Fig 1.
Biophysical characterization of 5D3 monocyte engagers.
(A, B, C) A single peak in the chromatograms of analytical SEC confirmed a monomeric form of each construct. The schemes of constructs provided on the right side of each chart show arrangement of BiP signal (grey), antibody domains (green), CP33 (beige), and SA-strep II tag (violet; created with BioRender.com). (D) Non-reduced samples separated in SDS PAGE gel stained by Coomassie Brilliant Blue G-250 show > 95% purity of all constructs. (E) The thermal stability of each construct was determined using nanoDSF. Melting temperatures of individual constructs were calculated from the first derivative of the fluorescence ratio at 350 nm and 330 nm (F350/F330).
Fig 2.
Characterization of purified 5D3-CP33 and ch5D3.
(A) Target specificity of 200 nM 5D3-CP33 to PSMA antigen was determined by flow cytometry using PSMA-positive PC3-PIP cells. PC3 cells served as PSMA-negative control. Approximately 30,000 live cells were included in analysis to generate histograms. (B) Specificity of 400 nM 5D3-CP33 to FcγRI/CD64 was estimated on CD64-positive HEK-293T-CD64 cells using flow cytometry. HEK-293T cells served as FcγRI/CD64-negative controls. Approximately 30,000 live cells were involved in the analysis. (C, D) Specific affinity of 5D3-CP33 to PSMA and FcγRI/CD64 was determined by flow cytometry using PC3-PIP cells and HEK-293T-CD64 cells, respectively. PC3 and HEK-293T cells were used as negative controls. (E, F) Estimation of specific affinity of ch5D3 run identically to 5D3-CP33 measurement. Individual KD values are shown in the upper left corner of charts.
Fig 3.
ROS production driven by 5D3-CP33.
(A) Comparison of the effect of 5D3-CP33 and ch5D3 on ROS production by activated U937 cells co-cultured with PSMA-positive PC3-PIP cells or PSMA-negative PC3 cells. (B) Quantification of produced ROS was visualized at 4-minute time-point to show in detail the difference between various concentrations of constructs and the ratio of ROS production in the presence of PSMA-positive and PSMA-negative cells. (C) ROS production by activated U937 cells in the presence of serial dilution of 5D3-CP33 co-cultured with PC3-PIP cells or PC3 cells. (D) Visualization at 4-minute time-point shows in detail concentration-dependent effect of 5D3-CP33 on ROS production in the presence of PSMA-positive and negative cells. The level of ROS was measured using the lucigenin-based chemiluminescence assay.
Fig 4.
Antibody-dependent cell-mediated phagocytosis (ADCP) of prostate cancer cells by U937 monocytes and human macrophages.
(A) PC3/PC3-PIP cells and U937 cells were labeled by DiO and DiD dye, respectively, co-incubated in the presence/absence of 111 nM 5D3-CP33 and analyzed by flow cytometer. The upper right quadrant of two-dimensional dot-plot charts shows double-positive events representing target cells engulfed by U937 monocytes. (B) U937/PC3-PIP co-cultures revealed a positive correlation between the level of ADCP and 5D3-CP33 concentration. (C) The chart shows a selective effect of 5 nM ch5D3 on the ADCP level in U937/PC3 and U937/PC3-PIP co-cultures, respectively. (D) Monocytes were isolated from human PBMCs and M1-polarized macrophages were derived by M-CSF and IFN-γ/LPS treatment. Macrophages (green) were mixed with PC3/PC3-PIP target cells (red) in the presence/absence of 111 nM 5D3-CP33. After 30 minute incubation samples were imaged by an automated digital fluorescence imaging system. Illustrative examples of phagocytosis are marked by white arrowheads. Scale bar: 100 µ m. (E) Time-lapse chart shows quantification of phagocytosis events and statistics (parametric T-test; ** P value < 0.005, *** P value < 0.0005).