Fab-PEG-Fab as a potential antibody mimetic.
IgG antibodies have evolved to be flexible so that they can bind to epitopes located over a wide spatial range. The two Fabs in an IgG antibody are linked together as if each Fab is at the end of a linear, flexible molecule. PEG was used as a scaffold molecule to link two Fabs together to give Fab-PEG-Fab molecules, or FpFs. Preparation of FpFs was achieved with reagents that undergo site-specific conjugation at each PEG terminus by bis-alkylation with the two cysteine thiols from a disulfide bond. This allowed each Fab to be conjugated to the PEG scaffold in essentially the same region that each Fab is linked in an IgG. Fabs were sourced directly (e.g., ranibizumab) or monoclonal IgG antibodies were proteolytically digested to obtain the Fabs. This allowed the resulting FpFs to be directly compared to parent IgGs. PEG scaffolds of 6, 10, and 20 kDa were used to make the corresponding FpFs. Dynamic light scatting data suggested the resulting FpFs were similar in size to an IgG antibody and about half the size of a 20 kDa PEGylated-Fab. The solution size of PEG-conjugated proteins is known to be dominated by the extended solution structure of PEG, so it is thought that the smaller size of the FpFs is due to interactions between the two Fabs. Anti-VEGF and anti-Her2 FpFs were prepared and evaluated. The FpFs displayed similar apparent affinities to their parent IgGs. Slower dissociation rates were observed for the anti-VEGF FpFs compared to bevacizumab. The anti-VEGF FpFs also displayed in vitro anti-angiogenic properties comparable to or better than bevacizumab. These first studies indicate that FpFs warrant further examination in a therapeutic indication where the presence of the Fc may not be required.
UCL School of Pharmacy, University College London , 29-39 Brunswick Square, London WC1N 1AX, United Kingdom.
Hanieh
H
Khalili
Antony
A
Godwin
Ji-Won
JW
Choi
Rebecca
R
Lever
Peng T
PT
Khaw
Steve
S
Brocchini
D000937:Antigen-Antibody Reactions; D040761:Biomimetic Materials / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology*; D008958:Models, Molecular; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry*
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011092:Polyethylene Glycols