HDX-MS implicates dynamic CD20 antigen binding to two discontinuous CDR regions in the NCD1.2 monoclonal antibody

ABSTRACT: A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3 and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Hydrogen deuterium exchange mass spectrometry was used to define two stages in the mode of binding of the CD20 epitope the antibody. In the early stage of the reaction, the antigen interacted with CDR3 (VH). In the equilibrium stages, stable binding occurred to CDR2 (VH) and CDR2 (VL), without any detectable CDR3 (VH) interactions. These data suggest that CDR3 (VH) functions as a transient antigen docking motif to nucleate the peptide into the antibody active site which resolves into antigen binding with the heavy and light chain CDR2 domains. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry to map the kinetic mode of antigen binding. These data support the further development of an engineered synthetic antibody for use as a canine lymphoma therapeutic that mimics the human anti-CD20 antibody therapeutic.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Canis Lupus X Canis Lupus Familiaris

TISSUE(S): B Cell

DISEASE(S): Lymphoma

SUBMITTER: Lenka Hernychova  

LAB HEAD: Ted Hupp

PROVIDER: PXD018550 | Pride | 2021-09-09

REPOSITORIES: pride