Quantitative methods for developing Fc mutants with extended half-lives.
Fc mutants with increased binding affinity for the neonatal receptor, FcRn, exhibit increased half-lives in vivo, and represent an attractive means for extending the half-lives of therapeutic antibodies. The half-lives of other therapeutic molecules (e.g., proteins) may also be extended by conjugating them to Fc fragments, thus decreasing the frequency of patient injections and allowing the administration of low and potentially nontoxic concentrations of the therapeutics. To investigate the possibility for further increasing the half-life of Fc, a pair of quantitative methods is presented to complement combinatorial screening and in vivo testing. Specifically, a simple molecular modeling procedure was developed to predict relative Gibbs free energies of binding values (DeltaDeltaGbind) between Fc and FcRn across different mutants and species. This procedure was found to reasonably reproduce experimental DeltaDeltaGbind values from our experiments and the literature, and may be used as an initial screen to explore Fc sequence space more fully prior to experimental testing. In addition, a mathematical model of Fc trafficking was formulated and combined with a cell-level pulse-chase assay to obtain a quantitative recycling parameter in human T84 cells. This Fc recycling parameter was found to be correlated with binding affinity, but captures the pH dependent nature of the interaction between Fc and FcRn and may serve as an additional screen following combinatorial experiments.
Kamei
Daniel T
DT
Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. kamei@seas.ucla.edu
Lao
Bert J
BJ
Ricci
Margaret Speed
MS
Deshpande
Rohini
R
Xu
Han
H
Tidor
Bruce
B
Lauffenburger
Douglas A
DA
D000818:Animals; D006207:Half-Life; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; C100688:Fc receptor, neonatal