Development of immunotherapy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common type of cancer globally. Although many different kinds of treatment are performed for HCC according to the practical guidelines, the prognosis of patients is not still satisfactory because the effects of treatments are limited for advanced tumors and the recurrence rate of HCC, even in early stages, is very high. Therefore, immunotherapy is highly anticipated as a new treatment method for HCC. For the development of a new HCC therapy, we attempted to establish immunotherapy using dendritic cells (DCs) and peptide vaccine. In several clinical trials that we performed, we confirmed that the immunotherapy was safe and well-tolerated by HCC patients. We observed that DC therapy prolonged the recurrence-free survival of patients compared with that of patients without DC infusion, as well as observing the radiological anti-tumor effect in HCC patients with peptide vaccine. In this chapter, we summarize the results of previous studies using DC and peptide vaccine, including our own data, and describe the prospects of immunotherapy for HCC. © 2015, Springer Japan. All rights reserved.[Book Chapter

C型慢性肝炎患者における肝組織中インターフェロンα/βレセプターの発現

取得学位 : 博士（医学）, 学位授与番号 : 医博乙第1490号, 学位授与年月日：平成11年4月21日,学位授与年：199

肝細胞癌に対する細胞性免疫治療に関する基礎的研究

金沢大学医薬保健研究域医学系肝細胞癌で発現が増加している癌抗原や他癌において報告されている癌関連抗原の細胞障害性T細胞(CTL)エピトープをもつ9-10アミノ酸からなるペプチドを合成した。次に肝細胞癌患者の末梢血リンパ球を用いてこれらのペプチドに対する免疫反応をenzyme linked immuno-spot (ELISPOT) assayとCTL assayで検討し、肝癌の免疫治療に有用と考えられるHLA-A24拘束性のCTLエピトープを同定した。具体的にはアルファフェトプロテイン(AFP)をはじめとして、14種類の癌関連抗原に対するエピトープが肝癌の免疫治療の候補となりうることを明らかにした。さらに38人の肝癌患者においてAFP特異的CTLと腫瘍因子などの臨床所見との関連を検討し、血中のAFPレベルや肝炎ウイルスの感染とは関係なくAFP特異的CTLの誘導が可能であることを明らかにした。末梢血におけるAFP特異的CTL数の検討では、これまでに報告されている大腸癌や悪性黒色腫の患者末梢血中に存在する癌抗原特異的CTL数と同等であり、免疫原性が低いとされていた肝細胞癌においても宿主の癌に対する免疫反応が存在することを証明した。またAFP特異的CTL数はTNM分類における腫瘍因子や進行度と有意な相関を示し、癌が進行した患者においてより顕著に反応が検出されるものの、肝癌発生初期には反応は弱く、このことが肝癌の伸展に関与している可能性を示していた。今回得られた知見はペプチドや樹状細胞を用いて肝癌に特異的なCTLを誘導することによって肝癌の免疫治療が可能であることを示唆するとともに、こうした免疫治療は肝癌の発生早期、もしくは既存の肝癌治療を行った後に再発を予防する目的で行うことが適当であることを予見するものであった。現在、今回の研究で同定したエピトープをもつペプチドとHLA-A24トランスジェニックマウスを用いて、免疫治療の効果と安全性を検討しており、一部のペプチドに関しては金沢大学医の倫理委員会における承認の下、肝癌患者における免疫治療の臨床試験を開始している。研究課題/領域番号:15790342, 研究期間(年度):2003 – 2004出典：「肝細胞癌に対する細胞性免疫治療に関する基礎的研究」研究成果報告書　課題番号15790342（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-15790342/)を加工して作

癌特異抗原を用いた肝癌の細胞性免疫療法に関する基礎的検討

金沢大学附属病院肝細胞癌患者の癌免疫療法に有用と考えられるHLA-A24拘束性細胞障害性T細胞(CTL)エピトープを16種類同定した。さらにこれらのエピトープをもつペプチドを作製し、今後の臨床試験に用いるものとして、16種類中1つのエピトープについて肝細胞癌患者に投与するための安全性と有効性を確認した。また本治療における免疫反応のモニタリングに必要な検査システムを確立した。具体的には以下の検討を行い、それぞれの結果を得た。(1)ペプチドを用いて肝癌患者のリンパ球を刺激し、インターフェロンガンマをはじめとするサイトカインの産生誘導能やT細胞の増殖能を測定し、肝癌の免疫治療に有用である可能性を持つ16種類のエピトープを同定した。本研究において同定されたペプチドは、肝癌患者末梢血リンパ球において高頻度にインターフェロンガンマ産生を誘導し、またペプチドで刺激することによって誘導したCTLはHLA-A24と癌抗原(ペプチドのアミノ酸配列を決めるもととなった抗原)を発現している肝癌細胞に対して、高い細胞障害活性を示した。(2)ヒト主要組織適合抗原(HLA)をもつ遺伝子改変マウス(HLAトランスジェニックマウス)を用いた動物モデルにおいて、上記エピトープを含む各種ペプチドを投与し、癌ワクチンとしての有用性と安全性を確認し、肝癌患者での臨床試験に用いるペプチドとして16種類のエピトープのうち1つを決定した。(3)この選定したエピトープをもつペプチドを人工的に臨床グレードで作製した。肝癌患者末梢血リンパ球から誘導した樹状細胞を同ペプチドでパルスし、患者体内に戻すことにより、本エピトープを発現している癌細胞を治療する臨床試験を行った。本研究では肝硬変の合併により肝臓の機能が低下した患者さんにおいても同治療方法が安全であることを確認した。さらに投与したペプチドに特異的な免疫反応が誘導されることを確認した。研究課題/領域番号:17790444, 研究期間(年度):2005 – 2006出典：「癌特異抗原を用いた肝癌の細胞性免疫療法に関する基礎的検討」研究成果報告書　課題番号17790444（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-17790444/)を加工して作

