1. PD1 vs ZsGreen
Pembrolizumab 1µg/ml
Isotype IgG1 1µg/ml
Mab005-IgG1 1µg/ml
SHR-1210-IgG1 1µg/ml
VEGFR2 vs ZsGreen
Pembrolizumab 1µg/ml
Isotype IgG1 1µg/ml
Mab005-IgG1 1µg/ml
SHR-1210-IgG1 1µg/ml
Supplemental Figure 1A

2. FZD5 vs ZsGreen
Pembrolizumab 1µg/ml
Isotype IgG1 1µg/ml
Mab005-IgG1 1µg/ml
SHR-1210-IgG1 1µg/ml
ULBP2 vs ZsGreen
Supplemental Figure 1B
Supplemental Figure 1. Analyses of binding specificity were performed on HEK293 cells transiently transfected with plasmids encoding either (A) human PD1, human VEGFR2, (B) human FZD5, human ULBP2. All plots show the target of interest transfected (grey line) versus ZS green marker-only transfected cells (black line). Transfected cells were stained using Mab005-IgG1, SHR-1210-IgG1, Pembrolizumab IgG1 null analog, and isotype IgG1. Each antibody was used in repeat staining at 1 g/ml. These analyses confirmed that all antibodies (other than the isotype control IgG1) exhibited binding to PD1, but no antibody exhibited measurable signal on ZS-green transfected cells. Both Mab005-IgG1 and SHR-1210-IgG1 also exhibited strong binding to all targets, while the isotype IgG1 and Pembrolizumab analog did not.

3. VEGFR2 vs ZsGreen
IgG1-06D02 1µg/ml
IgG1-12H04 1µg/ml
IgG1-05 1µg/ml
IgG1-08 1µg/ml
PD1 vs ZsGreen
Supplemental Figure 2A

4. FZD5 vs ZsGreen
IgG1-06D02 1µg/ml
IgG1-12H04 1µg/ml
IgG1-05 1µg/ml
IgG1-08 1µg/ml
ULBP2 vs ZsGreen
Supplemental Figure 2B
Supplemental Figure 2. Analyses of binding specificity were performed on HEK293 cells transiently transfected with plasmids encoding either (A) human PD1, human VEGFR2, (B) human FZD5, human ULBP2. All plots show the target of interest transfected cells (grey line) versus ZS green marker-only transfected cells (black line). Each antibody was used in repeat staining at 1 μg/ml. These analyses confirmed that all antibodies (other than the isotype control IgG1) exhibited binding to PD1, but no antibody exhibited measurable signal on ZS-green, VEGFR2, FZD5 or ULBP2 transfected cells.

5. Supplemental Figure 3. SHR-1210-IgG1, library-derived clones and control antibodies were examined for nonspecific binding to the negatively charged biomolecules Insulin and double-stranded DNA (dsDNA). All lead clones demonstrated binding scores below 10, similar to the negative control IgG1 Bevacizumab. Strong off-target binding (with scores >10) to insulin or dsDNA, as observed for Bococizumab, has been shown to be a high-risk indicator of poor pharmacokinetics of therapeutic antibodies, due to polyreactivity (Avery et al. mAbs, 2018).

6. SHR-1210-IgG1 on huVEGFR2-His
huVEGFR2-His 500 nM
huVEGFR2-His 250 nM
SHR-1210-IgG1 on rhVEGFR2-His
rhVEGFR2-His 500 nM
rhVEGFR2-His 250 nM
Mab005-IgG1 on huVEGFR2-His
Mab005-IgG1 on rhVEGFR2-His
Supplemental Figure 4. VEGFR2 binding for Mab005-IgG1 and SHR-1210-IgG1 by Biacore. Human and rhesus monomeric VEGFR2-his proteins were titrated (in nM) in direct binding against SHR-1210-IgG1 and Mab005-IgG1, captured by anti-human IgG1-Fc antibody. These analyses confirmed that both anti-PD1 antibodies exhibited binding to VEGFR2 recombinant protein, but binding was only observed at high concentrations of soluble analyte. As a result, reliable KD values could not be generated.