Yu Kato

Tumor cell expression of programmed cell death‐1 ligand 1 is a prognostic factor for malignant melanoma

Melanoma tends to be refractory to various immunotherapies because of tumor‐induced immunosuppression. To investigate the mechanism underlining the immunosuppression of melanoma patients, the authors focused on programmed cell death‐1 (PD‐1)/PD‐1 ligand 1 (PD‐L1) interaction between tumor cells and T cells.

Targeting of Tumor Cells for Human γδ T Cells by Nonpeptide Antigens

Human Vγ2/Vδ2+ γδ T cells respond to low molecular-mass nonpeptide Ags in a γδ TCR-dependent manner. Although requirements of Ag presentation have remained controversial, we have indicated that specific responses of the primary γδ T cells to pamidronate were dependent on monocytic adherent cells for Ag presentation. Here, we show that human tumor cells can efficiently present aminobisphosphonate and pyrophosphomonoester compounds to γδ T cells, inducing specific proliferation and IFN-γ production. γδ TCR dependency of the response to Ag-pulsed tumor cells was confirmed by using a Jurkat line transfected with a Vγ2/Vδ2 γδ TCR. Furthermore, γδ T cells exhibited markedly enhanced cytotoxicity against the Ag-pulsed tumor cells as compared with untreated tumor cells. Survey of a number of human tumor cell lines of different origins revealed that the majority of them became susceptible for γδ T cell-mediated cytotoxicity following the Ag pulsing except for breast cancer lines so far examined, while normal PHA blast cells remained resistant. The results not only imply a unique mode of nonpeptide Ag recognition by human γδ T cells but also may provide a novel strategic clue for immunotherapy of human malignancy.

PD-1 and LAG-3 inhibitory co-receptors act synergistically to prevent autoimmunity in mice

A new mouse model of spontaneous autoimmune disease reveals an important role for the inhibitory co-receptor LAG-3 in suppressing autoimmunity.

Requirement of Species-Specific Interactions for the Activation of Human γδ T Cells by Pamidronate

Human γδ T cells bearing Vγ2Vδ2-TCR recognize various kinds of small nonpeptide Ags, and activation of them by a nitrogen-containing bisphosphonate Ag, pamidronate, requires Ag presentation by cells other than γδ T cells, including many human tumor cells. Present results demonstrated that tumor cell lines of nonhuman origins pulsed with pamidronate failed to activate human γδ T cells without exception, whereas most if not all human tumor cell lines could do so. γδ T cells formed stable conjugates with pamidronate-pulsed human tumor cells and both conjugate formation and γδ T cell activation were inhibited significantly by anti-LFA-1 mAb, suggesting the requirement of LFA-1-mediated interaction with APC for efficient γδ T cell activation. Consistently, ICAM-1low tumor cell lines pulsed with pamidronate induced no or only weak activation of γδ T cells, whereas similarly treated ICAM-1high cell lines could activate them. One of the two ICAM-1low tumor cell lines pulsed with pamidronate induced strong γδ T cell activation after ICAM-1 gene transfer. However, another ICAM-1low human cell line as well as murine tumor cell lines pulsed with pamidronate remained totally defective in γδ T cell activation even after expression of human ICAM-1. These results suggested that activation of human γδ T cells by nonpeptide Ags required species-specific interactions in addition to LFA-1/ICAM-1-mediated cell adhesion with APC.

PD-1 deficiency results in the development of fatal myocarditis in MRL mice

The deficiency of programmed cell death 1 (PD-1, Pdcd1), a negative immuno-receptor belonging to the CD28/cytotoxic T lymphocyte antigen 4 (CTLA-4) family, can support various tissue-specific autoimmune conditions. Here, we analyzed the effect of PD-1 deficiency in MRL mice that is genetically predisposed to systemic autoimmunity. MRL-Pdcd1(-)(/-) mice developed a fatal myocarditis, which is reminiscent of CTLA-4-deficient (Ctla4(-)(/-)) mice. Massive infiltration of CD4(+) and CD8(+) T cells and myeloid cells was found in hearts of MRL-Pdcd1(-)(/-) mice concomitant with the production of high-titer auto-antibodies against cardiac myosin. In contrast to Ctla4(-)(/-) mice in which most of the CD4(+) T cells are non-specifically activated and invade various organs, T cells in the heart but not in the spleen and lymph nodes are activated in MRL-Pdcd1(-)(/-) mice, suggesting that myocarditis is mediated by antigen-specific autoimmune response. Heart infiltrating myeloid cells strongly suppressed the allogenic response of T cells in vitro, suggesting that these Mac1(+)Gr1(+) myeloid cells are phenotypically similar to myeloid suppressor cells, which can be found in tumor-bearing hosts. These findings unravel the hidden heart-specific autoimmune predisposition of MRL mice and provide MRL-Pdcd1(-)(/-) mice as a useful animal model of lymphocytic myocarditis.

