Incorporation of SKI-G-801, a Novel AXL Inhibitor, With Anti-PD-1 Plus Chemotherapy Improves Anti-Tumor Activity and Survival by Enhancing T Cell Immunity

Abstract

A recently developed treatment strategy for lung cancer that combines immune checkpoint inhibitors with chemotherapy has been applied as a standard treatment for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and it has improved the outcomes of chemotherapy. Maintenance treatment with anti-PD-1 antibody (aPD-1) enhances the effect of immunochemical combination therapy and improves therapeutic efficacy, which contributes toward a significant improvement in patient survival rates. The AXL receptor tyrosine kinase (AXL), which is expressed in tumor cells, plays an essential role in the resistance of cancers to chemotherapy and immunotherapy, and stimulates signaling associated with epithelial-mesenchymal transition (EMT) in metastatic cancer. AXL is thus an attractive target for controlling resistance to anti-tumor therapies. In this study, we examined the effect of AXL inhibitors on immune activation and tumor growth in TC1 and C3PQ mouse tumor models, in the context of clinical immunotherapy/chemotherapy and maintenance treatment, using an aPD-1 with/without pemetrexed. To determine the optimal timing for administration of SKI-G-801, an AXL inhibitor, we investigated its anti-tumor effects based on inclusion at the immunochemotherapy and maintenance therapy stages. We also performed flow cytometry-based immune profiling of myeloid cells and lymphoid cells at different points in the treatment schedule, to investigate the immune activation and anti-tumor effects of the AXL inhibitor. The addition of SKI-G-801 to the immune checkpoint inhibitor and chemotherapy stage, as well as the maintenance therapy stage, produced the best anti-tumor results, and significant tumor growth inhibition was observed in both the TC1 and C3PQ models. Both models also exhibited increased proportion of effector memory helper T cells and increased expression of CD86⁺ macrophages. Especially, regulatory T cells were significantly reduced in the TC1 tumor model and there was an increase in central memory cytotoxic T cell infiltration and an increased proportion of macrophages with high CD80 expression in the C3PQ tumor model. These results suggest increased infiltration of T cells, consistent with previous studies using AXL inhibitors. It is expected that the results from this study will serve as a stepping stone for clinical research to improve the existing standard of care.

Keywords: AXL receptor tyrosine kinase; chemotherapy; epithelial-mesenchymal transition; immunotherapy; non-small cell lung cancer.

Figure 1

Upfront AXL inhibitor add-on delayed tumor growth, enhanced tumor growth inhibition, and improves survival in combination with SoC chemotherapy for LUAD in TC1 model (A) Syngeneic TC1 tumor cells were injected into C57BL/6 mice and treatment was initiated when the tumor volume reached 100 mm³. [aPD-1 + Pem + CDDP] were administered as standard of care for chemotherapy, and aPD-1 and Pem were administered as standard of care for maintenance therapy. (B) AXL inhibition by SKI-G-801 with standard of care significantly delayed the growth of TC1 xenografts in C57BL/6 mice. Red arrows indicate sampling points during the treatment schedule (SKI-G-801 30 mg/kg, QD; Pem 100 mg/kg, BIW; CDDP 5 mg/kg, BIW; aPD-1 10 mg/kg, BIW). Mean tumor volumes for each group and SEM images are shown. (C) Tumor growth inhibition was enhanced by upfront addition of SKI-G-801, with all of the mice in the [aPD-1+PemCDDP+SKI-G-801] → [aPD-1+Pem+SKI-G-801] group showing more than 75% TGI. Dashed line indicates -75% TGI. (D) Upfront AXL inhibition significantly enhanced the overall survival of TC1 tumor-bearing C57BL/6 mice. Statistical significance between groups was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). LUAD, lung adenocarcinoma; aPD-1, anti-PD-1 antibody; Pem, pemetrexed; CDDP, cisplatin; SEM, standard error of the mean; TGI, tumor growth inhibition; ANOVA, analysis of variance.

Figure 2

Upfront AXL inhibitor add-on delays tumor growth, enhances tumor growth inhibition, and survival in combination with SoC chemotherapy for LUSC in C3PQ model. (A) Syngeneic C3PQ tumor cells were injected into BALB/c mice and treatment was initiated when the tumor volume reached 100mm³. aPD-1 + Pac + Carb were administered as standard of care for chemotherapy, and aPD-1 was administered as standard of care for maintenance therapy. (B) AXL inhibition by SKI-G-801 with standard of care significantly delayed the growth of C3PQ xenografts in BALB/c mice. Red arrows indicate sampling points during the treatment schedule (SKI-G-801 30 mg/kg, QD; Pac 20 mg/kg, QW; Carb 50 mg/kg, QW; aPD-1 10 mg/kg, BIW). Mean tumor volumes of each group and SEM images are shown. (C) Tumor growth inhibition was enhanced by upfront addition of SKI-G-801, with all of the mice in the [aPD-1+PacCarb+SKI-G-801] → [aPD-1+SKI-G-801] group showing more than 50% TGI. Dashed line indicates -50% TGI. (D) Upfront AXL inhibition conferred enhanced overall survival to C3PQ tumor-bearing BALB/c mice. Statistical significance between groups was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001). LUSC, lung squamous cell carcinoma; aPD-1, anti-PD-1 antibody; Pac, paclitaxel; Carb, carboplatin; SEM, standard error of the mean; TGI, tumor growth inhibition; ANOVA, analysis of variance.

