Fig 1.
Cell binding and effector functions activity of anti-CD4 IgG variants.
(A) Cell binding of anti-CD4 variants to CD4+ T cells. (B) ADCC activity of anti-CD4 variants against CD4+ T cells using KC1333 NK cells as effector cells. (C) Reporter-based ADCC activity of anti-CD4 variants against CD4+ T cells using NK92/NFAT cells as reporter cells. (D) CDC activity of anti-CD4 variants against CD4+ T cells using Rabbit complement at a final concentration of 11% (v/v). (E) ADCP activity of anti-CD4 variants against CD4+ T cells using human THP-1-derived macrophages as effector cells. NMGC represents isotype control antibody. Each point represents the mean values of triplicate wells and the standard deviation is represented by error bars. See (Table 1) for statistical analysis and P values.
Table 1.
Binding affinity of IgG to target antigens.
Table 2.
Effector function activity of CD4 affinity-reduced IgG variants.
Fig 2.
Cell binding and ADCC activity of anti-EGFR IgG variants.
(A-C) Cell binding of anti-EGFR variants to NCI-H358, SK-OV3 and MDA-MB-231 cells, expressing; low, medium and high levels of EGFR, respectively. (D-F) ADCC activity of anti-EGFR variants against NCI-H358, SK-OV3 and MDA-MB-231 cells using NK92/NFAT cells as reporter cells. NMGC represents isotype control antibody. Each point represents the mean values of triplicate wells and the standard deviation is represented by error bars. See (Table 4) for statistical analysis and P values.
Fig 3.
Cell binding and ADCC activity of anti-HER2 IgG variants.
(A-C) Cell binding of anti-HER2 variants to MDA-MB-361, SK-OV3 and BT474 cells, expressing; low, medium and high levels of HER2, respectively. (D-F) ADCC activity of anti-HER2 variants against MDA-MB-361, SK-OV3 and BT474 cells using NK92/NFAT cells as reporter cells. NMGC represents isotype control antibody. Each point represents the mean values of triplicate wells and the standard deviation is represented by error bars. See (Table 5) for statistical analysis and P values.
Table 3.
EGFR and HER2 receptor density on human tumor cell lines.
Table 4.
Reporter-based cytotoxicity of anti-EGFR IgG variants.
Table 5.
Reporter-based cytotoxicity of anti-HER2 IgG variants.
Fig 4.
Effect of E:T ratios and cellular internalization on ADCC activity.
(A) ADCC activity of anti-EGFR variants against SK-OV3 cells at varying E:T ratios. (B) Time course internalization of parental anti-EGFR and variants; VκS93A+VHP97A and VκF94A+VHP97A into MDA-MB-231 cells. NMGC represents isotype control antibody. Each point represents the mean values of triplicate wells and the standard deviation is represented by error bars.
Table 6.
Equilibrium binding of anti-EGFR IgGs to FcγRIIIa isoforms.
Fig 5.
(A) ADCC activity of parental ibalizumab and anti-CD4 variant VHY99A alone or in combination against CD4+ T cells. (B) ADCC activity of parental GA201 and anti-EGFR variant VκF94A+VHP97A alone or in combination against MDA-MB-231 cells. (C) ADCC activity of anti-HER2 B1D2 and C6.5 IgGs alone or in combination against BT474 cells. NMGC represents isotype control antibody. Each point represents the mean values of triplicate wells and the standard deviation is represented by error bars.
Fig 6.
Effect of bivalent vs. monovalent antigen binding on ADCC activity.
(A) ADCC activity of anti-CD4 ibalizumab formatted as either bivalent IgG or monovalent DuetMab against CD4+ T cells. (B) ADCC activity of anti-EGFR GA201formatted as either bivalent IgG or monovalent DuetMab against MDA-MB-231 cells. (C) ADCC activity of anti-HER2 B1D2 formatted as either bivalent IgG or monovalent DuetMab against BT474 cells. Each point represents the mean values of triplicate wells and the standard deviation is represented by error bars.
Fig 7.
Proposed model for how antibody binding affinity to target antigen regulates effector function potency.
Enhanced intrinsic affinity increases the likelihood for bivalent interaction with target cell. In contrast, a reduction in intrinsic affinity improves the probability for monovalent binding, leading to increased amount of antibody-Fc domains interacting with the target cell and hence better engagement with effector elements. However, additional reduction in intrinsic affinity, beyond a threshold of minimum affinity will result in poor cellular interaction and reduced effector functions.