Article Figures & Data
Figures
- Figure 1.
Impact of EGFR density on the apparent binding affinities of IgG to EGFR-overexpressing cells. A, low and high intrinsic affinity IgG (C10 and 2224) had their functional affinities measured on cells of varying EGFR density (B).
- Figure 2.
Impact of intrinsic and apparent antibody affinity and concentration on the ability of EGF to inhibit antibody binding to EGFR-overexpressing cells. A, at 1 nmol/L IgG, EGF inhibited the binding of IgG in proportion to their intrinsic affinities; At 10 nmol/L (B) and 100 nmol/L (C) IgG concentration, there was less effect of EGF on IgG binding, especially of the higher intrinsic affinity 2224 and to a lesser extent P2/4. D, EGF inhibited the binding of Fab in proportion to their intrinsic affinities. Comparing C to D, it is clear that an increase in apparent affinity due to IgG avidity significantly reduces the reduction of antibody binding by EGF.
- Figure 3.
Impact of intrinsic and apparent affinity on EGF induced EGFR phosphorylation. A, increasing scFv intrinsic affinity reduced EGF-induced EGFR phosphorylation; this effect was greatly increased by increasing functional affinity using an IgG constructed from the scFv V-genes. B, at lower antibody concentrations, no effect of scFv on EGF-induced EGFR phosphorylation was observed and the inhibitory effect of the IgG was significantly reduced. Anti-ErbB2 IgG C6.5 was used as the control antibody.
- Figure 4.
Inhibition of A431 cell proliferation by anti-EGFR scFv, IgG and Fab. A, inhibition of proliferation of A431 cells increased with increasing scFv intrinsic affinity, with an IC50 value of 35 nmol/L for the highest affinity 2224 scFv. B, inhibition of proliferation of A431 cells increased with increasing Fab intrinsic affinity, but this effect was less than that observed for the scFv. C, inhibition of proliferation of A431 cells increased with increasing IgG intrinsic affinity. 3D12, control anti-botulinum neurotoxin antibody.
Tables
- Table 1.
Comparison of affinities of scFv, Fab, and IgG for binding to EGFR-overexpressing A431 cells
Antibody scFv KD, nmol/L Fab KD, nmol/L IgG KD, nmol/L KD (Fab)/KD (IgG) C10 263.67 124.23 1.17 106 P3/5 88.24 58.24 0.5 116 P2/1 14.81 25.4 0.012 2,117 P2/4 15.39 25.2 0.0064 3,938 P2/2 17.01 18.1 0.0077 2,351 3524 7.47 15.4 0.012 1,903 2124 9.90 1.31 0.007 187 2224 0.94 1.2 0.007 171 C225 NA 0.013 0.006 2 NOTE: KD values for binding to cells were determined by fitting the data from Fig. 1 to the Lineweaver–Burk equation.
Abbreviation: NA, not applicable.
- Table 2.
Comparison of measured apparent IgG affinities for A431 cells versus affinities calculated by using a mathematical model
IgG Experimental KD, nmol/L Model KD, nmol/L Model KD (equilibrium), nmol/L KD (Fab)/KD (IgG) C10 1.17 1.5 1.5 82.8 P3/5 0.5 0.054 0.054 1,078 P2/1 0.012 0.0071 0.0051 4,980 P2/4 0.0064 0.0071 0.0051 4,941 P2/2 0.0077 0.0059 0.0034 5,323 3524 0.012 0.0057 0.0026 5,923 2124 0.007 0.0053 0.00033 3,969 2224 0.007 0.0053 0.00031 3,870 NOTE: Fab affinities from Table 1. KD (Fab)/KD (IgG) was corrected for nonequilibrium conditions.
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file, 62KB.
- Supplementary Figure 1 - PDF file, 450KB, Biphasic distribution of the increase in binding affinity for conversion from monovalent to bivalent forms for antibodies with different intrinsic affinities.
- Supplementary Figure 2 - PDF file, 95KB, Comparison of cell-binding curves for low and high affinity anti-EGFR IgG antibodies at the experimental incubation time and a hypothetical time.
- Supplementary Table 1 - PDF file, 51KB, Binding kinetics and affinities of IgG converted from affinity-matured scFv (2224 and P2/4) vs C225 as measured by KinExA.
- Supplementary Table 2 - PDF file, 56KB, Kinetic parameters used in the computational model of antibody binding.
- Supplementary Table 3 - PDF file, 52KB, Comparison of measured apparent IgG affinities vs affinities calculated by using a mathematical model on cells of different EGFR density (A431, MDAMB468, and MDAMB231).
Volume 11, Issue 7