Involvement of CD166 in the Activation of Human γδT Cells by Tumor Cells Sensitized with Nonpeptide Antigens

We previously reported that human Vγ2Vδ2-γδT cells were activated by many human tumor cell lines treated with pamidronate (PAM) in a γδTCR-dependent manner. In the present study, we indicated that a synthetic pyrophosphomonoester Ag, 2-methy-3-butenyl-1-pyrophosphate, could directly “sensitize” the tumor cells to activate γδT cells independently of the host metabolism, while the sensitizing effect of PAM was reported to be dependent on the pharmacological activity. Some exceptional tumor cells that failed to be sensitized by PAM were incapable of activating γδT cells by the treatment with 2-methy-3-butenyl-1-pyrophosphate either, suggesting a requirement of host factor(s) for the effective γδT cell activation in addition to the nonpeptide Ags. By screening mAbs against a large panel of tumor cell lines, we found that the expression of CD166 closely paralleled the capacity of activating γδT cells upon PAM treatment. The transfection of a CD166-negative tumor cell line with CD166 cDNA caused a marked enhancement of the capacity to activate γδ T cells following PAM treatment. On the contrary, down-regulation of the CD166 expression in a CD166-bearing tumor cell line by short hairpin RNA resulted in a significant reduction of PAM-induced γδΤ cell-stimulatory activity. γδT cells expressed CD6, a receptor of CD166, and CD6 and CD166 were recruited together to the center of synapse between γδ T cells and PAM-treated tumor cells, colocalizing with γδTCR/CD3. The results suggested that the engagement of CD6 with CD166 on tumor cells played an important role in the γδT cell activation by the tumor cells loaded with nonpeptide Ags either endogenously or exogenously.

Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway‐induced resistance to vascular endothelial growth factor receptor inhibitor

Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF‐induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

Identification of QTLs that modify peripheral neuropathy in NOD.H2b-Pdcd1-/-mice

The non-obese diabetic (NOD) mouse strain is prone to developing various autoimmune syndromes including type I diabetes mellitus (T1DM), sialadenitis, thyroiditis and pancreatitis. Although the genetic basis of T1DM has been extensively analyzed, genetic factors that modify the other autoimmune phenotypes are largely unknown. We have recently reported that NOD mice with anti-diabetogenic MHC haplotype (H-2(b)) and programmed cell death 1 (PD-1) deficiency (NOD.H2(b)-Pdcd1(-/-) mice) are protected from T1DM but develop various tissue-specific autoimmune diseases including peripheral neuropathy due to autoimmune neuritis, sialadenitis and gastritis. In the present study, we generated [(C57BL/6 x NOD.H2(b))(F1) x NOD-H2(b)](BC1)-Pdcd1(-/-) mice to screen non-MHC quantitative trait loci (QTLs) that modify autoimmune phenotypes other than T1DM. We identified seven QTLs for peripheral neuropathy and neuritis, one QTL for insulitis, four QTLs for gastritis, two QTLs for sialadenitis and seven QTLs for vasculitis throughout the genome and designated them as Annp loci for autoimmunity due to polymorphisms of non-MHC genes in NOD mice and PD-1 deficiency. Annp1, 5, 6 and 7 overlapped with reported loci for T1DM (Idd3, 9, 15 and 2, respectively), suggesting that these loci modify not only T1DM but also other autoimmune phenotypes. NOD allele was promotive at 9 of 14 Annp loci, while NOD allele was protective at the other loci. Half of Annp loci associated with a single phenotype, while the other seven loci associated with more than two phenotypes. These results indicate that NOD genetic background harbors various QTLs that modify autoimmune phenotypes either by organ-specific or by organ-non-specific manner.

