Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models

Figures & data

Figure 1. Structure of αCD25-CyPeg-Duo and cytotoxic mechanism of CD25-targeted NIR-DPR. (a) structure of αCD25-CyPeg-Duo. The N,N’-diethylethylenediamine linker is cleaved with 780 nm hv. (b) cytotoxic mechanism of CD25-targeted NIR-DPR. After binding of αCD25-CyPeg-Duo to CD25 on the surface of CD25-expressing cells, it is internalized into the cytoplasm. Light irradiation at 780 nm triggers payload uncaging, which results in apoptosis of CD25-expressing cells.

Figure 2. CD25-targeted NIR-DPR showed cytotoxic effects on CD25-expressing cells in vitro and Tregs ex vivo.

(a) Flow cytometric analysis of in vitro CD25 expression in HT2-A5E or EL4 cells. (b) Detection of αCD25-CyPeg-Duo bound to HT2-A5E or EL4 cells by flow cytometry. (c) and (d), In vitro apoptosis assay of HT2-A5E or EL4 cells after CD25-targeted NIR-DPR. Total apoptotic cells were calculated by summing early (Annexin V⁺/Fixable Viability Dye⁻) and late apoptotic cells (Annexin V⁺/Fixable Viability Dye⁺). (c) Representative dot plots. (d) Comparison of the percentage of total apoptotic cells (n = 4, mean ± SEM; one-way ANOVA followed by Tukey’s test; ***, p < 0.001; ****, p < 0.0001; ns, not significant). (e) Flow cytometric analysis of ex vivo CD25 expression in splenocytes. Foxp3⁺CD4⁺ T cells highly expressed CD25, whereas other immune cells did not show CD25 expression. (f)–(h), Ex vivo CD25-targeted NIR-DPR for splenocytes. (f) Representative dot plots of CD4⁺ T cells. The percentage of CD25⁺ Tregs in CD4⁺ T cells was shown. (g) Comparison of the percentage of CD25⁺ Tregs in CD4⁺ T cells (n = 4, mean ± SEM; one-way ANOVA followed by Tukey’s test; ****, p < 0.0001; ns, not significant). (h) Comparison of the percentage of each immune cells in CD45⁺ cells (n = 4, mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, not significant).

Figure 3. Delivery and biodistribution of intravenously administered αCD25-CyPeg-Duo in vivo.

(a)–(c), In vivo fluorescence imaging of αCD25-CyPeg-Duo. All images were acquired at the indicated timepoints after intravenously injecting αCD25-CyPeg-Duo into MB49-luc or LL/2-luc tumor-bearing mice. (a) Representative fluorescence images at 800 nm in a MB49-luc tumor-bearing mouse (A.U., arbitrary units). (b) Quantitative analysis of 800-nm fluorescence intensity at the tumor site after intravenously injecting αCD25-CyPeg-Duo (n = 7). (c) Quantitative analysis of target-to-background ratio (TBR) after intravenously injecting αCD25-CyPeg-Duo (n = 7). (d) In vivo delivery of digoxigenin (DIG)-conjugated anti-CD25 F(ab’)₂ to MB49-luc tumors. Tumors were harvested 24 hours after injecting CD25-DIG or control-DIG into mice and its distribution in MB49-luc tumors was examined by multiplex immunohistochemistry (images; ×200; scale bar, 20 μm). Antibody stainings of DIG, Foxp3, CD8, CD4, CD11b, and pan-cytokeratin (Pan-CK) are shown in red, yellow, magenta, green, cyan, and pink, respectively. Nucleus is stained with DAPI and shown in blue. The insets (a–g) are enlarged and displayed on the right side. They show representative images of a DIG⁺ Treg (a, white-filled arrowhead), DIG⁻ Treg (b, white hollow arrowhead), CD8⁺ T cell (c, gray-filled arrowhead), DIG⁺ Th (d, gray hollow arrowhead), DIG⁻ Th (e, black-filled arrowhead), myeloid cell (f, black hollow arrowhead), and cancer cell (g, red-filled arrowhead).

Figure 4. In vivo therapeutic efficacy of CD25-targeted NIR-DPR in a MB49-luc tumor mouse model.

