Research ArticleIMMUNOLOGY

B7-H3×4-1BB bispecific antibody augments antitumor immunity by enhancing terminally differentiated CD8+ tumor-infiltrating lymphocytes

See allHide authors and affiliations

Science Advances  15 Jan 2021:
Vol. 7, no. 3, eaax3160
DOI: 10.1126/sciadv.aax3160
  • Fig. 1 Functional characterization of B7-H3×4-1BB bsAb.

    (A and B) Dose-dependent costimulatory activity of hIgG1 isotype, 1D8, B5, and B5×1D8 on CD8 T cells stimulated with anti-CD3ε (1 μg/ml) and irradiated MC38hB7-H3. Flow cytometric analysis of surface expression on CD8α+ T cells (A) and IFN-γ secretion by ELISA (B) 72 hours after stimulation. (C) EC50 values for each parameter. (D) Flow cytometric analysis of surface expression on CD8α+ T cells stimulated with anti-CD3ε (1 μg/ml) and irradiated wild-type MC38 or MC38hB7-H3 with indicated antibodies (1 μg/ml) 72 hours after stimulation. (E) Representative ex vivo fluorescence images of spleen (S) and tumor (T) (left), and tumor/spleen ratio (right) from MC38hB7-H3 tumor–bearing mice 24 hours after intravenous injection of 37.5 μg of 680XL-labeled mAb (1D8 and B5) or 50.0 μg of 680XL-labeled B5×1D8 (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, two-way analysis of variance (ANOVA) with Bonferroni posttests compared with hIgG1 isotype group (A and B), two-way ANOVA with Bonferroni posttests (D), and one-way ANOVA with Bonferroni’s multiple comparison test (E). ns, not significant.

  • Fig. 2 Antitumor activity of B7-H3×4-1BB bsAb.

    (A) Experimental scheme of bsAb treatment in MC38hB7-H3 tumor–bearing C57BL/6 or 4-1BB KO mice (n = 7 to 14 per group). WT, wild type. (B to D) Tumor growth curves (B), survival curves (C), and serum ALT (D) for each group of C57BL/6 mice. (E and F) Tumor growth curves (E) and serum ALT (F) for C57BL/6 or 4-1BB KO mice. (G and H) Experimental scheme for rechallenge of long-term survivors from 4-1BB agonist treatments (B) with MC38 alone (G) or both MC38 and B16-F10 (H). Survival curves for mice inoculated MC38 alone (G, right). Tumor growth curves for individual mice inoculated MC38 (H, middle) and B16-F10 (H, right). (I to K) B16-F10hB7-H3 tumor–bearing C57BL/6 mice (n = 10 per group) treated with indicated antibodies. Tumor growth (I, right), survival (J), and serum ALT (K). (L to N) CT26hB7-H3 tumor–bearing BALB/c mice (n = 10 to 11 per group) treated with indicated antibodies. Tumor growth (L, right), survival (M), and serum ALT (N). mAb (10.0 μg) and bsAb (13.3 μg) were used in all experiments. Numbers in survival curves indicate tumor-free mice/total mice at the end of the experiment. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, two-way ANOVA with Bonferroni posttests (B, E, I, and L), one-way ANOVA with Bonferroni’s multiple comparison test (D, F, K, and N), and log-rank (Mantel-Cox) test (C, G, J, and M).

  • Fig. 3 Role of CD8 T cells for B7-H3×4-1BB bsAb–mediated antitumor activity.

    MC38hB7-H3 tumor–bearing C57BL/6 mice (n = 4 to 7 per group) were treated, as shown in Fig. 2A. Tumor tissues were analyzed 4 days after the last treatment. (A) Flow cytometric analysis of TIL composition (left) and cell count per milligram of the tumor (right). (B) CD8 T cell/Treg cell ratio. (C) TNF-α and IFN-γ in restimulated CD8 TILs. (D) Levels of TNF-α and IFN-γ in the tumor lysate by ELISA. (E and F) Flow cytometric analysis of Ki-67 (E) and GzmB (F) expression in CD8 TILs. The numbers in each plot indicate the percentage of cells expressing each molecule. Representatives of two independent experiments were shown. (G) Tumor growth from MC38hB7-H3 tumor–bearing C57BL/6 mice (n = 7 per group) treated with 10.0 μg of hIgG1 isotype or 13.3 μg of B5×1D8 and 200 μg of depletion antibody. Black and blue arrows indicate treatment points and depletion antibody injection points, respectively. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, unpaired Student’s t test (A to F) and two-way ANOVA with Bonferroni posttests compared with phosphate-buffered saline (PBS) + ISOTYPE–treated group (G).

