Antimesothelioma Immunotherapy by CTLA-4 Blockade Depends on Active PD1-Based TWIST1 Vaccination

Abstract

Checkpoint immunotherapy is a major breakthrough for cancer treatment, yet its efficacy is often limited against many types of malignancies, including malignant mesothelioma. Considering that the immunotherapeutic efficacy depends on immunosurveillance, we sought to develop an active immunization method to break immune tolerance to tumor self-antigen. Here, we demonstrated that TWIST1, the basic helix-loop-helix transcription factor, was associated with human mesothelioma tumorigenesis and required for the invasion and metastasis of mesothelioma in the immune-competent murine AB1 model. When conventional TWIST1 vaccines were not effective in vivo, programmed cell death protein 1 (PD1)-based vaccination provided prophylactic control by inducing long-lasting TWIST1-specific T cell responses against both subcutaneous and metastatic mesothelioma lethal challenges. Furthermore, while CTLA-4 blockade alone did not show any immunotherapeutic efficacy against established mesothelioma, its combination with PD1-based vaccination resulted in 60% complete remission. Mechanistically, these functional T cells recognized a novel highly conserved immunodominant TWIST1 epitope, exhibited cytotoxic activity and long-term memory, and led to durable tumor regression and survival benefit against established AB1 mesothelioma and 4T1 breast cancer. We concluded that PD1-based vaccination controls mesothelioma by breaking immune tolerance to the tumor self-antigen TWIST1. Our results warrant clinical development of the PD1-based vaccination to enhance immunotherapy against a wide range of TWIST1-expressing tumors.

Keywords: CTLA-4; DNA vaccine; PD1; TWIST1 antigen; cytotoxic T lymphocytes; immune checkpoint blockade; mesothelioma; soluble PD1-based vaccination.

© 2020 The Authors.

Figures

Figure 1

Expression of TWIST1 Promotes Invasion and Metastasis of AB1 Mesothelioma (A) TWIST1 expression in the mesothelioma cohort of TCGA (n = 87) by TNM stage. Stage I and II, n = 26. Stage III and IV, n = 61. (B) Kaplan-Meier overall survival curve of mesothelioma patients stratified by expression level of TWIST1, with weak (n = 45, TWIST1 ≤ 8.346) or strong (n = 42, TWIST1 > 8.346) expression of TWIST1. (C) Western blot analysis of TWIST1 in different murine tumor cell lines. The functional role of TWIST1 in AB1 cells was analyzed by gene overexpression (OE) and knockout (KO). (D) Western blot analysis of TWIST1 protein. WT, wild-type AB1 cells; OE, lentiviral vector-mediated TWIST1 OE; KO, CRISPR/Cas9-mediated TWIST1 KO. (E) qRT-PCR quantification of EMT-related molecules including vimentin, N-cadherin, and E-cadherin in WT, TWIST1 OE, or KO cells. Data shown are representative of two independent experiments. (F) Representative wells shown for colony-formation assay. (G) Matrigel cell invasion assay with representative images and quantification. Data in (F) and (G) shown are representative of two independent experiments. (H) Lung metastases after intravenous inoculation of 1 × 106 AB1 into BALB/c mice (n = 6). Left panel, survival curve. Right panel, representative images of lungs harvested at endpoint. Graphs show cumulative data from two separate experiments. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 2

PD1-Based Vaccination Enhanced TWIST1-Specific T Cell Responses (A) Schematic representation of TWIST1 DNA vaccine constructs. tPA, tissue plasminogen activator signal sequence. (B) Expression of TWIST1 DNA vaccine constructs after transfection in 293T cells. Cell lysates or culture supernatant of transfected 293T cells were subjected to western blot analysis using anti-TWIST1 antibody. (C) Flow cytometric analysis of binding between soluble proteins and mouse PD-L1/L2-transfected 293T cells. Transfection supernatant collected from sPD1-TWIST1-FLAG (red solid line)-, sTWIST1-FLAG (black dashed line)-, or mock (shaded region)-treated 293T cells were used to incubate 293T cells transiently transfected with mouse PD-L1 or PD-L2 expression vectors. Data shown are representative of two independent experiments. (D) DNA vaccination schedule. Groups of BALB/c mice (n = 4) received three DNA/EP vaccinations before being sacrificed for immunoassay at 2 weeks after the last vaccination. (E) ELISpot analysis of TWIST1-specific T cell responses. (F) Intracellular staining of IFN-γ-, IL-2-, and TNF-α-producing CD4+ and CD8+ T cells after DNA/EP vaccination. (G) Cytokine production following incubation of purified CD3+ T cells with WT or TWIST1 KO AB1 cells. Data in (E)–(G) shown are representative of two independent experiments. Data represents mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01.

