An Oncolytic Adenovirus Targeting Transforming Growth Factor β Inhibits Protumorigenic Signals and Produces Immune Activation: A Novel Approach to Enhance Anti-PD-1 and Anti-CTLA-4 Therapy

Abstract

In an effort to develop a new therapy for cancer and to improve antiprogrammed death inhibitor-1 (anti-PD-1) and anticytotoxic T lymphocyte-associated protein (anti-CTLA-4) responses, we have created a telomerase reverse transcriptase promoter-regulated oncolytic adenovirus rAd.sT containing a soluble transforming growth factor receptor II fused with human IgG Fc fragment (sTGFβRIIFc) gene. Infection of breast and renal tumor cells with rAd.sT produced sTGFβRIIFc protein with dose-dependent cytotoxicity. In immunocompetent mouse 4T1 breast tumor model, intratumoral delivery of rAd.sT inhibited both tumor growth and lung metastases. rAd.sT downregulated the expression of several transforming growth factor β (TGFβ) target genes involved in tumor growth and metastases, inhibited Th2 cytokine expression, and induced Th1 cytokines and chemokines, and granzyme B and perforin expression. rAd.sT treatment also increased the percentage of CD8⁺ T lymphocytes, promoted the generation of CD4⁺ T memory cells, reduced regulatory T lymphocytes (Tregs), and reduced bone marrow-derived suppressor cells. Importantly, rAd.sT treatment increased the percentage of CD4⁺ T lymphocytes, and promoted differentiation and maturation of antigen-presenting dendritic cells in the spleen. In the immunocompetent mouse Renca renal tumor model, similar therapeutic effects and immune activation results were observed. In the 4T1 mammary tumor model, rAd.sT improved the inhibition of tumor growth and lung and liver metastases by anti-PD-1 and anti-CTLA-4 antibodies. Analysis of the human breast and kidney tumors showed that a significant number of tumor tissues expressed high levels of TGFβ and TGFβ-inducible genes. Therefore, rAd.sT could be a potential enhancer of anti-PD-1 and anti-CTLA-4 therapy for treating breast and kidney cancers.

Keywords: TGFβ; adenovirus; breast cancer; immunotherapy; kidney cancer.

Figure 1.

Intratumoral inoculation of rAd.sT inhibits tumor growth and metastasis in mouse 4T1 and Renca tumor xenograft models. 4T1 cells were injected subcutaneously into the right flank (5 × 10⁶ cells/mouse) of 4–6-week-old female BALB/c mice (day 0). On day 7, tumor-bearing mice were divided into three groups, buffer, rAd.Null, and rAd.sT groups (n = 10/group). rAd.sT, rAd.Null (2.5 × 10¹⁰ VPs/100 μL), or PBS was injected intratumorally. On day 10, a repeat injection was administered. On days 7, 10, 14, 18, 21, and 25, the tumor sizes were measured (A). On day 25, six mice from each group were euthanized, and the lungs and tumors were removed. Tumor weight was measured (B). The tumor metastasis lesions in lungs were detected by H&E staining. The representative images are shown in (C), and the metastatic areas in the lung were calculated (D). The tumor tissues were stained with (H&E) and also subjected to immunohistochemistry for detecting sTGFβRIIFc expression (E). On day 25, the sera were collected, and the sTGFβRIIFc protein was measured by ELISA (F). Renca cells were injected under the right flank (5 × 10⁶ cells/mouse) of 4–6-week-old female BALB/c mice (day 0) to establish the subcutaneous renal model (n = 12/group). Tumors were treated with oncolytic adenoviruses as described above. The tumor sizes were measured at days 12, 15, 19, 22, 25, and 29 (G). On day 29, mice from each group were euthanized, and tumor weights were measured (H). Data are shown as mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001 versus buffer group; ^##p < 0.01 versus rAd.Null group. ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline; sTGFβRIIFc, soluble transforming growth factor receptor II fused with human IgG Fc fragment; VP, viral particle.

Figure 2.

rAd.sT downregulates protumorigenic/metastasis, reduces Th2 cytokines, and increases Th1 cytokine and chemokine expression in the tumor microenvironment. On day 12 (2 days following adenoviral treatments), tumors in 4T1 and Renca xenografts were removed, and total RNA was isolated. After cDNA was synthesized, the expression of metastasis-related genes (CTGF, PTHrP, CXCR4, and IL-11), angiogenesis-associated genes (VEGFA and VEGFR), and epithelial/mesenchymal transition markers (E-cadherin, N-cadherin, and vimentin) was analyzed by real-time RT-PCR, and normalized by β-actin (n = 4/group) (A). In the murine Renca model, tumors were also collected 2 days following treatments with viruses. The expression of CTGF, PTHrP, and IL-11 was detected by real-time RT-PCR (n = 4/group) (B). The expression of Th2 cytokines (TGFβ1, IL-6, and IL-10), Th1 cytokines (TNF-α, IL-2, IL-12, and IFN-γ), and chemokines (perforin and granzyme B) was detected by real-time RT-PCR in day 12 tumor tissues (C). (n = 4/group). Data are presented as mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001 versus buffer group. RT-PCR, reverse transcription PCR.