Abstract A92: Effects of lenvatinib on tumor-associated macrophages enhance antitumor activity of PD-1 signal inhibitors

[Background] Lenvatinib mesylate (lenvatinib) is an orally administrated multiple receptor tyrosine kinase inhibitor that selectively inhibits the kinase activities of VEGFR1-3, FGFR1-4, KIT, PDGF-Rα and RET, which are involved in tumor angiogenesis and proliferation of several cancer types such as thyroid cancer. In order to investigate whether lenvatinib has effects on immune cell population in tumors, we examined antitumor effects and immune modulating abilities of lenvatinib in syngeneic murine tumor models. [Materials and methods] We examined antitumor activity of lenvatinib against BNL 1ME A.7R.1 murine hepatocellular carcinoma cell line in syngeneic and immune-deficient mice model. Combination treatment with lenvatinib at 10mg/kg (qd) plus anti-PD-1 mAb at 500μg/ml (twice weekly) was conducted in LL/2 murine Lewis lung carcinoma cell line syngeneic model. For immune population analyses, lenvatinib at 10mg/kg was orally administrated in BNL 1ME A.7R.1 or LL/2 mice model. On Day 7 after starting treatment, percentages of immune populations were analyzed by flow cytometer. For cytokine analysis, IFNγ in CD8+ T cells was intracellularly stained and analyzed by flow cytometer. The expression of immune related molecules in tumors was analyzed by quantitative PCR. [Results] Lenvatinib showed more potent inhibition of BNL 1ME A.7R.1 tumor growth in immune functional mice than in immune-deficient mice when given the same dose. A flow-cytometry analysis of immune populations in both BNL 1ME A.7R.1 and LL/2 tumors indicated that lenvatinib significantly decreased the percentage of tumor associated macrophages (TAM) with CD45+CD11b+Ly6GlowLy6ClowF4/80+ markers. In addition, immune inhibitory molecules were downregulated in tumors. Lenvatinib significantly increased the percentage of IFNγ-secreting CD8+ T cells in regional lymph node. In LL/2 tumor model, the combination effect of lenvatinib with anti-PD-1 mAb showed a superior antitumor activity to each monotherapy. [Conclusion] These results indicate that lenvatinib has more potent antitumor effect in immune functional environment. It was noteworthy that lenvatinib decreased TAM population in tumors. Furthermore, lenvatinib enhanced antitumor activity of PD-1 signal inhibitors with combination treatment. Citation Format: Yu Kato, Kimiyo Tabata, Yusaku Hori, Sho Tachino, Kiyoshi Okamoto, Junji Matsui. Effects of lenvatinib on tumor-associated macrophages enhance antitumor activity of PD-1 signal inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A92.

Abstract 5172: E7386, an orally active CBP/beta-catenin modulator, induces T cells infiltration into tumor and enhances antitumor activity of anti-PD-1 mAb in Wnt1 tumor syngeneic mice model

Recently, immunotherapeutic approaches have come to forefront in melanoma, non-small cell lung cancer, bladder cancer, and so on. However, sufficient benefits from these treatments have been limited. T cell infiltrating into tumor tissues is reported as one of the response biomarker candidates for the immune checkpoint inhibitors. Recent studies have shown that the activation of the Wnt/beta-catenin signaling pathway results in T cell exclusion and resistance to immune checkpoint inhibitor in melanoma patients. We established the Wnt1 tumor syngeneic mice model (Wnt1 model) by implantation of mammary adenocarcinomas isolated from MMTV-Wnt1 transgenic mice. Here we demonstrate whether E7386, a first-in-class orally active CBP/beta-catenin modulator, affects the recruitment of T cells into tumor tissues, leading to the enhancement of anti-PD-1 mAb in Wnt/beta-catenin signaling pathway activating tumor model. The mice were treated with E7386 (50 mg/kg, orally, BID) and/or anti-mouse PD-1 mAb (10 mg/kg, intraperitoneal, twice a week) for three weeks. Tumor diameters are measured with digital calipers, and the tumor volume in mm3 is calculated. Immunohistochemical (IHC) analysis was evaluated for tumor-infiltrating T cells. E7386 showed significant antitumor activity in Wnt1 model, but anti-PD-1 mAb did not. In contrast, E7386 with anti-PD-1 mAb indicated prominent antitumor activity compared to each single treatment. Enhancement of body weight loss in combination group was not observed. In IHC analysis, infiltration of T cells was limited in vehicle control group, in contrast T cell infiltration into tumors was clearly observed in both E7386 treatment group and combination group. As a result, antitumor activity of immune checkpoint inhibitor was limited in Wnt-driven tumor model and E7386 as a novel CBP/beta-catenin modulator enhanced the antitumor activity of anti-PD-1 mAb via induction of tumor-infiltrating T cells. Citation Format: Yusaku Hori, Kazuhiko Yamada, Yu Kato, Yoichi Ozawa, Takenao Odagami, Junji Matsui, Tomohiro Matsushima, Kenichi Nomoto, Hiroyuki Kouji, Takashi Owa. E7386, an orally active CBP/beta-catenin modulator, induces T cells infiltration into tumor and enhances antitumor activity of anti-PD-1 mAb in Wnt1 tumor syngeneic mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5172. doi:10.1158/1538-7445.AM2017-5172