PD-1 blockade at the time of tumor escape potentiates the immune-mediated antitumor effects of a melanoma-targeting monoclonal antibody

Abstract

Tumor antigen-targeting monoclonal antibodies (TA-targeting mAbs) are used as therapeutics in many malignancies and their capacity to mobilize the host immunity puts them at the forefront of anti-cancer immunotherapies. Both innate and adaptive immune cells have been associated with the therapeutic activity of such antibodies, but tumor escape from mAb-induced tumor immune surveillance remains one of the main clinical issues. In this preclinical study, we grafted immunocompetent and immunocompromised mice with the B16F10 mouse melanoma cell line and treated them with the TA99 TA-targeting mAb to analyze the immune mechanisms associated with the tumor response and resistance to TA99 monotherapy. In immunocompetent mice TA99 treatment strongly increased the fraction of CD8 and CD4 effector T cells in the tumor compared with isotype control, highlighting the specific immune modulation of the tumor microenvironment by TA99. However, in most mice, TA99 immunotherapy could not prevent immune effector exhaustion and the recruitment of regulatory CD4 T cells and consequently tumor escape from immune surveillance. Remarkably, anti-PD-1 treatment at the time of tumor emergence restored the Th1 effector functions of CD4 and CD8 T cells as well as of natural killer and γδT cells, which translated into a significant slow-down of tumor progression and extended survival. Our findings provide the first evidence that PD-1 blockade at the time of tumor emergence can efficiently boost the host anti-tumor immune response initiated several weeks before by the TA-targeting mAb. These results are promising for the design of combined therapies to sensitize non-responder or resistant patients.

Keywords: anti-tumor immunity; combined therapies; immunomodulation; long-lasting effects; tumor escape; tumor immune microenvironment; tumor-targeting monoclonal antibodies.

Figure 1.

Long-term anti-tumor immunity in TA99-treated mice relies on a specific T-cell memory response. C57BL/6N (A, B), athymic nude (C, D) and NSG (E, F) mice engrafted with 5.10⁴ B16F10 melanoma cells received 6 injections (i.p.) of TA99 mAb (TA99), isotype control (IC) or PBS (PBS). The mean tumor sizes ± SEM (A, C, E) and Kaplan-Meier survival curves (B, D, F) are shown. Kaplan-Meier survival curves for tumor-free TA99-treated C57BL/6N (G) and athymic nude (H) mice after a second injection (s.c.) of 5.10⁴ B16F10 cells on the opposite flank (challenge). (I) Tumor-free TA99-treated C57BL/6N mice were challenged (s.c.) with 5.10⁵ MC38 colon adenocarcinoma cells (MC38). For G, H and I, a group of newly grafted C57BL/6N or athymic nude mice was used as control. *p < 0.05; **p < 0.01; ****p < 0.0001 (log-rank test for Kaplan Meier survival curves and non-parametric Mann-Whitney test for tumor growth).

Figure 2.

Long-term anti-tumor immunity in TA99-treated mice reduces B16F10 lung metastasis formation. 10⁵ B16F10 cells were injected (i.v.) in age-matched naive or tumor-free TA99-treated C57BL/6N mice that were killed 12 d later to quantify tumor foci in the lungs. Representative images of lungs (A) and quantification of total foci (B) and foci larger than 3 mm (C) in the lungs of naive (gray bars, n = 8) and tumor-free TA99-treated mice (black bars, n = 10). In B and C, data are the mean ± SEM. **p < 0.01 (non-parametric Mann-Whitney test).

Figure 3.

TA99 treatment induces cellular and humoral anti-tumor immune responses. Tumor-free TA99-treated C57BL/6N mice received a second s.c. injection of 5.10⁴ B16F10 cells on the opposite flank (challenged) and the CTL activity of CD8 splenocytes was evaluated 17 d later and compared with that of age-matched untreated and never grafted controls (naive). Representative flow cytometry histograms (A) and percentages of CTL activity (B) in naive (n = 9) and challenged (n = 9) mice. (C) Serum samples were collected from the same naive (n = 9) and TA99-treated mice before (pre-chall) and after challenge (post-chall) and anti-B16F10 IgGs quantified by ELISA. (D, E) Serum samples from naive mice (non-immune sera) or from tumor-free TA99-treated mice (immune sera) were used to treat C57BL/6N mice grafted with 5.10⁴ B16F10 cells for the first time (n = 6/group). The mean tumor size ± SEM (D) and Kaplan-Meier survival curves (E) are indicated. *p < 0.05; **p < 0.01 (log-rank test for Kaplan Meier survival curves, nonparametric Mann-Whitney test for tumor growth and non-parametric Kruskal-Wallis test for ELISA).