  • Fig. 4 Increased PD-1+Tim-3+ terminally differentiated CD8 T cells following B7-H3×4-1BB bsAb treatment.

    (A to C) MC38hB7-H3 tumor–bearing C57BL/6 mice (n = 6 to 14 per group) were treated as in Fig. 3, and TILs were analyzed by flow cytometry 4 days after the last treatment. (A) Surface expression of PD-1 and Tim-3 on CD8 TILs. Expression of Tim-3 and TCF1 (B), and T-bet and Eomes (C) in CD8 TILs. Mean fluorescence intensity (MFI) of Eomes in PD-1+ CD8 TILs was represented (C, right). The numbers in each plot indicate the percentage of cells expressing each molecule. Dots on the right are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, unpaired Student’s t test.

  • Fig. 5 Synergistic effect of B7-H3×4-1BB bsAb with PD-1 blockade on tumor growth and development of terminally differentiated CD8 T cells.

    (A) Experimental scheme of combination therapy of bsAb and anti–PD-1 in MC38hB7-H3 tumor–bearing C57BL/6 mice (n = 10 per group). mAb (37.5 μg) and bsAb (50.0 μg) were treated with rat IgG2a isotype (ISO) or anti–PD-1 (200 μg). (B to D) Tumor growth curves (B), survival curves (C), and tumor growth curves for individual mice (D). Numbers in each plot in (D) indicate tumor-free/total mice ratios. (E to H) Flow cytometric analysis of CD8 T cells (E), Ki-67 (F), and GzmB (G) expression on CD8 T cells, TCF1+PD-1+ stem-like CD8 T cells (H, left), Tim-3+PD-1+ CD8 T cells (H, middle), and Tim-3+PD-1+ CD8 T cell/TCF1+PD-1+ CD8 T cell ratio (H, right) at day 20. Data represent means ± SD of pooled biologically independent samples from three independent experiments (n = 6 to 9 per group) for (E) and (H). *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, two-way ANOVA with Bonferroni posttests (B), log-rank (Mantel-Cox) test (C), and one-way ANOVA with Bonferroni’s multiple comparison test (E to H).

  • Fig. 6 Antitumor efficacy of B7-H3×4-1BB bsAb in the human system.

    (A) Dose-dependent costimulatory activity of the urelumab analog and B5×1A10 on Jurkat-NFκB-luc2/h4-1BB reporter cells. Luminescence was measured 6 hours after stimulation. (B and C) Dose-dependent costimulatory activity of the urelumab analog, 1A10, and B5×1A10 on PBMCs stimulated with anti-human CD3 (5 μg/ml) and HCC1954 cells. IFN-γ secretion by ELISA (B) and optical cellular density by cell counting kit (C) 72 hours after stimulation. (D and E) MC38hB7-H3 tumor–bearing h4-1BB KI mice (n = 6 to 8 per group) were treated with indicated antibodies. Black arrows indicate treatment points. Tumor growth curves of individual mice are shown on the right. (F) Representative immunohistochemistry images (left) showing CD8 expression (brown) in tumor tissue from mice in (D). Infiltrating CD8+ cell counts in a high-power field (HPF; 20×) at randomly selected tumor areas (right). Nuclear staining with hematoxylin is in blue. Scale bars, 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, two-way ANOVA with Bonferroni posttests (D and E) and unpaired Student’s t test (F).

Supplementary Materials

  • Supplementary Materials

    B7-H3×4-1BB bispecific antibody augments antitumor immunity by enhancing terminally differentiated CD8+ tumor-infiltrating lymphocytes

    Gihoon You, Yangsoon Lee, Yeon-Woo Kang, Han Wook Park, Kyeongsu Park, Hyekang Kim, Young-Min Kim, Sora Kim, Ji-Hae Kim, Dain Moon, Hyejin Chung, Wonjun Son, Ui-jung Jung, Eunyoung Park, Shinai Lee, Yong-Gyu Son, Jaehyun Eom, Jonghwa Won, Yunji Park, Jaeho Jung, Seung-Woo Lee

    Download Supplement

    This PDF file includes:

    • Figs. S1 to S9

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article