A New, Triglycyl Peptide Linker Forantibody-Drug Conjugates (Adcs) with Improved

Supplementary Figures.

“A new, triglycyl peptide linker forantibody-drug conjugates (ADCs) with improved targetedkilling of cancer cells”

Supplementary Figures (S1-S4).Supplementary Figure S1:In vitro cytotoxic activities of tetraglycyl (Gly4), triglycyl (Gly3), diglycyl (Gly2), and valine-citrulline-glycine (VCG)-linked anti-EGFR ADCs and a triglycyl-linked non-binding antibody ADC in PC-9, Ca9-22, HSC-2, H1975, A-431, and OSC-19 cells (3-4 DAR ADCs). Supplementary Figure S2:In vitro cytotoxic activities of anti-EpCAM CX and SMCC ADCs toward a low EpCAM antigen-expressing cell line, RPMI 8226 (~5x104EpCAM per cell). The CX ADCs tested included a conjugate with a typical payload number (3.9 maytansinoid molecules per antibody molecule; 3.9 DAR) and conjugates with high payload numbers (8 and 9.6 DAR). The SMCC ADC had a typical payload number (4.3 DAR). Supplementary Figure S3: Binding-competition ELISA of catabolites of CX ADC (DM-CX1 and DM-CX2) and SMCC ADC (lysine-SMCC-DM1): inhibition by catabolites toward binding of anti-maytansine antibody to immobilized BSA-maytansinoid conjugate. Supplementary Figure S4: Test of bystander cytotoxic activity of anti-EGFR-CX-DM1 or SPDB-DM4 conjugate in mixed culture of EGFR-positive (Ca9-22) and EGFR-negative (Ramos) cells.

Supplementary Figure S1: In vitro cytotoxic activities of tetraglycyl (Gly4), triglycyl (Gly3), diglycyl (Gly2), and valine-citrulline-glycine (VCG)-linked anti-EGFR ADCs and a triglycyl-linked non-binding antibody ADC in PC-9, Ca9-22, HSC-2, H1975, A-431, and OSC-19 cells (3-4 DAR ADCs). Cells were incubated with conjugates at several concentrations for 5 days, after which cell viabilities were measured using WST-8 reagent.

Supplementary Figure S2: In vitro cytotoxic activities of anti-EpCAM CX and SMCC ADCs toward a low EpCAM antigen-expressing cell line, RPMI 8226 (~5x104EpCAM per cell). The CX ADCs tested included a conjugate with a typical payload number (3.9 maytansinoid molecules per antibody molecule; 3.9 DAR) and conjugates with high payload numbers (8 and 9.6 DAR). The SMCC ADC had a typical payload number (4.3 DAR). Cells were incubated with conjugates (or mixtures of conjugates with excess unconjugated anti-EpCAM antibody) at several concentrations for 5 days, after which cell viabilities were measured using WST-8 reagent.

Supplementary Figure S3: Binding-competition ELISA of catabolites of CX ADC (DM-CX1 and DM-CX2) and SMCC ADC (lysine-SMCC-DM1): inhibition by catabolites toward binding of anti-maytansine antibody to immobilized BSA-maytansinoid conjugate.

Supplementary Figure S4:Test of bystander cytotoxic activity of anti-EGFR-CX-DM1 or SPDB-DM4 conjugate in mixed culture of EGFR-positive (Ca9-22) and EGFR-negative (Ramos) cells. Cell mixtures (4000 Ca9-22 and 3000 Ramos cells) or Ramos cells alone were incubated in the presence or absence of conjugate (2 nM) for 5 days and then tested for cell survival using WST-8 reagent. The disulfide-linked SPDB-DM4 conjugate was used as a positive control for bystander cytotoxic activity. The SPDB-DM4 conjugate partially killed antigen-negative Ramos cells in mixed cell culture, in contrast to CX-DM1 conjugate which did not show bystander killing. The two conjugates killed EGFR-expressing Ca9-22 cellsbut not EGFR-negative Ramos cells in separate cell cultures.

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