Expression of chondroitin-glucuronate C5-epimerase and cellular immune responses in patients with hepatocellular carcinoma

Background & Aims: Chondroitin-glucuronate C5-epimerase is an enzyme that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulphate biosynthesis. It is also identified to be a tumour-associated antigen recognized by cytotoxic T cells (CTLs) and its enhanced expression in many cancers has been reported. In the present study, we investigated the usefulness of this molecule as an immunotherapeutic target in hepatocellular carcinoma (HCC). Methods: The expression of chondroitin-glucuronate C5-epimerase in hepatoma cell lines and HCC tissues was confirmed by immunofluorescence and immunohistochemical analysis. CTL responses were investigated by several immunological techniques using peripheral blood mononuclear cells (PBMCs) or tumour-infiltrating lymphocytes. To determine the safety of immunotherapy using chondroitin-glucuronate C5-epimerase-derived peptide, 12 patients with HCC were administered s.c. vaccinations of the peptides and analysed. Results: Chondroitin-glucuronate C5-epimerase was expressed in HCC cell lines and human tissues including alpha-foetoprotein (AFP)-negative individuals. Chondroitin-glucuronate C5-epimerase-specific CTLs could be generated by stimulating PBMCs of HCC patients with peptides and they showed cytotoxicity against HCC cells expressing the protein. The frequency of CTL precursors investigated by enzyme-linked immunospot (ELISPOT) assay was 0-34 cells/3 × 10 5 PBMCs and the infiltration of interferon-gamma-producing CTLs into the tumour site was confirmed. In the vaccination study, no severe adverse events were observed and the peptide-specific CTLs were induced in 4 of 12 patients tested. Conclusions: Chondroitin-glucuronate C5-epimerase is a potential candidate for tumour antigen with immunogenicity and the peptides derived from this antigen could be useful in HCC immunotherapy. © 2012 John Wiley & Sons A/S

Efficient generation of highly immunocompetent dendritic cells from peripheral blood of patients with hepatitis C virus-related hepatocellular carcinoma

Background & aims Immunotherapy using dendritic cells (DCs) is a promising cancer therapy. The success of this therapy depends on the function of induced DCs. However, there has been no consensus on optimal conditions for DC preparation in vitro for immunotherapy of hepatocellular carcinoma (HCC) patients. To address relevant issues, we evaluated the procedures to induce DCs that efficiently function in hepatitis C virus (HCV)-related HCC. Methods We studied immunological data from 14 HCC patients. The DC preparation and the surface markers were assessed by flow cytometric analysis. Four different additional activation stimuli (Method I, medium alone; Method II, with OK-432; Method III, with IL-1β + IL-6 + TNF-α; Method IV, with IL-1β + IL-6 + TNF-α + PGE2) were tested and the functions of DCs were confirmed by examination of the ability of phagocytosis, cytokine production and allogeneic mixed lymphocyte reaction (MLR). Results The numbers of DCs induced and their cytokine production ability were not different between healthy controls and HCC patients. T-cell stimulatory activity of DCs in MLR was significantly lower in HCC patients than in healthy controls. The maturation of DCs with OK-432 boosted production of cytokines and chemokines, such as IL-2, IL-12p70, IFN-γ, TNF-α, IL-13 and MIP1α, and restored T-cell stimulatory activity of DCs in MLR. Conclusions The clinically approved compound OK-432 is a candidate for highly immunocompetent DC preparation and may be considered as a key drug for immunotherapy of HCV-related HCC patients. © 2014 Published by Elsevier B.V

Peretinoin, an acyclic retinoid, improves the hepatic gene signature of chronic hepatitis C following curative therapy of hepatocellular carcinoma