Figure 4.

TA99 treatment increases the percentage of tumor infiltrating effector lymphocytes, but does not prevent their exhaustion. (A-C) Immune cells were isolated from spleens (white symbols) and tumors (black symbols) of mice treated with PBS (PBS, n = 9), IgG2a isotype control (IC, n = 9) or TA99 (TA99, n = 9). The ratio between effector memory (CD44⁺CD62L⁻) and naive (CD44⁻CD62L⁺) CD8⁺ T cell (A), and percentage of CD25⁺FOXP3⁻ effector CD4⁺ T cells (B) and CD25⁺FOXP3⁺ CD4⁺ Tregs (C) are indicated. (D, E) Expression of PD-1 and TIM3 in CD8 (D) and CD4 (E) T cells isolated as indicated in A and B. Top, representative dot-plots; bottom, individual data from 9 mice/group (same mice as in A-C). (F) Expression of PD-1 and TIM3 in PBMCs and TILs from 15 untreated patients with metastatic melanoma. The percentages of PD1⁺ and PD1⁺TIM3⁺ in the CD4 and CD8 subpopulations are shown. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant; determined with the non-parametric Kruskal-Wallis test (A, B, C, D, E) or the non-parametric Wilcoxon matched-pairs rank test (F).

Figure 5.

Anti-PD-1 treatment slows down tumor progression and increases survival of TA99-treated mice. C57BL/6N mice were grafted (s.c.) with 5.10⁴ B16F10 (A and B) or B16F1 (C and D) cells and treated with the TA99 mAb or the isotype control (IC). Upon tumor appearance (50 mm³), mice received anti-PD-1 antibodies (i.p.) twice a week (IC+αPD-1, n = 8 or TA99+αPD-1, n = 8) or PBS (IC, n = 6 or TA99, n = 6) for 3 weeks. The mean tumor sizes ± SEM (A and C) and Kaplan-Meier survival curves (B and D) are indicated. Day 0 stands for the day of tumor appearance. ***p < 0.001; **p < 0.01 determined with mixed-effects ML regression test for tumor growth (A and C) and the log-rank test for Kaplan Meier survival curves (B and D).

Figure 6.

PD-1 blockade synergizes with TA99 therapy to enhance effector T cell functions. Upon tumor appearance, TA99-treated and isotype control (IC)-treated mice received anti-PD-1 antibodies (i.p.) twice per week (TA99+αPD1, n = 9; IC+αPD1, n = 9) or PBS (TA99, n = 7; IC, n = 7). When tumors reached 200–300 mm³,cytokine production was analyzed in spleen and tumor by flow cytometry. (A and B) Percentage of IFNγ (top) and IFNγ plus IL-2 (bottom)-producing CD4 (A) and CD8 (B) T cells isolated from spleens and tumors. (C and D) Percentage of NK (C) and γδ T cells (D) that produce IFNγ (left) and expressing CD107a (right) in tumors; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (non-parametric Kruskal-Wallis test).

Figure 7.

PD-1 blockade synergizes with TA99 treatment to enhance the cellular and humoral adaptive memory responses. Tumor-free TA99-treated mice were challenged (s.c.) with 5.10⁴ B16F10 cells. Upon tumor appearance, mice received 2 i.p. injections, anti-PD-1 antibodies (TA99+αPD1, n = 5) or PBS (TA99, n = 7). The CTL activity of CD8 splenocytes was evaluated 17 d after the challenge and compared with that of splenocytes from age-matched non-grafted and untreated mice (naive, n = 8). Representative flow cytometry histograms (left) and percentages of CTL activity (right) in spleen (A), draining (B) and non-draining lymph nodes (C) from naive and TA99-treated mice treated or not with anti-PD-1 antibodies. (D) Concentration of anti-B16F10 IgGs determined by ELISA in serum samples from naive, TA99-treated and TA99+αPD1-treated mice on the day of sacrifice for the CTL assay. (E) Tumor growth in TA99-treated and TA99+αPD1-treated animals. *p < 0.05; ns, not significant; determined with the non-parametric Kruskal-Wallis (A, B, C, D) and the non-parametric Mann-Whitney test (E).