TW202037392A - Combination therapies comprising dendritic cells-based vaccine and immune checkpoint inhibitor - Google Patents

Abstract

The present disclosure provides a method for treating HCC comprising co-administering to the patient, dendritic cells-based vaccine in combination with an immune checkpoint inhibitor. The treatment with DC vaccine in combination with immune checkpoint inhibitor has significantly improved overall survival in subjects.

Description

包含基於樹突細胞之疫苗與免疫檢查點抑制劑之組合治療Combination therapy including dendritic cell-based vaccine and immune checkpoint inhibitor

本發明係關於醫藥領域。特定言之，本發明係關於使用基於樹突細胞之疫苗及免疫檢查點抑制劑治療癌症(較佳為肝細胞癌(HCC))。The invention relates to the field of medicine. In particular, the present invention relates to the use of dendritic cell-based vaccines and immune checkpoint inhibitors to treat cancer (preferably hepatocellular carcinoma (HCC)).

肝細胞癌(HCC)為最常見的肝膽(肝臟、膽囊及膽管)癌症。HCC之發病機制與慢性B型肝炎病毒(HBV)及C型肝炎病毒(HCV)感染以及肝硬化誘發之肝臟病狀相關。HCC之風險因素包括經B型肝炎病毒(HBV)或C型肝炎病毒(HCV)感染、酒精性肝硬化及其他肝臟病狀，諸如血色素沉著症或晚期原發性膽汁性肝硬化症(PBC)。已研發用於治療HCC之多種治療方法，包括手術切除、肝臟移植及多種非手術治療選擇，諸如射頻切除、經導管肝動脈化療栓塞、全身化療及靶向療法。Hepatocellular carcinoma (HCC) is the most common hepatobiliary (liver, gallbladder, and bile duct) cancer. The pathogenesis of HCC is related to chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection and liver disease induced by cirrhosis. HCC risk factors include hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, alcoholic cirrhosis and other liver conditions, such as hemochromatosis or advanced primary biliary cirrhosis (PBC) . A variety of treatment methods have been developed for the treatment of HCC, including surgical resection, liver transplantation, and a variety of non-surgical treatment options, such as radiofrequency resection, transcatheter hepatic artery chemoembolization, systemic chemotherapy and targeted therapy.

US 20180207253提供一種在患有癌症之患者體內引發免疫反應之方法，該方法包括向該患者投與含有活化T細胞群體之組合物，該等活化T細胞選擇性地識別患者中異常表現由GVYDGEEHSV之胺基酸序列組成之肽的癌細胞，其中該肽與MHC分子複合。US 20180258169係關於抗緊密連接蛋白1單株抗體及其醫藥組合物用於預防及/或治療肝細胞癌之用途。US 20180162941提供一種方法，該方法包含確定T細胞上PD-1之狀態，及基於某些細胞上PD-1之水準變化，確定酪胺酸激酶之有效性，且可作出關於包含較低劑量之酪胺酸激酶抑制劑與PD-1抑制劑之組合治療的指示。US 20180207253 provides a method for initiating an immune response in a patient suffering from cancer, the method comprising administering to the patient a composition containing a population of activated T cells, the activated T cells selectively identifying abnormalities in the patient by GVYDGEEHSV A cancer cell composed of a peptide of amino acid sequence, in which the peptide is complexed with MHC molecules. US 20180258169 relates to the use of anti- Claudin 1 monoclonal antibodies and pharmaceutical compositions thereof for the prevention and/or treatment of hepatocellular carcinoma. US 20180162941 provides a method that includes determining the status of PD-1 on T cells, and determining the effectiveness of tyrosine kinase based on changes in the level of PD-1 on certain cells, and making a decision about the inclusion of lower doses Indication of combined therapy of tyrosine kinase inhibitor and PD-1 inhibitor.

然而，此等治療展現有限的存活益處。HCC呈現手術治療之後的高復發速率以及對常用化學治療及靶向藥物的高耐受性，導致不佳患者存活率。However, these treatments exhibit limited survival benefits. HCC presents a high recurrence rate after surgery and high tolerance to commonly used chemotherapy and targeted drugs, resulting in poor patient survival.

本文中提供一種用於治療HCC之方法，該方法包含向患者投與基於樹突細胞之疫苗與免疫檢查點抑制劑之組合。組合例如根據本文所揭示之臨床給藥方案(根據特定給藥時程給予之特定劑量)共投與(或用於共投與)。Provided herein is a method for treating HCC, which method comprises administering a combination of a dendritic cell-based vaccine and an immune checkpoint inhibitor to a patient. The combination is, for example, co-administered (or used for co-administration) according to the clinical dosing schedule disclosed herein (a specific dose given according to a specific administration schedule).

在一個實施例中，基於樹突細胞之疫苗係以約1×10⁵ 個細胞/劑量/天至約1×10⁸ 個細胞/劑量/天範圍內之劑量投與。一個實施例包括以約1×10⁶ 個細胞/劑量/天之劑量投與基於樹突細胞之疫苗。基於樹突細胞之疫苗的特定實施例包括但不限於不成熟樹突細胞、成熟樹突細胞、骨髓樹突細胞(cDC)、漿細胞樣樹突細胞(pDC)及骨髓源性樹突細胞。In one embodiment, the dendritic cell-based vaccine is administered at a dose ranging from about 1×10 ⁵ cells/dose/day to about 1×10 ⁸ cells/dose/day. One example includes administration of a dendritic cell-based vaccine at a dose of about 1×10 ⁶ cells/dose/day. Specific examples of dendritic cell-based vaccines include, but are not limited to, immature dendritic cells, mature dendritic cells, bone marrow dendritic cells (cDC), plasmacytoid dendritic cells (pDC), and bone marrow-derived dendritic cells.

在一個實施例中，免疫檢查點抑制劑係以約50微克/劑量/天至約400微克/劑量/天範圍內之劑量投與。一個實施例包括以約100微克/劑量/天或約200微克/劑量/天之劑量投與免疫檢查點抑制劑。免疫檢查點抑制劑之某些實施例包括針對免疫檢查點蛋白質之抗體，諸如針對細胞毒性T-淋巴細胞抗原4 (CTLA-4或CD152)或計劃性細胞死亡配位體-1 (PDL-1)或計劃性細胞死亡蛋白1 (PD-1)之抗體。In one embodiment, the immune checkpoint inhibitor is administered at a dose ranging from about 50 micrograms/dose/day to about 400 micrograms/dose/day. One embodiment includes the administration of an immune checkpoint inhibitor at a dose of about 100 micrograms/dose/day or about 200 micrograms/dose/day. Some examples of immune checkpoint inhibitors include antibodies against immune checkpoint proteins, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4 or CD152) or planned cell death ligand-1 (PDL-1 ) Or planned cell death protein 1 (PD-1) antibody.

