Fig. 7

Blocking the B7-CTLA-4 interaction does not contribute to anti-CTLA-4 mAbs elicited cancer immunotherapeutic activity and intratumorial Treg depletion. a Comparable immunotherapeutic effect despite vastly different blocking activity by two anti-CTLA-4 mAbs. 5 × 10⁵ or 1 × 10⁶ MC38 tumor cells were injected (s.c.) into Ctla4^h/h mice (n = 5–6), and mice were treated (i.p.) with 100 μg (left), 30 μg (middle) or 10 μg (right) Ipilimumab, L3D10 or control hIgG-Fc per mouse on days 7, 10, 13 and 16, as indicated by arrows. Data represent mean ± S.E.M. of 5–6 mice per group. Statistical analyses were performed by two-way repeated measures ANOVA (treatment × time). For 100 μg treatments, Ipilimumab vs hIgG-Fc: P < 0.0001; L3D10 vs hIgG-Fc: P < 0.0001; Ipilimumab vs L3D10: P = 0.0699. For 30 μg treatments, Ipilimumab vs hIgG-Fc: P < 0.0001; L3D10 vs hIgGFc: P < 0.0001; Ipilimumab vs L3D10: P = 0.9969. For 10 μg treatments, Ipilimumab vs hIgG-Fc: P < 0.0001; L3D10 vs hIgG-Fc: P < 0.0001; Ipilimumab vs L3D10: P = 0.9988. Data are representative of 3–5 independent experiments. b Ipilimumab and L3D10 have similar therapeutic effect for B16 melanoma growth. 1 × 10⁵ B16 tumor cells were injected (s.c.) into Ctla4^h/h mice (n = 4–5), and mice were treated (i.p.) with 100 μg (left) or 250 μg (right) Ipilimumab, L3D10 or control hIgG-Fc on day 11, 14, 17(left) or on day 2, 5 and 8 (right), as indicated by arrows. For the left panel, Ipilimumab vs hIgG-Fc: P = 0.0265; L3D10 vs hIgG-Fc: P = 0.0487; Ipilimumab vs L3D10: P = 0.302. For the right panel, Ipilimumab vs hIgG-Fc: P = 0.00616; L3D10 vs hIgG-Fc: P = 0.0269: Ipilimumab vs L3D10: P = 0.370, Data represent mean ± S.E.M. of 4–5 mice per group. c–f Blocking B7-CTLA-4 interaction does not contribute to selective depletion of Treg cells in tumor microenvironment in the Ctla4^h/h mice. L3D10 and Ipilimumab did not delete Treg cells in the spleen (c) of mice at 3 days after third treatment. Data shown are the percentage of Foxp3⁺ cells among CD4 T cells in Ctla4^h/h mice. n = 6 mice for each group. Both L3D10 and Ipilimumab depleted Treg cells in tumors transplanted into the Ctla4^h/h mice, as determined by % Treg cells among CD4 T cells (d, upper), absolute Treg cell number (d, lower) and CD8/Treg ratios (e). Summary data from two experiments involving seven mice per group are presented in d (upper panel) and e. The numbers of Foxp3⁺ cells (d, lower panel) in the tumor from Ctla4^h/h mice were counted by flow cytometry on 3 days after the third antibody treatment. n = 5 for each group. Statistical analyses were performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. f Blocking B7-CTLA-4 interaction does not contribute to increased IFNγ-producing cells among tumor-infiltrating CD4 (left) or CD8 (right) T cells. Summary data are from two experiments involving seven mice per group. Single-cell suspensions of collagenase-digested tumors were prepared between 13 or 16 days and cultured in the presence of Golgi blocker for 4 h and stained for intracellular cytokines. g–j In Ctla4^h/m mice where neither antibody blocks the B7-CTLA-4 interaction, both L3D10 and Ipilimumab induce robust tumor rejection and intratumorial Treg depletion. As in a, except that heterozygous mice that express both mouse and human CTLA-4 were used. g, h Both higher doses (g, 100 μg/mouse/ injection) and lower doses (h, 10 μg/mouse/injection) of antibody treatments showed effective therapeutically effects. In g, Ipilimumab vs hIgG-Fc: P < 0.0001; L3D10 vs hIgG-Fc: P < 0.0001; Ipilimumab vs L3D10: P = 0.4970. Data are representative of five independent experiments. Treg cells were selectively depleted in the tumor (i) but not in the spleen (j) of Ctla4^h/m mice that neither antibodies significantly blocked B7-CTLA-4 interaction in vivo. Data (mean ± S.E.M.) shown in c, d, e and i are the percentage of Treg cells at 18 (experiment 1) or 20 days (experiment 2) after tumor cell challenge and 11 or 13 days after initiation of 3 or 4 anti-CTLA-4 mAb treatments as indicated in arrows. Statistical significance in c–f and i, j was determined using Mann–Whitney test. k Anti-FcR mAb administration abrogated the therapeutic effect of Ipilimumab. 5 × 10⁵ MC38 tumor cells were injected (s.c.) into Ctla4^h/h mice, and mice were treated (i.p.) with 30 μg Ipilimumab alone, or 30 μg Ipilimumab (black arrow) plus 1 mg 2.4G2 (red arrow) or control hIgG-Fc on days 7, 10, 13 and 16, as indicated. Statistical analyses were performed by two-way repeated measures ANOVA (treatment × time). Ipilimumab vs hIgG-Fc: P = 0.0003; Ipilimumab plus 2.4G2 vs hIgG-Fc: P = 0.6962; Ipilimumab plus 2.4G2 vs Ipilimumab: P = 0.0259