(a) Diagram of NIR light irradiation. The red circle represents where NIR light was irradiated. (b)–(g) CD25-targeted NIR-DPR decreased the proportion of intratumoral Tregs and impaired their functions. Mice with MB49-luc tumors were treated with CD25-targeted NIR-DPR. The tumors, tumor-draining lymph nodes (TDLNs), and spleens were harvested and analyzed by flow cytometry 24 hours after NIR light irradiation. (b) Representative dot plots of CD4⁺ T cells in the tumor. (c) Comparison of the percentage of CD25⁺ Tregs in CD4⁺ T cells in the tumor, TDLN, and spleen (n = 4, mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01; ns, not significant). (d) Comparison of the ratio of CD8⁺ T cells to CD25⁺ Tregs in the tumor (n = 4, mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01). (e)–(g) Flow cytometric histograms of Ki-67 (e), IL-10 (f), and PD-1 (g) on intratumoral CD25⁺ Tregs and comparison of their expressions (n = 4, mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01; ns, not significant; MFI, mean fluorescence intensity). (h) Treatment schedule. (i) Representative fluorescent imaging at 800 nm before and after treatment in MB49-luc tumor-bearing mice. A.U., arbitrary units. (j) Changes in 800-nm fluorescence intensity at the tumor site before and after treatment (n = 10; mean ± SEM; repeated measures two-way ANOVA followed by Sidak’s test; ****, p < 0.0001). (k) Luciferase activity after treatment (n = 10; mean ± SEM; repeated measures two-way ANOVA followed by Tukey’s test; ns, not significant). (l) Tumor growth curves after treatment (n = 10; mean ± SEM; repeated measures two-way ANOVA followed by Tukey’s test; *, p < 0.05 vs. the Control group; ^††, p < 0.01 vs. the αCD25-CyPeg-Duo IV group). (m) Survival curves after treatment (n = 10, log-rank test with Bonferroni correction; **, p < 0.01; ns, not significant).

Figure 5. In vivo therapeutic efficacy of CD25-targeted NIR-DPR combined with PD-1 blockade.

(a) and (b) The balance of PD-1 expression on between CD8⁺ T cells and CD25⁺ Tregs in murine tumors. Tumors were harvested and analyzed by flow cytometry. Relative fluorescence intensity (RFI) was calculated as the ratio of the mean fluorescence intensity of anti-PD-1 antibody to that of the isotype control. (a) PD-1 expression on intratumoral CD8⁺ T cells or CD25⁺ Tregs (n = 5, mean ± SEM). (b) Comparison of CD8⁺/Treg PD-1 ratio. CD8⁺/Treg PD-1 ratio was calculated as the ratio of PD-1 RFI in CD8⁺ T cells to PD-1 RFI in CD25⁺ Tregs (n = 5, mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01; ****, p < 0.0001). (c) Treatment schedule in a MB49-luc tumor model. (d) Representative fluorescent imaging at 800 nm before and after treatment in MB49-luc tumor-bearing mice. A.U., arbitrary units. (e) Changes in 800-nm fluorescence intensity at the tumor site before and after treatment in MB49-luc tumor-bearing mice (n = 9–10; mean ± SEM; repeated measures two-way ANOVA followed by Sidak’s test; ns, not significant). (f) Representative bioluminescence images before and after treatment in a MB49-luc tumor model. (g) Luciferase activity after treatment in a MB49-luc tumor model (n = 9–10; mean ± SEM; repeated measures two-way ANOVA followed by Sidak’s test; *, p < 0.05 vs. the Control group). (h) Tumor growth curves after treatment in a MB49-luc tumor model (n = 9–10; mean ± SEM; repeated measures two-way ANOVA followed by Tukey’s test; **, p < 0.01 vs. the Control group; ^††, p < 0.01 vs. the NIR-DPR group; ^‡, p < 0.05 vs. the PD-1 blockade group). (i) Survival curves after treatment in a MB49-luc tumor model (n = 9–10, log-rank test with Bonferroni correction; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant). (j) Tumor growth curves after treatment in a MC38 tumor model (n = 9–10; mean ± SEM; repeated measures two-way ANOVA followed by Tukey’s test; *, p < 0.05 vs. the Control group; ^†, p < 0.05 vs. the NIR-DPR group). (k) Survival curves after treatment in a MC38 tumor model (n = 9–10, log-rank test with Bonferroni correction; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant). In addition, complete remission (CR) rates were compared (Chi-square test; *, p < 0.05). (l) Tumor growth curves after treatment in a LL/2-luc tumor model (n = 10; mean ± SEM; repeated measures two-way ANOVA followed by Tukey’s test; **, p < 0.01 vs. the Control group). (m) Survival curves after treatment in a LL/2-luc tumor model (n = 10, log-rank test with Bonferroni correction; ***, p < 0.001; ns, not significant).