藉由輸注或注射共投與基於樹突細胞之疫苗及免疫檢查點抑制劑。基於樹突細胞之疫苗及免疫檢查點抑制劑可提供為在相同時間或在不同時間投與之獨立藥劑。共投與通常係週期性地重複，其可視需要重複例如1至35個週期。在一個實施例中，投與週期包含每隔一天投與該基於樹突細胞之疫苗及該免疫檢查點抑制劑，持續總共三個劑量。共投與可包括以相同或不同劑型同時投與治療劑(基於樹突細胞之疫苗及免疫檢查點抑制劑)，或分開投與治療劑。Co-administration of dendritic cell-based vaccines and immune checkpoint inhibitors by infusion or injection. Dendritic cell-based vaccines and immune checkpoint inhibitors can be provided as independent agents administered at the same time or at different times. Co-administration is usually repeated periodically, which may be repeated for 1 to 35 cycles as needed. In one embodiment, the administration cycle includes administration of the dendritic cell-based vaccine and the immune checkpoint inhibitor every other day for a total of three doses. Co-administration may include simultaneous administration of therapeutic agents (dendritic cell-based vaccines and immune checkpoint inhibitors) in the same or different dosage forms, or separate administration of therapeutic agents.

亦提供一種用於投與基於樹突細胞之疫苗及免疫檢查點抑制劑之組合之醫藥套組，其包含關於投與基於樹突細胞之疫苗及免疫檢查點抑制劑的印刷說明書，以及呈用於至少一個週期之劑量單位的基於樹突細胞之疫苗及免疫檢查點抑制劑之組合。A medical kit for administering a combination of a dendritic cell-based vaccine and an immune checkpoint inhibitor is also provided, which includes printed instructions on the administration of a dendritic cell-based vaccine and immune checkpoint inhibitor, and presentation A combination of a dendritic cell-based vaccine and an immune checkpoint inhibitor in a dosage unit of at least one cycle.

除非另作定義，否則本文所使用之所有技術及科學術語均具有與一般熟習本發明所屬之技術者通常所理解相同的含義。一般而言，本文所用之命名法及下文將描述之實驗方法為此項技術中熟知且常用之彼等。Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by those familiar with the present invention. Generally speaking, the nomenclature used herein and the experimental methods described below are well-known and commonly used in the art.

如本文所使用，在上下文中所使用之術語「一(a/an)」及「該」及類似參考物可理解為涵蓋單數及複數兩者。As used herein, the terms "a/an" and "the" and similar references used in the context can be understood to encompass both the singular and the plural.

如本文所使用，術語「個體」、「個人」或「患者」在本文中可互換使用，且係指脊椎動物，較佳哺乳動物，更佳人類。As used herein, the terms "individual", "individual" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, and more preferably humans.

如本文所使用，除非上下文另外清楚指示，否則術語「疾病」、「病症」及「病狀」可互換使用。As used herein, unless the context clearly dictates otherwise, the terms "disease", "condition" and "condition" are used interchangeably.

如本文所使用，「組合」係指兩個或更多個項目之間的任何結合。組合可為兩個或更多個獨立項目，諸如兩種組合物或兩個集合，可為其混合物，諸如兩個或更多個項目之單一混合物或其任何變化形式。組合之要素通常在功能上關聯或相關。As used herein, "combination" refers to any combination between two or more items. The combination may be two or more separate items, such as two compositions or two collections, may be a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of the combination are usually functionally related or related.

如本文所使用，術語「治療(treatment)」或「治療(treating)」應理解為包括在治療、緩解或改善損傷、病理學或病狀方面成功之任何標誌。此可包括諸如以下之參數：症狀減輕、緩解、減少，退化或衰退之速率減緩，使得最終退化點衰弱程度較小；改善患者的身體或心理健康；或預防疾病發作。As used herein, the term "treatment" or "treating" should be understood to include any sign of success in treating, alleviating or ameliorating an injury, pathology, or condition. This may include parameters such as: symptom relief, relief, reduction, slowing of the rate of degeneration or decline, so that the final degeneration point is less debilitating; improving the patient's physical or mental health; or preventing the onset of disease.

如本文所使用，術語「治療有效量」在參考疾病/病狀之症狀使用時係指改善、減輕或消除疾病/病狀之一或多個症狀或預防或延遲症狀發作之化合物的量及/或濃度。As used herein, the term "therapeutically effective amount" when used with reference to the symptoms of a disease/condition refers to the amount of a compound that improves, alleviates, or eliminates one or more symptoms of the disease/condition or prevents or delays the onset of symptoms and/ Or concentration.

如本文所使用，藉由「組合」或「與……組合」並不意欲暗示療法或治療劑必須同時投與及/或調配用於一起傳送，儘管此等傳送方法屬於本文所述之範疇內。組合中之治療劑可與一或多種其他額外療法或治療劑同時投與，在其之前投與或在其之後投與。治療劑或治療方案可以任何次序投與。As used herein, the use of "combination" or "combination with" is not intended to imply that therapies or therapeutic agents must be administered and/or formulated for simultaneous delivery, although these delivery methods fall within the scope of this article . The therapeutic agents in the combination can be administered simultaneously with one or more other additional therapies or therapeutic agents, before or after them. The therapeutic agents or treatment regimens can be administered in any order.

