79 research outputs found
Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo
Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins
Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding of Designed Bispecific Antibody Therapeutics
Bispecific antibodies (bsAbs) represent a critically
important
class of emerging therapeutics capable of targeting two different
antigens simultaneously. As such, bsAbs have been developed as effective
treatment agents for diseases that remain challenging for conventional
monoclonal antibody (mAb) therapeutics to access. Despite these advantages,
bsAbs are intricate molecules, requiring both the appropriate engineering
and pairing of heavy and light chains derived from separate parent
mAbs. Current analytical tools for tracking the bsAb construction
process have demonstrated a limited ability to robustly probe the
higher-order structure (HOS) of bsAbs. Native ion mobility-mass spectrometry
(IM-MS) and collision-induced unfolding (CIU) have proven to be useful
tools in probing the HOS of mAb therapeutics. In this report, we describe
a series of detailed and quantitative IM-MS and CIU data sets that
reveal HOS details associated with a knob-into-hole (KiH) bsAb model
system and its corresponding parent mAbs. We find that quantitative
analysis of CIU data indicates that global KiH bsAb stability occupies
an intermediate space between the stabilities recorded for its parent
mAbs. Furthermore, our CIU data identify the hole-containing half
of the KiH bsAb construct to be the least stable, thus driving much
of the overall stability of the KiH bsAb. An analysis of both intact
bsAb and enzymatic fragments allows us to associate the first and
second CIU transitions observed for the intact KiH bsAb to the unfolding
Fab and Fc domains, respectively. This result is likely general for
CIU data collected for low charge state mAb ions and is supported
by data acquired for deglycosylated KiH bsAb and mAb constructs, each
of which indicates greater destabilization of the second CIU transition
observed in our data. When integrated, our CIU analysis allows us
to link changes in the first CIU transition primarily to the Fab region
of the hole-containing halfmer, while the second CIU transition is
likely strongly connected to the Fc region of the knob-containing
halfmer. Taken together, our results provide an unprecedented road
map for evaluating the domain-level stabilities and HOS of both KiH
bsAb and mAb constructs using CIU
- …