Immune consequences of tyrosine kinase inhibitors that synergize with cancer immunotherapy

Abstract

Combination therapy for the treatment of cancer is becoming increasingly essential as we gain improved understanding of the complexity of cancer progression and the mechanisms by which cancer cells become resistant to single-agent therapy. Recent studies, both clinical and preclinical, have suggested that immunotherapy is a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still needed. A number of recent studies have indicated that antiangiogenic tyrosine kinase inhibitors (TKIs) target multiple components of the tumor microenvironment and are an ideal class of agents for synergizing with cancer immunotherapy. TKIs are well known to modulate tumor endothelial cells, leading to vascular normalization; however, these agents have also been recently shown to decrease tumor compactness and tight junctions, thereby reducing solid tumor pressure and allowing for improved perfusion of collapsed vessels and increased tumor oxygenation. In addition, some TKIs are capable of inducing immunogenic modulation, whereby tumor cells are sensitized to killing by T lymphocytes. Moreover, a number of TKIs have been shown to be involved in immune subset conditioning, increasing the frequency and function of effector immune elements, while decreasing the number and function of immune suppressor cells. The alteration of the immune landscape, direct modification of tumor cells, and improved vascular perfusion leads to improved antitumor efficacy when antiangiogenic TKIs are combined with immunotherapy. Collectively, the data presented in this review support the clinical combination of multi-targeted antiangiogenic TKIs, including but not limited to cabozantinib, sunitinib, and sorafenib, as well as to other antiangiogenic therapies, such as the anti-VEGF antibody bevacizumab, with cancer vaccines for improved treatment of solid tumors.

Keywords: cabozantinib; combination therapy; immune subset conditioning; immunogenic modulation; immunotherapy; sunitinib.

Figures

Figure 1. Cabozantinib treatment improves tumor-cell sensitivity to T cell-mediated lysis

Tumor cells were treated with cabozantinib for 24 hours, labeled with indium111, then incubated with T cells specific for the indicated antigen for 18 hours at an effector:target ratio of 30:1. Murine tumor lines MC38-CEA (A) and LLC-CEA (B) were incubated with CEA-specific murine T cells. Human tumor lines SW620 (C) and H441 (D) were incubated with MUC1-specific human T cells. After 18 hours, supernatants were harvested and analyzed for indium111 release. Percent lysis was calculated as follows: [(experimental cpm – spontaneous cpm) / (maximum cpm – spontaneous cpm)] × 100. Error bars depict mean ± standard error derived from triplicate measurements. * = P < 0.01 as determined by Student’s t test. Data adapted from [12].

Figure 2. Antiangiogenic TKIs reduce tumor vascularity, leading to increased oxygenation

(A) C57/BL6 mice were treated as indicated beginning 14 days post-implantation of MC38-CEA cells. Mice receiving vaccine received a priming vaccination of MVA-CEA/TRICOM on day 14 and a booster vaccination of rF-CEA/TRICOM on day 21. Tumors were harvested 28 days post-tumor implantation. Images show representative tumor areas at 20x magnification obtained using an Aperio ScanScope. (B) Seven days post-implantation of MC38-CEA cells, indicated mice began receiving sunitinib. MVA-CEA/TRICOM was given on day 14. Tumors were harvested 21 days post-tumor implantation. Images show representative tumor areas at 40x magnification obtained using an Aperio ScanScope. In both cases, tumors were harvested and double-stained for the vascular markers CD31 and CD105 using DAB for visualization. Representative images adapted from [12] and [15] are shown. (C) C57/BL6 mice were treated as in (B). IHC analysis of hypoxia was performed using Hypoxyprobe-DAB. Images show representative tumors at 2x magnification obtained using an Aperio ScanScope. Data adapted from [15].

Figure 3. TKIs alone and with a cancer vaccine alter immune-cell function

For cabozantinib studies, C57/BL6 CEA-Tg mice received cabozantinib beginning on day 0. Mice treated with vaccine received MVA-CEA/TRICOM on day 0 and rF-CEA/TRICOM on days 7 and 14. On day 35, spleens were harvested and a single cell suspension of splenocytes was obtained. (A) CEA peptide was incubated with the splenocytes for 7 days after which cells were incubated with fresh, irradiated, naïve splenocytes and either CEA or HIV-gag peptide for 24 hours at 37°C. After 24 hours, a cytometric bead array was used to analyze the supernatants for murine IFN-γ. CEA-specific cytokine production was determined by subtracting cytokine production induced by the HIV-gag peptide from that induced by the CEA peptide. Error bars depict the mean ± standard error. * = P +CD4+CD25+FoxP3+cells) were isolated from spleens using negative selection. Tregs were cultured with CD4+ T cells from naïve mice, antigen-presenting cells (APCs, irradiated allogeneic splenocytes) and soluble anti-CD3 for 72 hours. The background level of CD4+ T cell proliferation was determined by incubating the naïve CD4+ T cells with APCs and anti-CD3 in the absence of purified Tregs. Error bars depict the mean ± standard error. * = P < 0.01 compared to the proliferation of CD4+ T cells incubated with Tregs from untreated mice; ns = no significant difference between CD4+ T cell proliferation in the absence of Tregs and the proliferation of CD4+ T cells incubated with Tregs isolated from mice treated with the indicated therapy. Significance was determined Student’s t test. Data adapted from [12]. For sunitinib studies, C57/BL6 mice received sunitinib beginning on day 0. Mice treated with vaccine received MVA-CEA/TRICOM on day 7, and rF-CEA/TRICOM on day 14. On day 35, spleens were harvested and a single cell suspension of splenocytes was obtained. (C) CD4+ lymphocytes were isolated from spleens and cocultured with APCs and 6.25 g/mL CEA protein for 5 days at 37°C. One μCi [3H] thymidine was added to each well for the last 24 hours. Mean cellular proliferation was dictated by [3H] thymidine incorporation. Error bars depict the mean ± standard error. * = P < 0.01 relative to control and single agents as determined by Student’s t test. (D) Treg functional assay performed as in (B) using splenic Tregs purified from mice treated with sunitinib ± vaccine. Data adapted from [14].

Figure 4. Combining TKIs with a cancer vaccine results in improved antitumor efficacy

(A) Mice were treated as indicated (n = 10/group) beginning 4 days post-implantation of MC38-CEA cells. Ratios denote the number of tumor-free mice at day 35. (B) Indicated groups (n = 12) began receiving sunitinib 7 days post-implantation of MC38-CEA cells. Vaccine administration began 14 days post-implantation. Ratios denote the number of tumor-free mice at day 42. Tumor volumes were calculated as follows: (length × width2)/2. Data adapted from [12] and [14], respectively.