樹突細胞(DC)為人類免疫系統中最強效之抗原呈遞細胞。在不成熟狀態下，DC存在於血液及組織中，會抽取源於病毒感染、致瘤或外來細胞之抗原。當攝取可呈遞抗原後，DC經歷成熟及抗原加工並遷移至淋巴結，DC呈遞抗原至淋巴結並活化T細胞及產生介白素12 (IL-12)以促進T細胞增殖，從而觸發抗原特異性免疫反應以摧毀靶細胞。基於此等特徵，刺激腫瘤特異性免疫反應之基於DC之免疫療法已作為用於HCC之有前景的治療策略而出現(Palucka K, Banchereau J. Cancer immunotherapy via dendritic cells. Nat Rev Cancer 2012;12: 265-277 ； Shang N, Figini M, Shangguan J, Wang B, Sun C, Pan L, Ma Q, Zhang Z. Dendritic cells based immunotherapy. Am J Cancer Res 2017;7: 2091-2102 )。已進行若干臨床試驗以評估基於DC之疫苗治療HCC患者之功效，例如用自體人類腫瘤細胞或人類肝腫瘤細胞株HepG2細胞之全蛋白溶解物，以及用衍生自諸如α-胎蛋白及磷脂醯肌醇蛋白聚醣-3之已知腫瘤抗原之肽脈衝的DC (Butterfield LH, Ribas A, Potter DM, Economou JS. Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer. Cancer Immunol Immunother 2007;56: 1931-1943 ； Sawada Y, Yoshikawa T, Nobuoka D, Shirakawa H, Kuronuma T, Motomura Y, Mizuno S, Ishii H, Nakachi K, Konishi M, Nakagohri T, Takahashi S, Gotohda N, Takayama T, Yamao K, Uesaka K, Furuse J, Kinoshita T, Nakatsura T. Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival. Clin Cancer Res 2012;18: 3686-3696 )。總體而言，此等臨床試驗證明基於DC之疫苗在治療HCC患者中為安全且有前景的。然而，當前DC疫苗接種之總體結果尚未產生顯著改善之臨床結果。因此，需要新策略來提高DC疫苗接種誘導之針對HCC之免疫反應的有效性。Dendritic cells (DC) are the most potent antigen-presenting cells in the human immune system. In the immature state, DCs are present in blood and tissues, and antigens derived from virus infection, tumorigenesis, or foreign cells are extracted. After ingesting presentable antigen, DC undergoes maturation and antigen processing and migrates to lymph nodes. DC presents antigen to lymph nodes and activates T cells and produces interleukin 12 (IL-12) to promote T cell proliferation, thereby triggering antigen-specific immunity React to destroy target cells. Based on these characteristics, DC-based immunotherapy to stimulate tumor-specific immune response has emerged as a promising treatment strategy for HCC ( Palucka K, Banchereau J. Cancer immunotherapy via dendritic cells. Nat Rev Cancer 2012; 12: 265-277 ; Shang N, Figini M, Shangguan J, Wang B, Sun C, Pan L, Ma Q, Zhang Z. Dendritic cells based immunotherapy. Am J Cancer Res 2017;7: 2091-2102 ). Several clinical trials have been conducted to evaluate the efficacy of DC-based vaccines in the treatment of HCC patients, such as the use of autologous human tumor cells or human liver tumor cell line HepG2 cell whole protein lysates, and the use of proteins derived from such as α-fetoprotein and phospholipid Peptide-pulsed DC ( Butterfield LH, Ribas A, Potter DM, Economou JS. Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer. Cancer Immunol Immunother 2007;56: 1931-1943 ; Sawada Y, Yoshikawa T, Nobuoka D, Shirakawa H, Kuronuma T, Motomura Y, Mizuno S, Ishii H, Nakachi K, Konishi M, Nakagohri T, Takahashi S, Gotohda N, Takayama T, Yamao K, Uesaka K, Furuse J, Kinoshita T, Nakatsura T. Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival. Clin Cancer Res 2012;18: 3686-3696 ). Overall, these clinical trials prove that DC-based vaccines are safe and promising in the treatment of HCC patients. However, the current overall results of DC vaccination have not yet produced significantly improved clinical results. Therefore, new strategies are needed to improve the effectiveness of the immune response against HCC induced by DC vaccination.

在一個實施例中，本發明提供一種用於治療HCC之方法，該方法包含向患者共投與基於樹突細胞之疫苗與免疫檢查點抑制劑之組合。用DC疫苗與免疫檢查點抑制劑(諸如PD-1/PD-L1抗體)之組合治療會顯著提高個體之總存活率。值得注意的是，用DC疫苗及免疫檢查點抑制劑(諸如PD-1/PD-L1抗體)之組合治療係以劑量依賴性方式致使個體之總存活期比任一單獨治療長。DC疫苗與免疫檢查點抑制劑(諸如PD-L1抗體)之組合治療展現比與免疫檢查點抑制劑(諸如PD-1抗體)組合更高之總存活率。本發明證明DC疫苗與免疫檢查點抑制劑(諸如PD-1/PD-L1抗體)之組合治療具有作為HCC之新穎治療策略之極大前景。In one embodiment, the present invention provides a method for treating HCC, the method comprising co-administering a combination of a dendritic cell-based vaccine and an immune checkpoint inhibitor to a patient. Combination treatment with DC vaccine and immune checkpoint inhibitors (such as PD-1/PD-L1 antibody) can significantly increase the overall survival rate of individuals. It is worth noting that the combined treatment with DC vaccine and immune checkpoint inhibitors (such as PD-1/PD-L1 antibody) causes the overall survival of the individual to be longer than any single treatment in a dose-dependent manner. Combination therapy of DC vaccine and immune checkpoint inhibitor (such as PD-L1 antibody) exhibits a higher overall survival rate than combination with immune checkpoint inhibitor (such as PD-1 antibody). The present invention proves that the combination therapy of DC vaccine and immune checkpoint inhibitor (such as PD-1/PD-L1 antibody) has great prospects as a novel treatment strategy for HCC.

癌症患者中之樹突細胞(DC)疫苗接種旨在誘導或加強針對腫瘤抗原之有效抗腫瘤免疫反應。在一個實施例中，基於樹突細胞之疫苗係以約1×1×10⁵ 個細胞/劑量/天至約1×10⁸ 個細胞/劑量/天範圍內之劑量投與。在一個實施例中，基於樹突細胞之疫苗係以約1×10⁶ 個細胞/劑量/天之劑量投與。Dendritic cell (DC) vaccination in cancer patients aims to induce or enhance an effective anti-tumor immune response against tumor antigens. In one embodiment, the dendritic cell-based vaccine is administered at a dose ranging from about 1×1×10 ⁵ cells/dose/day to about 1×10 ⁸ cells/dose/day. In one embodiment, the dendritic cell-based vaccine is administered at a dose of about 1×10 ⁶ cells/dose/day.

在一個實施例中，基於樹突細胞之疫苗的實例包括但不限於不成熟樹突細胞、成熟樹突細胞、骨髓樹突細胞(cDC)、漿細胞樣樹突細胞(pDC)及骨髓源性樹突細胞。In one embodiment, examples of vaccines based on dendritic cells include, but are not limited to, immature dendritic cells, mature dendritic cells, bone marrow dendritic cells (cDC), plasmacytoid dendritic cells (pDC), and bone marrow-derived Dendritic cells.

