WT IgG1 and modified anti-CD3ε antibody ADI-26906 were tested for binding to human THP-1 and PC3 cells. In brief, 100 nM IgG was incubated with the cells for 30 min on ice. Following two washes with PBSF (PBS + 0.1% bovine serum albumin (BSA)), IgGs were detected with goat anti-Human IgG R-PE secondary reagent (Southern Biotech, #2040–09) and analyzed by flow cytometry using a BD FACSCanto II (BD Bioscience). FCS Express software (De Novo Software, version 5) was used for data analysis, and binding was expressed as median fluorescent intensity (MFI).
Increasing the sensitivity of binding to THP-1 was accomplished by precomplexing variant antibodies with a CD3ε N-terminal peptide (residues 21–47) first conjugated to BSA (hereafter referred to as CD3ε-BSA, procured from New England Peptide (Gardner, MA, USA). Approximately 100 nM of each Fc variant antibody was incubated with 10 nM CD3ε-BSA at room temperature for 20 min. The samples were then mixed with THP-1 cells (200,000 cells per well) and incubated on ice for 30 min. Following three washes with PBSF, the cells were incubated with 100 μL of goat anti-human IgG PE antibody (Southern Biotech) at a dilution of 1:200 for 20 min at room temperature. The cells were then washed twice with PBSF and resuspended in 100 μL PBSF and then analyzed on a FACS Canto (BD Biosciences). Median fluorescence intensity (MFI) was recorded.
Increasing the sensitivity of binding to THP-1 was accomplished by precomplexing variant antibodies with a CD3ε N-terminal peptide (residues 21–47) first conjugated to BSA (hereafter referred to as CD3ε-BSA, procured from New England Peptide (Gardner, MA, USA). Approximately 100 nM of each Fc variant antibody was incubated with 10 nM CD3ε-BSA at room temperature for 20 min. The samples were then mixed with THP-1 cells (200,000 cells per well) and incubated on ice for 30 min. Following three washes with PBSF, the cells were incubated with 100 μL of goat anti-human IgG PE antibody (Southern Biotech) at a dilution of 1:200 for 20 min at room temperature. The cells were then washed twice with PBSF and resuspended in 100 μL PBSF and then analyzed on a FACS Canto (BD Biosciences). Median fluorescence intensity (MFI) was recorded.