Inhibition of Notch4 using Novel Neutralizing Antibodies Reduces Tumor Growth in Murine Cancer Models by Targeting the Tumor Endothelium.

Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed on tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancer. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms.

Lenvatinib plus anti-PD-1 antibody combination treatment activates CD8+ T cells through reduction of tumor-associated macrophage and activation of the interferon pathway.

Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting mainly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors. We investigated the immunomodulatory activities of lenvatinib in the tumor microenvironment and its mechanisms of enhanced antitumor activity when combined with a programmed cell death-1 (PD-1) blockade. Antitumor activity was examined in immunodeficient and immunocompetent mouse tumor models. Single-cell analysis, flow cytometric analysis, and immunohistochemistry were used to analyze immune cell populations and their activation. Gene co-expression network analysis and pathway analysis using RNA sequencing data were used to identify lenvatinib-driven combined activity with anti-PD-1 antibody (anti-PD-1). Lenvatinib showed potent antitumor activity in the immunocompetent tumor microenvironment compared with the immunodeficient tumor microenvironment. Antitumor activity of lenvatinib plus anti-PD-1 was greater than that of either single treatment. Flow cytometric analysis revealed that lenvatinib reduced tumor-associated macrophages (TAMs) and increased the percentage of activated CD8+ T cells secreting interferon (IFN)-γ+ and granzyme B (GzmB). Combination treatment further increased the percentage of T cells, especially CD8+ T cells, among CD45+ cells and increased IFN-γ+ and GzmB+ CD8+ T cells. Transcriptome analyses of tumors resected from treated mice showed that genes specifically regulated by the combination were significantly enriched for type-I IFN signaling. Pretreatment with lenvatinib followed by anti-PD-1 treatment induced significant antitumor activity compared with anti-PD-1 treatment alone. Our findings show that lenvatinib modulates cancer immunity in the tumor microenvironment by reducing TAMs and, when combined with PD-1 blockade, shows enhanced antitumor activity via the IFN signaling pathway. These findings provide a scientific rationale for combination therapy of lenvatinib with PD-1 blockade to improve cancer immunotherapy.

HLA-DPB1*05: 01-restricted WT1332-specific TCR-transduced CD4+ T lymphocytes display a helper activity for WT1-specific CTL induction and a cytotoxicity against leukemia cells.

Wilms tumor gene 1 (WT1) is overexpressed in various malignant neoplasms, and has been demonstrated as an attractive target for cancer immunotherapy. We previously reported the identification of a WT1 protein-derived, 16-mer helper peptide WT1332 that could elicit Th1-type CD4+ T-cell response and bind to multiple HLA class II molecules. In this study, we examined the feasibility of adoptive therapy using CD4+ T cells that were transduced an HLA-DPB1*05:01-restricted, WT1332-specific T-cell receptor (TCR). HLA-DPB1*05:01-restricted, WT1332-specific TCR-transduced CD4+ T cells were successfully generated using lentiviral vector and exhibited strong proliferative response and Th1-type cytokine production in response to WT1332 peptide, WT1 protein, or WT1-expressing tumor cell lysate. Furthermore, the WT1332-specific TCR-transduced CD4+ T cells lysed HLA-DPB1*05:01-positive, WT1-expressing human leukemia cells through granzyme B/perforin pathway. Furthermore, stimulation of peripheral blood mononuclear cells with both HLA-A*24:02-restricted cytotoxic T lymphocytes-epitope peptide (modified 9-mer WT1235 peptide, WT1235m) and WT1332 helper peptide in the presence of WT1332-specific TCR-transduced CD4+ T cells strikingly enhanced the induction of WT1235m-specific cytotoxic T lymphocytes. Thus, these results demonstrated the feasibility of immunotherapy based on adoptive transfer of WT1332-specific TCR-transduced CD4+ T cells for the treatment of leukemia.

Functional human Th17 clones with WT1-specific helper activity.

Th17 plays important roles in the pathogenesis of various inflammatory and autoimmune diseases. Although the importance of Th17 in tumor immunity has also been suggested, precise roles of tumor-associated antigen-specific Th17 still remain poorly understood, especially in humans. We previously identified WT1332, a 16-mer helper epitope derived from tumor-associated antigen Wilms' tumor gene 1 (WT1) product, and WT1332-specific Th1 clones were established. In the present study, WT1-specific Th17 clones were established by the stimulation of peripheral blood mononuclear cells with the WT1332 helper peptide under human Th17-polarizing conditions. The WT1-specific Th17 clone exhibited the helper function for proliferation of conventional CD4(+) T cells in the antigenic stimulation-specific manner. This is the first report of establishment of functional Th17 clones with both antigen (WT1332) specificity and antigen-specific helper activity. Th17 clones established here and the method to establish antigen-specific Th17 clones should be a useful tool to further analyze the roles of human Th17 in tumor immunity.

Recognition of a natural WT1 epitope by a modified WT1 peptide-specific T-cell receptor.

Wilms' tumor gene WT1 is highly expressed in leukemia and in various types of solid tumors and exerts an oncogenic function. Thus, WT1 protein is a most promising tumor-associated antigen. We have been successfully performing WT1 vaccination with a 9-mer modified WT1(235) peptide, which has one amino acid substitution (M→Y) at position 2 of 9-mer natural WT1(235) peptide (235-243 a.a.), for close to 700 HLA-A*24:02-positive patients with leukemia or solid tumors. Although vaccination of modified WT1(235) peptide induced natural WT1(235) peptide-recognizing cytotoxic T-lymphocytes (CTLs) and exerted cytotoxic activity towards leukemia and solid tumor cells that expressed the natural WT1(235) peptide (epitope) but not the vaccinated modified WT1(235) peptide (epitope), the molecular basis has remained unclear. In this study, we established a modified WT1(235) peptide-specific CTL clone, we isolated T-cell receptor (TCR) genes from it and transduced the TCR genes into CD8(+) T-cells. The TCR-transduced CD8(+) T-cells produced interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in response to stimulation not only with the modified WT1(235) peptide but also with the natural WT1(235) peptide and lysed modified or natural WT1(235) peptide-pulsed target cells and endogenously WT1-expressing leukemia cells in a HLA-A*24:02-restriction manner. These results provided us, for the first time at molecular basis, with a proof-of-concept of modified WT1(235) peptide-based immunotherapy for natural WT1(235) peptide-expressing malignancies.