在本文所提供之組合治療中，免疫檢查點抑制劑可為針對免疫檢查點蛋白之抗體，諸如針對細胞毒性T-淋巴細胞抗原4 (CTLA-4或CD152)或計劃性細胞死亡配位體-1 (PDL-1)或計劃性細胞死亡蛋白1 (PD-1)之抗體。In the combination therapy provided herein, immune checkpoint inhibitors may be antibodies against immune checkpoint proteins, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4 or CD152) or planned cell death ligand- 1 (PDL-1) or planned cell death protein 1 (PD-1) antibody.

在一個實施例中，免疫檢查點抑制劑係以約50微克/劑量/天至約400微克/劑量/天範圍內之劑量投與。在一個實施例中，免疫檢查點抑制劑係以約100微克/劑量/天或約200微克/劑量/天之劑量投與。In one embodiment, the immune checkpoint inhibitor is administered at a dose ranging from about 50 micrograms/dose/day to about 400 micrograms/dose/day. In one embodiment, the immune checkpoint inhibitor is administered at a dose of about 100 micrograms/dose/day or about 200 micrograms/dose/day.

抗CTLA4抗體係指特異性結合至細胞毒性T-淋巴組織結合蛋白4 (CTLA4)或其可溶片段且阻斷配位體與CTLA4之結合，藉此引起CTLA4之競爭性抑制及CTLA4介導之T細胞活化抑制之抑制的任何抗體。因此，抗CTLA4抗體為CTLA4抑制劑。本文提及之抗CTLA4抗體包括全長抗體及其衍生物，諸如其特異性結合至CTLA4之抗原結合片段。例示性抗CTLA4抗體包括但不限於伊匹單抗(Ipilimumab)或曲美單抗(Tremelimumab)或其衍生物或抗原結合片段。Anti-CTLA4 antibody system refers to specific binding to cytotoxic T-lymphoid tissue binding protein 4 (CTLA4) or its soluble fragments and blocking the binding of ligands to CTLA4, thereby causing competitive inhibition of CTLA4 and CTLA4-mediated T Any antibody that inhibits cell activation. Therefore, anti-CTLA4 antibodies are CTLA4 inhibitors. The anti-CTLA4 antibodies mentioned herein include full-length antibodies and derivatives thereof, such as antigen-binding fragments thereof that specifically bind to CTLA4. Exemplary anti-CTLA4 antibodies include, but are not limited to, Ipilimumab or Tremelimumab or derivatives or antigen-binding fragments thereof.

抗PD-1抗體係指特異性結合至計劃性細胞死亡蛋白1 (PD-1)或其可溶性片段且阻斷配位體與PD-1之結合，從而引起PD-1之競爭性抑制及PD-1介導之T細胞活化抑制之抑制的任何抗體。因此，抗PD-1抗體為PD-1抑制劑。本文所提及之抗PD-1抗體包括全長抗體及其衍生物，諸如其特異性結合至PD-1之抗原結合片段。例示性抗PD-1抗體包括但不限於納武單抗(Nivolumab)、MK-3475、皮立珠單抗(Pidilizumab)或其衍生物或抗原結合片段。The anti-PD-1 anti-system refers to specifically binding to planned cell death protein 1 (PD-1) or its soluble fragments and blocking the binding of ligands to PD-1, thereby causing competitive inhibition of PD-1 and PD Any antibody that inhibits the suppression of -1 mediated T cell activation. Therefore, anti-PD-1 antibodies are PD-1 inhibitors. The anti-PD-1 antibodies mentioned herein include full-length antibodies and derivatives thereof, such as antigen-binding fragments that specifically bind to PD-1. Exemplary anti-PD-1 antibodies include, but are not limited to, Nivolumab, MK-3475, Pidilizumab or derivatives or antigen-binding fragments thereof.

抗PD-L1抗體係指特異性結合至計劃性死亡配位體1 (PD-L1)或其可溶性片段且阻斷配位體與PD-1之結合，從而引起PD-1之競爭性抑制及PD-1介導之T細胞活性抑制之抑制的抗體。因此，抗PD-L1抗體為PD-1抑制劑。本文所提及之抗PD-L1抗體包括全長抗體及其衍生物，諸如其特異性結合至PD-L1之抗原結合片段。例示性抗PD-L1抗體包括但不限於BMS-936559、MPDL3280A、MEDI4736或其衍生物或抗原結合片段。The anti-PD-L1 antibody system refers to specifically binding to planned death ligand 1 (PD-L1) or its soluble fragments and blocking the binding of the ligand to PD-1, thereby causing competitive inhibition of PD-1 and Antibody that inhibits PD-1 mediated inhibition of T cell activity. Therefore, anti-PD-L1 antibodies are PD-1 inhibitors. The anti-PD-L1 antibodies mentioned herein include full-length antibodies and derivatives thereof, such as antigen-binding fragments that specifically bind to PD-L1. Exemplary anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A, MEDI4736 or derivatives or antigen-binding fragments thereof.

在一個實施例中，基於樹突細胞之疫苗及免疫檢查點抑制劑係作為抗腫瘤療法之部分組合投與。較佳藉由輸注或注射投與該組合。藉由注射或輸注投與之途徑包括靜脈內、腹膜內、肌肉內、鞘內及皮下。基於樹突細胞之疫苗及免疫檢查點抑制劑可提供為在相同時間或在不同時間投與之獨立藥劑。在一個實施例中，基於樹突細胞之疫苗及免疫檢查點抑制劑提供為在不同時間投與之獨立藥劑。當分開且在不同時間投與時，較佳投與基於樹突細胞之疫苗，隨後投與免疫檢查點抑制劑。In one embodiment, the dendritic cell-based vaccine and immune checkpoint inhibitor are administered as part of the combination of anti-tumor therapy. The combination is preferably administered by infusion or injection. The route of administration by injection or infusion includes intravenous, intraperitoneal, intramuscular, intrathecal and subcutaneous. Dendritic cell-based vaccines and immune checkpoint inhibitors can be provided as independent agents administered at the same time or at different times. In one embodiment, dendritic cell-based vaccines and immune checkpoint inhibitors are provided as independent agents to be administered at different times. When administered separately and at different times, it is preferable to administer a dendritic cell-based vaccine followed by an immune checkpoint inhibitor.