Figure 6. Early host immune responses to CD25-targeted NIR-DPR combined with PD-1 blockade in a MB49-luc tumor mouse model.

Mice with MB49-luc tumors were treated with CD25-targeted NIR-DPR or PD-1 blockade according to the schedule shown in Figure 5c. Immune cell populations in the tumor and tumor-draining lymph node (TDLN) were evaluated by flow cytometry three days after NIR light irradiation. (a) CD25⁺ Treg percentage in the tumor. The percentage of CD25⁺ Tregs in CD45⁺ cells was compared (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test; ns, not significant). (b) and (c) Expression of functional markers of CD25⁺ Tregs in the tumor. Ki-67 (b) and IL-10 (c) expressions on Tregs were compared (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01; MFI, mean fluorescence intensity). (d) and (e) Expression of dendritic cell (DC) maturation markers in the tumor (d) and TDLN (e). CD40, CD80, and CD86 expressions on DCs were compared (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant; MFI, mean fluorescence intensity). (f) and (g) Expression of CD8⁺ T cell activation markers in the tumor (f) and TDLN (g). CD69 and CD25 expressions on CD8⁺ T cells were compared (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant).

Figure 7. Enhanced anti-cancer immunity was successfully established after CD25-targeted NIR-DPR combined with PD-1 blockade in a MB49-luc tumor mouse model.

(a)–(c), Tumor-infiltrating CD8⁺ T cells seven days after CD25-targeted NIR-DPR combined with PD-1 blockade in a MB49-luc tumor mouse model. Mice with MB49-luc tumors were treated with CD25-targeted NIR-DPR or PD-1 blockade according to the schedule shown in Figure 5c. Tumors were harvested seven days after NIR light irradiation, and tumor-infiltrating lymphocytes were evaluated by multiplex immunohistochemistry. (a) Representative images (images; ×200; scale bar, 20 μm). Antibody staining of Granzyme B (GZMB), CD8, CD4, and pan-cytokeratin (Pan-CK) is shown in yellow, magenta, green, and pink, respectively. Nucleus are stained with DAPI and shown in blue. The insets (a and b) displayed on the bottom show representative images of a GZMB⁻CD8⁺ T cell (white-filled arrowhead) and GZMB⁺CD8⁺ T cell (white hollow arrowhead). (b) Comparison of CD8⁺ T cell density (left) and GZMB⁺CD8⁺ T cell density (right) in the tumor area (n = 5; mean ± SEM; one-way ANOVA followed with Tukey’s test; *, p < 0.05; **, p < 0.01; ***, p < 0.001). (c) Comparison of CD8⁺ T cell density (left) and GZMB⁺CD8⁺ T cell density (right) in the stroma area (n = 5; mean ± SEM; one-way ANOVA followed with Tukey’s test; ns, not significant). (d)–(e) Effect of CD25-targeted NIR-DPR combined with PD-1 blockade on CD8⁺ memory T cell populations within the tumor-draining lymph node (TDLN). Mice with MB49-luc tumors were treated with CD25-targeted NIR-DPR and PD-1 blockade. The TDLN was harvested 14 days after NIR light irradiation and analyzed by flow cytometry. (d) Representative dot plots of CD8⁺ T cells. CD8⁺ T cells were categorized into effector memory T cell (Tem), central memory T cell (Tcm), and naïve T cell (Tn) based on the expression of CD44 and CD62L. (e) Comparison of each CD8⁺ memory T cell percentage (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test; *, p < 0.05; ns, not significant).

Data availability statement

The data that support the findings of this study are available from the first or corresponding author, [HF or HK], upon reasonable request.