BACKGROUND: The acyclic retinoid, peretinoin, has been shown to be effective for suppressing hepatocellular carcinoma (HCC) recurrence after definitive treatment in a small-scale randomized clinical trial. However, little has been documented about the mechanism by which peretinoin exerts its inhibitory effects against recurrent HCC in humans in vivo. METHODS: Twelve hepatitis C virus-positive patients whose HCC had been eradicated through curative resection or ablation underwent liver biopsy at baseline and week 8 of treatment with either a daily dose of 300 or 600 mg peretinoin. RNA isolated from biopsy samples was subjected to gene expression profile analysis. RESULTS: Peretinoin treatment elevated the expression levels of IGFBP6, RBP1, PRB4, CEBPA, G0S2, TGM2, GPRC5A, CYP26B1, and many other retinoid target genes. Elevated expression was also observed for interferon-, Wnt-, and tumor suppressor-related genes. By contrast, decreased expression levels were found for mTOR- and tumor progression-related genes. Interestingly, gene expression profiles for week 8 of peretinoin treatment could be classified into two groups of recurrence and non-recurrence with a prediction accuracy rate of 79.6% (P<0.05). In the liver of patients with non-recurrence, expression of PDGFC and other angiogenesis genes, cancer stem cell marker genes, and genes related to tumor progression was down-regulated, while expression of genes related to hepatocyte differentiation, tumor suppression genes, and other genes related to apoptosis induction was up-regulated. CONCLUSIONS: Gene expression profiling at week 8 of peretinoin treatment could successfully predict HCC recurrence within 2 years. This study is the first to show the effect of peretinoin in suppressing HCC recurrence in vivo based on gene expression profiles and provides a molecular basis for understanding the efficacy of peretinoin

Antitumor effect after radiofrequency ablation of murine hepatoma is augmented by an active variant of CC chemokine ligand 3/macrophage inflammatory protein-1á

金沢大学医薬保健研究域医学系Several chemokines are used for immunotherapy against cancers because they can attract immune cells such as dendritic and cytotoxic T cells to augment immune responses. Radiofrequency ablation (RFA) is used to locally eliminate cancers such as hepatocellular carcinoma (HCC), renal cell carcinoma, and lung cancer. Because HCC often recurs even after an eradicative treatment with RFA, additional immunotherapy is necessary. We treated tumor-bearing mice by administering ECI301, an active variant of CC chemokine ligand 3, after RFA. Mice were injected s.c. with BNL 1ME A.7R.1, a murine hepatoma cell line, in the bilateral flank. After the tumor became palpable, RFA was done on the tumor of one flank with or without ECI301. RFA alone eliminated the treated ipsilateral tumors and retarded the growth of contralateral non-RFA-treated tumors accompanied by massive T-cell infiltration. Injection of ECI301 augmented RFA-induced antitumor effect against non-RFA-treated tumors when administered to wild-type or CCR5-deficient but not CCR1-deficient mice. ECI301 also increased CCR1-expressing CD11c+ cells in peripheral blood and RFA-treated tumors after RFA. Deficiency of CCR1 impairs accumulation of CD11c+, CD4+, and CD8+ cells in RFA-treated tumors. Furthermore, in IFN-ã-enzyme-linked immunospot assay, ECI301 augmented tumor-specific responses after RFA whereas deficiency of CCR1 abolished this augmentation. Thus, we proved that ECI301 further augments RFA-induced antitumor immune responses in a CCR1-dependent manner. ©2010 AACR

Myeloid-derived suppressor cells correlate with patient outcomes in hepatic arterial infusion chemotherapy for hepatocellular carcinoma

Hepatic arterial infusion chemotherapy (HAIC) has been employed as an alternative therapy to sorafenib for the patients with advanced hepatocellular carcinoma (HCC). In this study, we performed a comparative analysis of various immune cell responses including tumor-associated antigen (TAA)-specific T cells, regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in advanced HCC patients treated with HAIC. Thirty-six HCC patients were examined in the study. Interferon gamma enzyme-linked immunospot assays were performed to examine the frequency of TAA-specific T cells. The frequencies of Tregs and MDSCs were examined by multicolor fluorescence-activated cell sorting analysis. The treatment with HAIC using interferon (IFN)/5-fluorouracil (FU) or IFN/FU + cisplatin modulated the frequencies of various immune cells. In 22.2 % of patients, the frequency of TAA-specific T cells increased after HAIC. Although the frequency of Tregs decreased after HAIC, it was not associated with the prognosis of patients. An analysis of prognostic factors for overall survival identified diameter of the tumor (<3.0 cm), absence of major portal vein invasion, absence of distant metastasis, Union Internationale Contre Le Cancer tumor lymph node metastasis stage (I or II), neutrophil lymphocytic ratio (<2.1) and the frequency of MDSCs (<30.5 %) as factors that prolonged overall survival time after HAIC. Even in the group adjusted with progressive levels of tumors, patients with a low frequency of MDSCs had a significantly longer overall survival time. In conclusion, the frequency of MDSCs before the treatment is a prognostic factor in HAIC against HCC. © 2016 Springer-Verlag Berlin Heidelber