Figure 3

SKI-G-801 treatment increases memory function of the immune system. Analysis of T cell subset of TC1 tumor model (A) and C3PQ tumor model (B) by flow cytometry. (A) The effector memory subset of CD4⁺ T cells was significantly increased in the maintenance state of the [aPD-1+PemCDDP] → [SKI-G-801] group and the [aPD-1+PemCDDP+SKI-G-801] → [aPD-1+Pem+SKI-G-801] group compared to the treatment naïve group. The effector memory subset of CD8⁺ T cells showed an increase and CD4⁺ regulatory T cells showed a decreasing trend after chemotherapy, but these results were not significant. (B) The effector memory subset of CD4⁺ T cells was significantly increased in the maintenance state of all groups compared to the treatment naïve group. In addition, the effector memory subset of CD8⁺ T cells was increased in the maintenance state of the [aPD-1+PacCarb] → [aPD-1+SKI-G-801] group and the [aPD-1+PacCarb+SKI-G-801] → [aPD-1+SKI-G-801] group compared to the treatment naïve group. CD4⁺ regulatory T cells showed no meaningful changes. (*p ≤ 0.05, ***p ≤0.001).

Figure 4

Antigen-presenting machinery increases after SKI-G-801 treatment. Analysis of myeloid cell subset of TC1 tumor model (A) and C3PQ tumor (B) by flow cytometry. (A) M1-like macrophages were significantly increased in the maintenance state of the [aPD-1+PemCDDP] → [aPD-1+Pem+SKI-G-801] group, the [aPD-1+PemCDDP] → [SKI-G-801] group, and the [aPD-1+PemCDDP+SKI-G-801] → [aPD-1+Pem+SKI-G-801] group compared to the treatment naïve group. The macrophage total also increased in the maintenance state of the [aPD-1+PemCDDP] → [SKI-G-801] group and the [aPD-1+PemCDDP+SKI-G-801] → [aPD-1+Pem+SKI-G-801] group compared to the treatment naïve group. The number of CD86 expressing macrophages was significantly high in all groups compared to the treatment naïve group. (B) M1-like macrophages showed a decreasing trend in all groups, but total macrophage count was significantly increased in the maintenance state of the [aPD-1+PacCarb] → [aPD-1+SKI-G-801] group compared to the treatment naïve group. In addition, the number of CD86 and CD80 expressing macrophages increased in the maintenance state of the [aPD-1+PacCarb] → [aPD-1+SKI-G-801] group and the [aPD-1+PacCarb+SKI-G-801] → [aPD-1+SKI-G-801] group compared to treatment naïve state. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001).

Figure 5

Immunohistochemistry with anti-Foxp3 reveals tumor infiltration by regulatory T cells. Positive Foxp3 expression was observed in the [aPD-1+PemCDDP] → [aPD-1+Pem] (A) and [aPD-1+PemCDDP+SKI-G-801] → [aPD-1+Pem+SKI-G-801] (E) groups in TC1 tumor tissues. (B), Higher magnification of images in (A, F), higher magnification of images in (E). In addition, positive Foxp3 expression was visible in the [aPD-1+ PacCarb] → [aPD-1] (C) and [aPD-1+ PacCarb+SKI-G-801] → [aPD-1+SKI-G-801] (G) groups in C3PQ tumor tissues. (D), Higher magnification of images in (C, H), higher magnification of images in (G). (I), the number of Foxp3⁺ cells per mm² of TC1 tumor tissues and (J), the number of Foxp3⁺ cells per mm² of C3PQ tumor tissues. *p < 0.05. Scale bars in (A, C, E, G): 100µm. (*p ≤ 0.05).

Figure 6

Immunological benefits of SKI-G-801 addition to chemotherapies in TC1 and C3PQ tumor models. The differences in immune cell changes between TC1 and C3PQ after treatment in each group is shown. The number of effector memory Th cells and CD86⁺ high macrophages was similar in TC1 and C3PQ. In particular, Tregs were downregulated in TC1, and central memory of Tc cells were upregulated in C3PQ. ↑: p < 0.05, ↑↑: p < 0.01.