title: Comparing domain interactions within antibody Fabs with kappa and lambda light chains.
abstract: IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Raheleh
authors.initials: R
authors.lastname: Toughiri
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Xiufeng
authors.initials: X
authors.lastname: Wu
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Diana
authors.initials: D
authors.lastname: Ruiz
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Flora
authors.initials: F
authors.lastname: Huang
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Crissman
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Dickey
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Froning
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Conner
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Cujec
authors.affiliation: a Eli Lilly and Company, Lilly Biotechnology Center , 10300 Campus Point Drive, San Diego , CA 92130 , USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Demarest
mesh_terms: D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry*; D007146:Immunoglobulin lambda-Chains / Q000737:chemistry*; D000072417:Protein Domains
chemical_list: D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains
keywords: Antibody; Fab; kappa light chain; lambda light chain; stability