Transcutaneous Imiquimod Combined With Anti-Programmed Cell Death-1 Monoclonal Antibody Extends the Survival of Mice Bearing Renal Cell Carcinoma

Abstract

Purpose: Imiquimod (IQM), an imidazoquinoline derivative, is an immunomodulator that activates an adaptive immune response. IQM is applied topically for genital warts and actinic keratosis. Programmed cell death-1 (PD-1) suppresses activated T cells by binding to programmed cell death-ligand 1 and programmed cell death-ligand 2, braking antitumor immunity. Anti-PD-1 therapy has been used for various malignant neoplasms including renal cell carcinoma (RCC). Whether combination therapy with transcutaneous administration of IQM cream and intraperitoneal administration of anti-PD-1 monoclonal antibody (mAb) suppresses mouse RCC cells growing in subcutaneous tissue was investigated.

Methods: Female BALB/c mice were implanted subcutaneously with 2 × 10⁵ RENCA mouse RCC cells and treated with a transcutaneously applied cream containing IQM and intraperitoneal administration of anti-PD-1 mAb beginning 5 days after cell implantation. Tumor burden and survival of the mice were determined. RENCA tumor-specific IgG production and a minor CD8⁺ T cell subset derived from the spleen of the mice bearing RENCA tumor were detected by flow cytometry. The tumor and spleen weights of mice treated with IQM, anti-PD-1 mAb, and their combination were compared.

Results: Combination therapy with IQM and anti-PD-1 mAb significantly suppressed tumor growth compared to each monotherapy and prolonged the survival of the mice. The combination therapy produced more RENCA tumor-specific IgG than either IQM or anti-PD-1 mAb alone. The percentage of the CD44^highCD62L^low CD8⁺ T cell subset (effector memory T cells) among splenocytes from mice treated with IQM therapy increased. The CD44^lowCD62L^low CD8⁺ T cell subset (pre-effector-like T cells) of mice treated with anti-PD-1 mAb increased. A negative correlation between tumor and spleen weights was suggested in mice treated with therapies containing IQM.

Conclusions: The present results show that combination therapy with IQM and anti-PD-1 mAb might be a promising novel therapeutic strategy for advanced RCC.

Keywords: IgG; T cell; imiquimod; immune checkpoint inhibitor; programmed cell death‐1; renal cell carcinoma; spleen.

FIGURE 1

RENCA mouse RCC cells were implanted subcutaneously on the back of BALB/c mice. Therapies were then started 4 days after implantation and continued for 42 days. Tumor volume was measured every 6 days after initiation of therapy with control vehicle, IQM, anti‐PD‐1 mAb, and the combination of IQM and anti‐PD‐1 mAb. The combination therapy significantly suppressed tumor growth when compared with control vehicle, IQM, and anti‐PD‐1 mAb on days 24 and 30 (●, Control; ■, IQM; △, anti‐PD‐1 mAb; ○, combination. *p‐value < 0.05).

FIGURE 2

Kaplan–Meier survival curves of the mice treated with control vehicle, IQM, anti‐PD‐1 mAb, and the combination are shown. The mice were continuously observed after the therapy was completed at 42 days. The mice treated with IQM, anti‐PD‐1, and combination therapy showed a tendency for longer survival. Notably, one of the ten mice treated with the combination therapy survived to the end of observation at 150 days after therapy was started (a). Macroscopic findings of the mouse treated with IQM and anti‐PD‐1 mAb showing complete remission (b). The RENCA cells show adequate tumorigenesis at 30 days after combination therapy was started. The tumor is crusting and falling without ulceration at 40 days. At 50 days, the tumor has completely disappeared. RENCA cells were subcutaneously reimplanted in the mouse showing complete remission in the first in vivo animal experiment. The tumor never engrafts during observation without any therapy (1st reimplantation). Furthermore, RENCA cells were reimplanted in the same mouse in the third in vivo experiment. Small crusting without tumor is seen (2nd reimplantation).

FIGURE 3

RENCA tumor‐specific IgG production in the serum of mice treated with control vehicle, IQM, anti‐PD‐1 mAb, and the combination was determined by flow cytometry. IgG production is shown in the histogram. The IgG production of the mice without tumor (unimmunized) is shown as the gray curve in the histogram. The curve of combination therapy (black bold curve) is shifted to the far right, meaning that the IgG production of the mice treated with the combination is higher than that of the mice treated with control vehicle (black dotted curve), IQM (black curve), and anti‐PD‐1 mAb (black broken curve). The mice bearing RENCA tumors produced tumor‐specific IgG, even those treated with control vehicle.

FIGURE 4

Flow cytometry gating strategy to identify minor CD8⁺ T cell subset of splenocytes from the mice bearing RENCA tumor (a). Representative dot plots showing the expressions of CD44 and CD62L on CD8⁺ T cells from the splenocytes of mice treated with control vehicle and combination (a). Based on the expressions of CD44 and CD62L, four subsets were gated: CD44^highCD62L^low subset (Q1, effector memory T cells); CD44^highCD62L^high subset (Q2, central memory T cells); CD44^lowCD62L^high subset (Q3, naïve T cells); and CD44^lowCD62L^low subset (Q4, pre‐effector‐like T cells). Numbers in the plots indicate proportions of gated cell populations. Comparison of the percentage of minor CD8⁺ T cell subsets in the therapies with control vehicle, IQM, anti‐PD‐1 mAb, and combination therapy (b). The IQM increases the Q1 and Q2 subsets; the anti‐PD‐1 mAb increases the Q4 subset; the combination of IQM and anti‐PD‐1 mAb increases both the Q1 and Q4 subsets, compared to the control vehicle.

FIGURE 5

Representative dot plots of flow cytometry showing the expressions of IFN‐γ and Granzyme B on CD8⁺ T cells from the splenocytes of mice treated with control vehicle and combination therapy. Numbers in the plots indicate proportions of gated cell populations. The combination of IQM and anti‐PD‐1 mAb increases the IFN‐γ and/or the Granzyme B subsets (Q1, Q2, and Q3) compared with the control vehicle.

FIGURE 6

Correlations of tumor and spleen weights in the mice treated with the therapies containing IQM (a) and without IQM (b). The therapies containing IQM (IQM and Combination) show a significantly negative correlation between tumor and spleen weights (Pearson's correlation coefficient = −0.794, p‐value = 0.002), whereas the mice treated without IQM (control vehicle and anti‐PD‐1 mAb) do not. Pearson's correlation coefficient and p‐values of individual therapies are shown in the lower table (c).