適合於向患者投與之藥物較佳呈用於輸注或注射之液體形式。一般而言，藥劑通常包含醫藥學上可接受之載劑。如本文所使用，術語「醫藥學上可接受」意謂由美國藥典(Pharmacopeia)或另一公認藥典中所列之政府監管機構批准適用於動物，尤其用於人類。術語「載劑」係指與治療劑一起投與之稀釋劑、佐劑、賦形劑或媒劑。此類醫藥載劑可為無菌液體，諸如水及油，包括石油、動物、植物或合成來源之油，諸如花生油、大豆油、礦物油、芝麻油及其類似物。水或水溶液生理鹽水及右旋糖及甘油水溶液可用作載劑，尤其用於可注射溶液。The drugs suitable for administration to patients are preferably in liquid form for infusion or injection. Generally speaking, the medicament usually contains a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means that it is approved for use in animals, especially humans, by a government regulatory agency listed in the United States Pharmacopeia (Pharmacopeia) or another recognized pharmacopeia. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solutions, physiological saline and aqueous dextrose and glycerol solutions can be used as carriers, especially for injectable solutions.

在一個實施例中，共投與通常係週期性地重複，其可視需要重複例如1至35個週期。在一個實施例中，投與週期包含每隔一天投與基於樹突細胞之疫苗及免疫檢查點抑制劑，持續總共三個劑量。In one embodiment, co-administration is usually repeated periodically, which may be repeated for 1 to 35 cycles as needed. In one embodiment, the administration cycle includes administration of the dendritic cell-based vaccine and immune checkpoint inhibitor every other day for a total of three doses.

共投與可包括以相同或不同劑型同時投與治療劑(基於樹突細胞之疫苗及免疫檢查點抑制劑)或分開投與治療劑。舉例而言，基於樹突細胞之疫苗可與免疫檢查點抑制劑同時投與。或者，基於樹突細胞之疫苗可與免疫檢查點抑制劑組合投與，其中基於樹突細胞之疫苗及免疫檢查點抑制劑兩者經調配用於分開投與且係同時或依序地投與。舉例而言，可首先投與基於樹突細胞之疫苗，隨後投與免疫檢查點抑制劑。或者，可首先投與免疫檢查點抑制劑，隨後投與基於樹突細胞之疫苗。Co-administration may include simultaneous administration of therapeutic agents (dendritic cell-based vaccines and immune checkpoint inhibitors) in the same or different dosage forms or separate administration of therapeutic agents. For example, vaccines based on dendritic cells can be administered simultaneously with immune checkpoint inhibitors. Alternatively, the dendritic cell-based vaccine can be administered in combination with an immune checkpoint inhibitor, wherein both the dendritic cell-based vaccine and the immune checkpoint inhibitor are formulated for separate administration and are administered simultaneously or sequentially . For example, a vaccine based on dendritic cells can be administered first, followed by immune checkpoint inhibitors. Alternatively, the immune checkpoint inhibitor can be administered first, followed by the dendritic cell-based vaccine.

在本發明之另一態樣中，提供一種用於投與基於樹突細胞之疫苗與免疫檢查點抑制劑之組合之醫藥套組，該套組包含關於根據上文所陳述之給藥時程投與基於樹突細胞之疫苗及免疫檢查點抑制劑的印刷說明書，以及呈用於至少一個週期之劑量單位的基於樹突細胞之疫苗及免疫檢查點抑制劑之組合，其中各劑量單位含有適量的如上文所定義之用於治療的基於樹突細胞之疫苗及免疫檢查點抑制劑。In another aspect of the present invention, there is provided a medical kit for administering a combination of a dendritic cell-based vaccine and an immune checkpoint inhibitor, the kit containing information about the administration schedule according to the above statement Printed instructions for administration of dendritic cell-based vaccines and immune checkpoint inhibitors, and a combination of dendritic cell-based vaccines and immune checkpoint inhibitors in dosage units for at least one cycle, wherein each dosage unit contains an appropriate amount Dendritic cell-based vaccines and immune checkpoint inhibitors for treatment as defined above.

共投與可在最大耐受劑量內連續或週期性地進行。Co-administration can be carried out continuously or periodically within the maximum tolerated dose.

儘管出於清楚理解之目的已藉助於說明及實例相當詳細地提供本發明，但對熟習此項技術者將顯而易見的是，可在不脫離本發明之精神或範疇的情況下實踐各種改變及修改。因此，前述描述及實例不應解釋為限制性的。實例 Although the present invention has been provided in considerable detail by means of descriptions and examples for the purpose of clear understanding, it will be obvious to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the present invention . Therefore, the foregoing description and examples should not be construed as limiting. Instance

材料及方法Materials and methods

原位In situ HCCHCC 小鼠模型之建立Establishment of mouse model

如所描述地建立原位HCC小鼠模型。³¹ 簡言之，用異氟醚麻醉8週齡免疫勝任型C57BL/6雄性小鼠且對其進行中線剖腹術。將購自Cell Lines Service (Eppelheim, Germany)且維持在DMEM培養基(Invitrogen, Carlsbad, CA, USA)(補充有10%胎牛血清(FBS) (Gibco, Grand Island, NY, USA)及1×青黴素/鏈黴素(P/S) (Invitrogen))中之2×10⁶ 個小鼠肝腫瘤Hep-55.1C細胞直接注入至小鼠之左肝葉中。在止血之後，使腹部在兩層中閉合。在手術之後，追蹤小鼠之總存活率且在小鼠死亡時記錄小鼠腫瘤負荷。所有動物實驗均在China Medical University, Taichung, Taiwan之機構動物護理及使用委員會之批准下進行。An orthotopic HCC mouse model was established as described. ³¹ Briefly, 8-week-old immunized competent C57BL/6 male mice were anesthetized with isoflurane and subjected to midline laparotomy. Will be purchased from Cell Lines Service (Eppelheim, Germany) and maintained in DMEM medium (Invitrogen, Carlsbad, CA, USA) (supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1× penicillin 2×10 ⁶ mouse liver tumor Hep-55.1C cells in /streptomycin (P/S) (Invitrogen)) were directly injected into the left liver lobe of the mouse. After hemostasis, close the abdomen in two layers. After the operation, the overall survival rate of the mice was tracked and the tumor burden of the mice was recorded when the mice died. All animal experiments were conducted under the approval of the Institutional Animal Care and Use Committee of China Medical University, Taichung, Taiwan.