Figure 3

PD1-Based Vaccination Inhibited Growth of Primary AB1 Mesothelioma (A) Schematic representation of treatment schedule. (B and C) Vaccinated mice (n = 8) were assessed by tumor growth (B) and survival (C) after WT AB1 challenge. Graphs show cumulative data from two separate experiments. Alternatively, sPD1-TWIST1-vaccinated mice (n = 5 each group) were challenged s.c. with 1 × 106 TWIST1 OE, KO, or WT AB1 cells, respectively. (D and E) Tumor growth (D) and survival (E) were calculated. (F) 120 days after the last vaccination, mice were challenged with 1 × 106 WT AB1 cells and measured for tumor growth (G) and tumor-free survival (H). (I) Mice were sacrificed at day 30 post-tumor-inoculation for analysis of naive (CD44loCD62Lhi) and effector memory (CD44hiCD62Llo) splenic T cells. (J and K) Tumor-free (n = 3) and tumor-bearing (n = 4) mice receiving sPD1-TWIST1 vaccination were assessed by frequencies of CD8+ naive and effector memory T cells (J) and production of TNF-α and IL-2 in CD4+ and CD8+ effector memory T cells (K). Numbers within each plot represent cell proportions. Data represents mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 4

sPD1-TWIST1 Vaccine Inhibited AB1 Lung Metastasis (A and B) Following the same vaccination regimen in Figure 3A, vaccinated BALB/c mice (n = 4) received 1 × 106 AB1 cells i.v. 2 weeks after last vaccination, and tumor growth (A) was monitored by bioluminescence and shown with representative bioluminescence images (B). (C and D) On day 28, the body weight of mice was measured (C), and then mice were sacrificed to collected lungs for (D) macroscopic evaluation and H&E staining. (E) Assessment of effector function of T cell subsets (upper) and immunosuppressive cell subsets (lower) in spleen at the endpoint. Data represents mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5

Checkpoint Modulation Enhances the Antitumor Activity of sPD1-TWIST1 Vaccination for Curing Established Mesothelioma BMDCs were pulsed with TWIST1 or OVA peptides, followed by incubation with 1.0, 10, or 100 μg/mL α-CTLA-4 antibody. CD3+ T cells purified from splenocytes of sPD1-TWIST1-, sTWIST1-, or sham-vaccinated tumor-bearing mice (Figure 3B) were added into the co-culture. Proliferation of T cells were analyzed from 6-day BMDC-T cell co-cultures. (A) Representative plots showing proliferation of CD3+ T cells from sPD1-TWIST1-vaccinated mice. Numbers in each plot indicating CFSElow populations. (B) Proliferation of CD3+ T cells from sPD1-TWIST1, sTWIST1, or sham-vaccinated mice. iso, mouse IgG2b isotype control. Graphs show cumulative data from two separate experiments. (C) Schematic representation of therapeutic study. (D and E) Tumor growth measurement (D) and tumor-free survival curve (E) after therapeutic vaccination. Mice were sacrificed when tumor size was >15 mm. (F and G) TWIST1-specific T cell responses across all groups (F) or TWIST1-, AH1-specific T cell responses in combined sPD1-TWIST1 vaccination and α-CTLA-4 therapy group (G) were quantified by IFN-γ ELISpot assay. Arrow indicated individual tumor-free mouse in that group. Graphs show cumulative data from two separate experiments. At least eight mice were used in each group. Groups of female BALB/c mice (n = 5) were inoculated with 2 × 105 4T1 cells in the mammary gland, followed by vaccination three times in 10-day intervals, starting at 1 day post-tumor-inoculation. Anti-CTLA4 antibody at a dose of 200 μg per injection were administrated i.p. starting from 2 days post-tumor-inoculation and every 4 days afterward. (H) 4T1 Primary tumor growth curve (left) and tumor weights (right) harvested at day 27 post-4T1-inoculation. (I) Enumeration of clonogenic metastatic cells in the lungs (left) and representative images of clonogenic colonies after 14 days incubation (right, ×200 dilution factor). (J) Representative dot plots and percentages of IFN-γ+TNF-α+ CD8+ T cells in spleens were measured at the endpoint. Data represents mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 6

Combination Therapy Induced Durable T Cell Immunity Responsive to an Immunodominant TWIST1 Epitope (A and B) 60 days after tumor ablation, protected mice (n = 5) in the combined sPD1-TWIST1 and α-CTLA-4 group were rechallenged s.c. and measured for tumor growth (A) with representative bioluminescence images of AB1-Luc tumors (B). (C) Cytotoxicity assay of T cells toward AB1 cells at different effector:target (E:T) ratios. T cells were isolated from spleen of mice receiving combined sPD1-TWIST1 vaccination and α-CTLA-4 therapy after initial complete tumor rejection. The experiment was repeated two times. (D) Schematic representation (upper) and tumor growth curve (lower) for T cell adoptive transfer. T cells from either naive or vaccinated/protected mice were adoptively transferred to SCID mice bearing 7-day-old AB1-Luc tumors and assessed for tumor growth. Cy (cyclophosphamide) at a dose of 150 mg/kg was administrated i.p. at day 6. (E and F) Characterization of TWIST1 immunodominant epitopes using minipools spanning the entire TWIST1 sequence (E) or 15-mer peptides in minipool37-39 (F). Data shown are representative of two independent experiments. Data represents mean ± SEM. ∗p