Supplementary Figures and Figure Legends

Figure S1. The chemical structure of DS-8201a

Figure S2. Importance of targeted delivery by the ADC

The antitumor effect was examined in a syngeneic mouse model with CT26.WT-hHER2 (s.c. inoculation) upon treatment with DS-8201a (10 mg/kg, once a week, twice, i.v.) and isotype non-targeted control ADC (10 mg/kg, once a week, twice, i.v.) at the time points indicated by arrows. When the tumors were established, the treatments were initiated (day 0). The graph shows the mean tumor volumes and standard errors (n = 10). The mean tumor volumes at day 10 of vehicle-treated, control ADC-treated and DS-8201a-treated groups were 1210 mm3, 899 mm3 and 202 mm3, respectively. A Wilcoxon rank sum test was conducted to provide statistical analysis of the tumor volumes and compare the control ADC-treated and DS-8201a-treated groups on day 10.

Figure S3. Reduced antitumor effect of DS-8201a in an immunocompromised mouse model

The antitumor effect was examined in a nude mouse model with CT26.WT-hHER2 cells (s.c. inoculation) after the treatment with DS-8201a (10 mg/kg, once a week, twice, i.v.). DS-8201a was administered at the time points indicated by arrows. Treatment was initiated after tumors were established (day 0). The graph shows mean tumor volumes and standard errors (n = 12). The mean tumor volumes at day 13 of vehicle-treated and DS-8201a-treated groups were 2748 mm3 and 2489 mm3, respectively. Student’s t-test was conducted for the comparison between DS-8201a-treated and vehicle-treated groups at day 13. There was no significant difference.

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Figure S4. Cell growth inhibition activity of DS-8201a and DXd

A, Expression of exogenously introduced human HER2 gene was confirmed in mouse EMT6-hHER2 cells. B, Tumor cells were treated with DS-8201a, control ADC, anti-hHER2 antibody, and human IgG1 isotype control for six days, and cell viability (%) was determined. C, Tumor cells were treated with DXd for six days, and cell viability (%) was determined. The graphs show means and standard errors (from triplicate).

Figure S5. Increased CD8+ cell infiltration into tumors

Representative CD8 staining of tumors from A, vehicle-treated and B, DS-8201a-treated (10 mg/kg, once, i.v.) mice. Scale bars = 250 μm. C, The number of CD8+ cells/tumor area (μm2). The graph shows the mean and standard errors (n = 3). The mean number of CD8+ cells/tumor area (μm2) of vehicle-treated and DS-8201a-treated groups were 6.51 and 9.79, respectively.

Figure S6. Reduced antitumor effect of DS-8201a by CD8 depletion

The antitumor effect was examined in a mouse syngeneic model with CT26.WT-hHER2 (s.c. inoculation). The mice were treated with vehicle, DS-8201a (10 mg/kg, once a week, twice, i.v.), anti-CD4 antibody Ab (200 μg/kg, once a week, twice, i.v.), and anti-CD8 Ab (200 μg/kg, once a week, twice, i.v.) alone or in combination at the time points indicated by arrows. A, The mean tumor volumes and standard errors are represented on the graph (n = 10). The mean tumor volumes at day 11 of vehicle-treated, DS-8201a-treated, DS-8201a and anti-CD4 antibody-treated and DS-8201a and anti-CD8 antibody-treated groups were 2173 mm3, 651 mm3, 561 mm3 and 2247 mm3, respectively. The data of vehicle groups on the right and left panels are identical. B, Spider plots of individual data. The tumor growth curves of anti-CD4 Ab-treated and anti-CD8 Ab-treated were are interrupted in the middle of the study as some of mice had to be euthanized for ethical reason.

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Figure S7. DXd, the payload of DS-8201a directly increased expression of MHC class I

CT26.WT-hHER2 cells was cultured in the presence of payloads for 24 h, and MHC class I expression was determined by flow cytometry. A, MHC class I expression in the presence of vehicle [dimethyl sulfoxide, (DMSO)] or DXd. B, The effect of DXd on MHC class I expression compared with ADC payloads of DM1, DM4, and MMAE. Adjusted mean fluorescence intensity (MFI) that was determined by calculating the MFI of MHC class I and subtracting the MFI of the isotype control and standard errors are shown (triplicate). Asterisks (*) indicate statistically significant differences from DMSO control, as determined by a Dunnett's multiple comparison test (***: P < 0.001, **: P < 0.01). It should be noted that the compounds of each concentration were compared with DMSO control in B.

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