腫瘤體積及組織病理學之量測Measurement of tumor volume and histopathology

當小鼠死亡時，記錄小鼠之體重且分離肝臟用於成像。腫瘤體積係根據等式V = 1/2 (L × W² )計算，其中V為腫瘤體積，L為長度且W為寬度。為了評估腫瘤組織病理學，將福馬林固定及石蠟包埋之肝臟組織切成4 μm厚以用於蘇木精及曙紅(H&E)染色。When the mouse died, the weight of the mouse was recorded and the liver was isolated for imaging. The tumor volume is calculated according to the equation V = 1/2 (L × W ² ), where V is the tumor volume, L is the length and W is the width. In order to evaluate the tumor histopathology, the formalin-fixed and paraffin-embedded liver tissue was cut into 4 μm thick for hematoxylin and eosin (H&E) staining.

HCCHCC 細胞溶解物脈衝之成熟Maturation of lysate pulse DC (BMDC)DC (BMDC) 之生成The generation

在此研究中，DC來源於小鼠骨髓。首先，骨髓自6週齡C57BL/6小鼠之股骨及脛骨獲得且用膠原蛋白酶消化，耗盡紅血球，穿過100 μm過濾器，且隨後離心以收集細胞集結粒。隨後，將細胞集結粒再懸浮且以2×10⁵ 個細胞/毫升之密度在補充有10% FBS (Gibco)、1× P/S (Invitrogen)、1×最低必需培養基非必需胺基酸(Invitrogen)、1 mM丙酮酸鈉(Invitrogen)、100 ng/mL人類顆粒球巨噬細胞群落刺激因子(GM-CSF)(Sino Biological Inc., Beijing, China)及10 ng/mL介白素(IL)-4 (Sino Biological Inc.)之RPMI-1640培養基(Invitrogen)中在37℃下在潮濕的5% CO₂ 氛圍中培養6天。培養基及細胞介素在培養第3天再新。在第6天，自培養物中之非黏著及鬆散黏著細胞收集不成熟DC (IMDC)。為了產生BMDC，隨後將IMDC以1×10⁶ 個細胞/毫升之密度在添加有1 mg凍融Hep-55.1C腫瘤細胞溶解物之上述培養基中培養30分鐘，隨後添加50 ng/mL脂多醣(LPS) (Sigma, Louis, MO, USA)後再維持一天。在第7天，所有培養細胞收集為BMDC且用作DC疫苗。In this study, DC was derived from mouse bone marrow. First, bone marrow was obtained from the femur and tibia of 6-week-old C57BL/6 mice and digested with collagenase to deplete red blood cells, passed through a 100 μm filter, and then centrifuged to collect cell aggregates. Subsequently, the pellet was resuspended and cells in / ml of the density of 2 × 10 ⁵ cells with a 10% FBS (Gibco), 1 × P / S (Invitrogen) supplemented, 1 × nonessential amino acids Minimal Essential Medium ( Invitrogen), 1 mM sodium pyruvate (Invitrogen), 100 ng/mL human granulocyte macrophage colony stimulating factor (GM-CSF) (Sino Biological Inc., Beijing, China), and 10 ng/mL interleukin (IL )-4 (Sino Biological Inc.) in RPMI-1640 medium (Invitrogen) at 37°C in a humid 5% CO ₂ atmosphere for 6 days. The medium and cytokines were renewed on the third day of culture. On day 6, immature DC (IMDC) was collected from non-adherent and loosely adherent cells in the culture. In order to produce BMDC, IMDC was subsequently cultured at a density of 1×10 ⁶ cells/ml in the above medium supplemented with 1 mg of freeze-thaw Hep-55.1C tumor cell lysate for 30 minutes, and then 50 ng/mL lipopolysaccharide ( LPS) (Sigma, Louis, MO, USA) for another day. On day 7, all cultured cells were collected as BMDC and used as DC vaccine.

DCDC 表型之流動式細胞測量術分析Phenotype analysis by flow cytometry

將IMDC及BMDC用磷酸鹽緩衝鹽水(PBS)洗滌，等分成份(5×10⁵ 個細胞/100微升)，且隨後在暗處在室溫下用購自BD Biosciences (San Jose, CA, USA)之以下抗體染色30分鐘，最終濃度為5 μg/mL：螢光異硫氰酸鹽(FITC)結合之CD11c (553801)、FITC結合之CD40 (553790)、FITC結合之CD80 (553768)及FITC結合之CD86 (553691)。作為陰性或無抗體對照組，細胞亦用對應FITC結合之同型匹配對照抗體染色或保持未染色。在染色之後，用PBS洗滌細胞兩次且藉由BD LSRII流式細胞儀(BD Biosciences)分析。使用FlowJo軟體(Tree Star, San Carlos, CA, USA)進行資料分析。將針對FITC-CD11c呈陽性之細胞視為已成功地自骨髓細胞分化之DC。將針對FITC-CD40、FITC-CD80及FITC-CD86呈陽性之細胞視為已經歷成功成熟之DC。The IMDC and BMDC were washed with phosphate buffered saline (PBS), aliquoted (5×10 ⁵ cells/100 microliters), and then used in the dark at room temperature purchased from BD Biosciences (San Jose, CA, USA) the following antibodies were stained for 30 minutes at a final concentration of 5 μg/mL: fluorescent isothiocyanate (FITC) bound CD11c (553801), FITC bound CD40 (553790), FITC bound CD80 (553768) and FITC combined with CD86 (553691). As a negative or no antibody control group, the cells were also stained with an isotype-matched control antibody corresponding to FITC binding or kept unstained. After staining, the cells were washed twice with PBS and analyzed by BD LSRII flow cytometer (BD Biosciences). Use FlowJo software (Tree Star, San Carlos, CA, USA) for data analysis. Cells that are positive for FITC-CD11c are regarded as DCs that have successfully differentiated from bone marrow cells. Cells that are positive for FITC-CD40, FITC-CD80 and FITC-CD86 are regarded as DCs that have undergone successful maturation.

FITCFITC 結合之聚葡萄糖Bound polydextrose (FITC-(FITC- 聚葡萄糖Polydextrose )) 攝取分析Uptake analysis

將IMDC及BMDC用1 mg/mL FITC-聚葡萄糖(MW4000；Sigma)以1×10⁶ 個細胞/毫升之密度在補充有10% FBS (Gibco)及1× P/S (Invitrogen)之RPMI-1640培養基(Invitrogen)中在暗處在37℃下培育30分鐘以允許吞噬作用，或在冰上以停止吞噬作用，作為陰性對照。培育之後，將細胞用冰冷PBS洗滌兩次且藉由BD LSRII流式細胞儀(BD Biosciences)分析。使用FlowJo軟體(Tree Star)進行資料分析。將FITC陽性細胞視為已成功吞噬聚葡萄糖之細胞。獨立地進行三次實驗。Use 1 mg/mL FITC-polydextrose (MW4000; Sigma) for IMDC and BMDC at a density of 1×10 ⁶ cells/ml supplemented with 10% FBS (Gibco) and 1× P/S (Invitrogen) RPMI- Incubate in 1640 medium (Invitrogen) in the dark at 37°C for 30 minutes to allow phagocytosis, or on ice to stop phagocytosis, as a negative control. After incubation, the cells were washed twice with ice-cold PBS and analyzed by BD LSRII flow cytometer (BD Biosciences). Use FlowJo software (Tree Star) for data analysis. FITC-positive cells were regarded as cells that had successfully swallowed polydextrose. Three experiments were performed independently.

