Figure 3.
Dendritic cell activation and the T cells’ contribution to TS-L6 efficacy.
a, Dendritic cells from two healthy donors were activated by NCI-N87, which had been pre-treated with TS-L6. NCI-N87 was pre-treated with 5 nmol/L or 10 nmol/L TS-L6 for 2 days, and then co-cultured with dendritic cells for another 2 days to activate dendritic cells. The dendritic cell activating markers CD80, CD86, MHC-I, and MHC-II increased after dendritic cells were incubated with tumor cells committed to ICD induced by TS-L6. Three independent tests were performed, and the data are shown as mean and SD.
b, MF-6 exhibited greater potency in upregulating DAMPs, MHC-I, MHC-II, and PD-L1 on the tumor cell surface compared with DXd. After being treated with serial dilutions of MF-6 or DXd, the molecules, including calreticulin, HSP70, HSP90, MHC-I, MHC-II and PD-L1 on NCI-N87 surface increased when MF-6 reached 1-2 nmol/L, but those molecules had not increased when DXd reached up to 4 nmol/L. Three independent tests were performed, and the data are shown as mean and SD.
c, In vivo efficacy of TS-L6 was assessed with nude mice (left) and naïve BALB/c mice (right) bearing CT26.WT-HER2 tumors. In both nude and naïve BALB/c mice, administration of 20 mg/kg (mpk) TS once weekly × 2 (QW × 2) did not show any efficacy. Both 10 mg/kg and 20 mg/kg TS-L6 administrated once weekly × 2 inhibited tumor growth in nude and naïve mice, but TS-L6 was more efficacious in naïve mice. In nude mice, 10 mg/kg (TGI = 65.9 ± 17.0%, P<0.001) and 20 mg/kg TS-L6 (TGI = 76.8 ± 10.9%, P<0.001) showed tumor inhibition but no CRs were reached. In naïve mice, the tumor growth in the 10 mg/kg (TGI = 95.8 ± 14.4%, P<0.001) and 20 mg/kg TS-L6 (TGI = 107.9 ± 4.2%, P<0.001) groups was significantly inhibited with 29% (2/7) and 75% (6/8) CRs, respectively. The tumor volumes of drug-treated groups were statistically compared with the volumes of the saline group on the day when the saline group mice were euthanized due to large tumors. Black arrows represent drugs administrated. The tumor volume data are shown as mean and SEM.
d, TS-L6in vivo efficacy increased when combined with anti-mCD4 (left) and decreased with mCD8 (right) antibody. When cytotoxic CD8+ T cells were depleted using an anti-mCD8 monoclonal antibody, the efficacy of the TS-L6 was abrogated. Compared with TS-L6 treatment, the combination of TS-L6 and anti-mCD8 antibody significantly reduced antitumor efficacy (TGI = 88.5 ± 24.9% vs 28.7 ± 17.4%, P<0.001). When we ablated CD4+ T cells using a depleting anti-mCD4 monoclonal antibody, the TGI was 47.6% at day 11 in anti-mCD4 antibody group, but all eight mice reached CRs in the TS-L6 combined with anti-mCD4 antibody group. The tumor volumes of the combination drug-treated groups were statistically compared with the volumes of the single drug-treated group at the time when the single drug-treated mice were euthanized due to large tumors. Black and white arrows represent ADC and anti-mCD4/CD8 antibody administrated, respectively. The tumor volume data are shown as mean and SEM.
e,fIn vivo efficacy of TS-L6 combined with atezolizumab (E) and anti-mCTLA-4 antibody (F) indicated an increased combination efficacy with immune checkpoint inhibitors. Even at a relatively low dose of 5 mg/kg for TS-L6, both 7.5 mg/kg and 15 mg/kg atezolizumab significantly enhanced ADC efficacy to 75% (6/8) and 100% (8/8) CRs 29 days post-administration, respectively. When we combined TS-L6 with anti-mCTLA-4 antibody, the efficacy of TS-L6 was enhanced with 84.3 ± 29.6% TGI (tumor volume P<0.01 compared with TS-L6 treatment TGI 27.5 ± 28.8%) 12 days post-administration and 43% (3/7) CRs after 15 days, whereas the anti-mCTLA-4 antibody alone showed limited efficacy with only 13.1 ± 53.7% TGI (tumor volume P>0.05 compared with saline treatment) after 12 days. Black and white arrows represent ADC and immune checkpoint inhibitors administrated, respectively. The tumor volume data are shown as mean and SEM.
ns, *, ** and *** represent no significance, P<0.05, 0.01, and 0.001, respectively.