com.
authors.forename: Rob C
authors.initials: RC
authors.lastname: Roovers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D002161:Camelids, New World / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015850:Interleukin-6 / Q000276:immunology; D018448:Models, Immunological; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: C508600:IL6 protein, human; D007140:Immunoglobulin Fab Fragments; D015850:Interleukin-6; D019151:Peptide Library; D011994:Recombinant Proteins
keywords: Lama glama; affinity selection; antibody; in vivo potency; phage display