IL-12IL-12 產生之偵測Generated detection

以1×10⁶ 個細胞/毫升之密度在補充有10% FBS (Gibco)及1× P/S (Invitrogen)之RPMI-1640培養基(Invitrogen)中培養IMDC及BMDC。在培養1天之後，收集上清液且藉由遵循製造商之說明書使用小鼠IL-12 (p70) ELISA Set (BD Biosciences)針對IL-12 p70進行酶聯結免疫吸附劑分析法(ELISA)來量測。一式三份地獨立進行三次實驗。IMDC and BMDC were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS (Gibco) and 1×P/S (Invitrogen) at a density of 1×10 ⁶ cells/ml. After culturing for 1 day, collect the supernatant and perform an enzyme-linked immunosorbent assay (ELISA) against IL-12 p70 using the mouse IL-12 (p70) ELISA Set (BD Biosciences) following the manufacturer’s instructions. Measure. Three experiments were performed independently in triplicate.

TT 細胞增殖分析Cell proliferation analysis

為了分離T細胞，使用與關於骨髓細胞在上文所描述相同之步驟自6週齡C57BL/6小鼠獲得脾細胞，隨後藉由用Ficoll-Paque PLUS (密度1.077 g/mL；GE Healthcare, Uppsala, Sweden)之密度梯度離心分離。DC及T細胞之共培養係如下進行：將細胞培養***物(孔徑0.4 μm；Falcon, Oxnard, CA, USA)置放於24孔盤之各孔上，隨後將DC及T細胞接種至孔中且分別以2×10⁵ 個細胞/孔及1×10⁵ 個細胞/***物之密度***於補充有10% FBS (Gibco)及1× P/S (Invitrogen)之RPMI-1640培養基(Invitrogen)中。在培養3天之後，從各***物收集T細胞且立即藉由Countess Automated Cell Counter (Invitrogen)計數。一式三份地獨立進行三次實驗。To isolate T cells, spleen cells were obtained from 6-week-old C57BL/6 mice using the same procedure as described above for bone marrow cells, followed by Ficoll-Paque PLUS (density 1.077 g/mL; GE Healthcare, Uppsala , Sweden) density gradient centrifugation. The co-culture of DC and T cells is performed as follows: cell culture inserts (pore size 0.4 μm; Falcon, Oxnard, CA, USA) are placed on each well of a 24-well plate, and then DC and T cells are seeded into the wells And insert into RPMI-1640 medium (Invitrogen) supplemented with 10% FBS (Gibco) and 1× P/S (Invitrogen) at a density of 2×10 ⁵ cells/well and 1×10 ⁵ cells/insert respectively in. After 3 days of culture, T cells were collected from each insert and immediately counted by Countess Automated Cell Counter (Invitrogen). Three experiments were performed independently in triplicate.

BMDCBMDC 及and PD-1/PD-L1PD-1/PD-L1 抗體之活體內投與In vivo administration of antibodies

如上文所描述地形成BMDC。具有嚴格品質控制措施之免疫檢查點抑制劑、InVivoPlus抗小鼠PD-1 (BP0146)及PD-L1 (BP0101)單株抗體係購自Bio X Cell (West Lebanon, NH, USA)。在腫瘤細胞注射後第7天，將原位HCC小鼠隨機分成以下10個治療組(6隻小鼠/組)中之一者：媒劑對照組、BMDC (1×10⁶ 個細胞/劑量)、抗PD-1 (100微克/劑量)、抗PD-1 (200微克/劑量)、抗PD-L1 (100微克/劑量)、抗PD-L1 (200微克/劑量)、BMDC (1×10⁶ 個細胞/劑量)加抗PD-1(100微克/劑量)、BMDC (1×10⁶ 個細胞/劑量)加抗PD-1 (200微克/劑量)、BMDC (1×10⁶ 個細胞/劑量)加抗PD-L1 (100微克/劑量)及BMDC (1×10⁶ 個細胞/劑量)加抗PD-L1 (200微克/劑量)治療組。將BMDC皮下注射至小鼠之腹股溝區域(淋巴結附近)中。將抗PD-1抗體及抗PD-L1抗體經腹膜內注射至小鼠中。將無菌PBS用作媒劑對照且經皮下及腹膜內兩者注射至對照小鼠中，以及分別經腹膜內及皮下注射至BMDC治療之小鼠及抗PD-1/抗PD-L1治療之小鼠中。在腫瘤細胞注射之後第7天開始所有治療且在各組小鼠中每隔一天重複，持續總共三個劑量。在治療之後，追蹤小鼠直至死亡時間以確定存活天數。The BMDC is formed as described above. Immune checkpoint inhibitors with strict quality control measures, InVivoPlus anti-mouse PD-1 (BP0146) and PD-L1 (BP0101) monoclonal antibody systems were purchased from Bio X Cell (West Lebanon, NH, USA). On the 7th day after tumor cell injection, the orthotopic HCC mice were randomly divided into one of the following 10 treatment groups (6 mice/group): vehicle control group, BMDC (1×10 ⁶ cells/dose) ), anti-PD-1 (100μg/dose), anti-PD-1 (200μg/dose), anti-PD-L1 (100μg/dose), anti-PD-L1 (200μg/dose), BMDC (1× 10 ⁶ cells/dose) plus anti-PD-1 (100 μg/dose), BMDC (1×10 ⁶ cells/dose) plus anti-PD-1 (200 μg/dose), BMDC (1×10 ⁶ cells) /Dose) plus anti-PD-L1 (100μg/dose) and BMDC (1×10 ⁶ cells/dose) plus anti-PD-L1 (200μg/dose) treatment group. BMDC was injected subcutaneously into the inguinal area (near the lymph nodes) of the mice. Anti-PD-1 antibody and anti-PD-L1 antibody were injected intraperitoneally into mice. Sterile PBS was used as a vehicle control and injected subcutaneously and intraperitoneally into control mice, and intraperitoneally and subcutaneously injected into BMDC-treated mice and anti-PD-1/anti-PD-L1 treated mice, respectively. In the mouse. All treatments were started on the 7th day after tumor cell injection and repeated every other day in each group of mice for a total of three doses. After treatment, the mice were tracked until death to determine the number of days to survive.