Figure 3.

rAd.sT increases CD8⁺ T lymphocytes and CD4⁺ T memory cells, and downregulates MDSCs in the peripheral blood. On day 12, anticoagulant heparin-treated peripheral blood samples were collected from murine 4T1 and Renca tumor models. Blood cells were labeled with APC-conjugated anti-mouse CD3e, FITC-conjugated anti-mouse CD4, and PE-conjugated anti-mouse CD8 antibodies. The percentage of CD8⁺ T lymphocytes and CD4⁺ T lymphocytes was analyzed by flow cytometry (A). Blood cells were also labeled with FITC-conjugated anti-mouse CD4, PerCP-Cy™5.5-conjugated anti-mouse CD44, and APC-conjugated anti-mouse CD62L antibodies. The percentage of T memory cells (CD44^HighCD62L^High) among CD4⁺ T lymphocytes was detected by flow cytometry. The representative images are shown (B), and the statistical results are presented in (D). Blood samples were labeled with FITC-conjugated anti-mouse Ly-6G and APC-conjugated anti-mouse CD11b, and analyzed by flow cytometry. The representative images are shown (C), and the statistical analysis of the data is presented in (D). Data are shown as mean ± s.e.m. *p < 0.05; ***p < 0.001 versus buffer group; ^#p < 0.05; ^##p < 0.01 versus rAd.Null group. n = 4 per group. MDSCs, myeloid-derived suppressor cells.

Figure 4.

Effects of rAd.sT treatment on Tregs and DCs in the mouse spleen. Following various treatments, 4T1-bearing mice were euthanized at days 18 and 31. The spleens were removed and single-cell suspensions were prepared. After treating with RBC lysis buffer, splenocytes were counted. Then, cells were labeled with FITC-conjugated anti-mouse CD4 antibody, PE-conjugated anti-mouse CD25 antibody, and APC-conjugated anti-mouse FoxP3 antibody. The total number of CD25⁺FoxP3⁺ Tregs in spleen (A) and the percentage of Tregs in CD4⁺ (B) were analyzed by flow cytometry. Moreover, cells were labeled with FITC-conjugated anti-mouse CD11c and PE-conjugated anti-mouse CD86 antibodies. The percentage of DCs (CD11c⁺CD86⁺) was examined by flow cytometry. The representative images on day 18 are shown in (C). Quantification of DCs in days 18 and 31 samples is shown in (D, E). Data are shown as mean ± s.e.m. *p < 0.05; **p < 0.01 versus buffer group. n = 4 per group. DCs, dendritic cells; Tregs, regulatory T lymphocytes.

Figure 5.

Combination therapy of rAd.sT with anti-PD-1 and anti-CTLA-4 antibodies in 4T1 xenograft. 4T1 cells were injected subcutaneously in female mice. On day 6, when the tumors were palpable, the tumor size was measured. On day 7, rAd.sT (2.5 × 10¹⁰ VPs in 50 μL) was administered directly into the tumors. A repeat viral dose was given on day 9. On days 8, 10, 12, and 14, anti-PD-1 and anti-CTLA-4 antibodies were administered intraperitoneally (10 mg/kg mouse weight). The tumor growth was monitored (A), and mice were euthanized on day 30. The lungs were excised. Tumor lesions in the lungs were counted (B), and the representative images are shown (C). Tissues slices were subjected to H&E staining to confirm the metastatic lesions (C). Data are shown as mean ± s.e.m. **p < 0.01, ***p < 0.001 versus corresponding groups in (A); *p < 0.05; ***p < 0.001 versus buffer group in (B). n = 8 per group. anti-CTLA-4, anticytotoxic T lymphocyte-associated protein; anti-PD-1, antiprogrammed death inhibitor-1.

Figure 6.

TGFβ and TGFβ regulatory genes are upregulated in breast and renal cancer patients. Surgical specimens were obtained from 34 breast cancer patients and 16 renal cancer patients for clinical and pathological examination. (A) TGFβ mRNA expression in the tumors. The TGFβ expression was measured by real-time RT-PCR in the tumor tissues and distal normal tissues. The relative expression of TGFβ in tumor tissues was normalized by that in normal tissues of the same patient. (B–E) Expression of TGFβ target genes in tumor tissues expressing higher TGFβ levels. The mRNA expression of CXCR4, PTHrP, and CTGF in breast cancer (B, D) and renal cancer patients (C, E) was detected by real-time RT-PCR. Data are shown as mean ± s.e.m. *p < 0.05; ***p < 0.001 versus normal tissues. CTGF, connective tissue growth factor; PTHrP, parathyroid hormone-related protein; TGFβ, transforming growth factor β.