統計分析Statistical Analysis

IMDC與BMDC之間的攝取聚葡萄糖、產生IL-12及刺激T細胞增殖之能力的差異顯著性係藉由未配對t -測試測定。資料表示為三個獨立實驗之平均值±平均值標準差(SEM)誤差槓。不同治療組小鼠之間的總存活率之差異顯著性係藉由克魯斯卡爾-沃利斯單因素方差分析(Kruskal-Wallis one-way ANOVA)繼之以鄧氏多重比較測試(Dunn's multiple comparisons test)來測定。將小於0.05之P 值視為顯著的(*P ＜ 0.05，**P ＜ 0.01，***P ＜ 0.001)。實例 1 原位 HCC 小鼠在肝臟中產生腫瘤且在接種腫瘤細胞之後約 32 至 38 天死亡 The significance of the difference in the ability of IMDC and BMDC to take up polydextrose, produce IL-12, and stimulate T cell proliferation was determined by an unpaired t -test. The data is expressed as the mean±standard deviation of the mean (SEM) error bars of three independent experiments. The significant difference in overall survival rates between mice in different treatment groups was determined by Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparison test (Dunn's multiple comparison test). comparisons test) to determine. P value less than 0.05 is considered significant (* P <0.05, ** P <0.01, *** P <0.001). Example 1 HCC mice in situ developed tumors in the liver and died about 32 to 38 days after tumor cell inoculation

如材料及方法部分中所述地建立原位HCC小鼠模型(圖1)。如圖2A中所示，原位HCC小鼠(Hep-55.1C小鼠，n=6)在接種腫瘤細胞之後分別具有36 (SEM，1.00)及36.5 (範圍，32至38)天之平均值及中值存活時間。當小鼠死亡時，觀察到在所有六隻小鼠之肝臟中原位地產生HCC腫瘤(圖2B)。腫瘤體積與體重之比(平均值±SEM (中值，範圍)為215.90±11.02 mm³ /g (217.3，178.4至248.5) (圖2C)。藉由H&E染色評估腫瘤組織病理學(圖2D)。實例 2  BMDC 展示適當形態及表型 The in situ HCC mouse model was established as described in the materials and methods section (Figure 1). As shown in Figure 2A, in situ HCC mice (Hep-55.1C mice, n=6) had an average of 36 (SEM, 1.00) and 36.5 (range, 32 to 38) days after tumor cell inoculation, respectively And median survival time. When the mice died, it was observed that HCC tumors were produced in situ in the livers of all six mice (Figure 2B). The ratio of tumor volume to body weight (mean±SEM (median, range) is 215.90±11.02 mm ³ /g (217.3, 178.4 to 248.5) (Figure 2C). Tumor histopathology was evaluated by H&E staining (Figure 2D) example 2 BMDC show appropriate morphology and phenotype

BMDC係如材料及方法部分中所描述地形成(圖3A)。如圖3B中所示，與培養第1天相比，細胞在第3天逐漸增加且開始在懸浮液中形成群落。在培養第6天，細胞體積明顯擴大且懸浮細胞開始形成樹突突起(一種經典樹突細胞形態)，變成IMDC。在與Hep-55.1C腫瘤細胞溶解物及LPS一起培育隔天(第7天)之後，IMDC成熟為具有進一步伸長之樹突突起之BMDC。BMDC is formed as described in the materials and methods section (Figure 3A). As shown in Figure 3B, the cells gradually increased on the 3rd day and started to form colonies in the suspension compared to the first day of culture. On the 6th day of culture, the cell volume expanded significantly and the suspended cells began to form dendritic processes (a classic dendritic cell morphology), becoming IMDC. After incubating with Hep-55.1C tumor cell lysate and LPS on the next day (day 7), IMDC matured into BMDC with further elongated dendritic processes.

為了評估BMDC之表型，進行流動式細胞測量術以分析DC表面標記物(包括標識標記物CD11c及成熟標記物CD40、CD80及CD86)之表現。如圖4中所示，與表現高含量之此四種分子之BMDC相比，IMDC表現高含量之CD11c但表現低含量之CD40、CD80及CD86。資料指示吾人製備之BMDC具有高純度及成熟度。實例 3  BMDC 展現具有減少之抗原攝取及增強的產生 IL-12 及促進 T 細胞增殖之能力的最佳成熟 In order to evaluate the phenotype of BMDC, flow cytometry was performed to analyze the performance of DC surface markers (including identification markers CD11c and mature markers CD40, CD80 and CD86). As shown in FIG. 4, compared to BMDC, which exhibits high content of these four molecules, IMDC exhibits high content of CD11c but low content of CD40, CD80, and CD86. The data indicate that the BMDC prepared by us has high purity and maturity. Example 3 BMDC exhibits optimal maturation with reduced antigen uptake and enhanced ability to produce IL-12 and promote T cell proliferation

DC成熟過程與DC攝取抗原之能力的損失相關。¹² 為了比較IMDC與BMDC之間的抗原攝取能力，將細胞與FITC-聚葡萄糖一起培育，隨後進行流動式細胞測量術分析。如所預期，與IMDC相比，BMDC展現顯著降低水準之FITC-聚葡萄糖攝取(平均值±SEM，2.60±0.60%相對於15.25±0.15%；P =0.0023) (圖5A及圖5B)。The DC maturation process is related to the loss of the ability of the DC to take up antigen. ¹² In order to compare the antigen uptake ability between IMDC and BMDC, cells were incubated with FITC-polydextrose, and then analyzed by flow cytometry. As expected, BMDC exhibited significantly reduced levels of FITC-polydextrose uptake (mean ± SEM, 2.60 ± 0.60% vs. 15.25 ± 0.15%; P = 0.0023) compared to IMDC (Figure 5A and Figure 5B).

成熟DC可合成高水準之IL-12，其在DC與T細胞之間的接觸期間介導T細胞活化及增殖。¹¹ 隨後，吾人評估BMDC分泌IL-12及刺激T細胞增殖之能力。如圖5C中所示，與IMDC相比，IL-12濃度在BMDC之培養上清液中顯著升高(平均值±SEM，5078.0±73.7 pg/mL相對於166.3±25.7 pg/mL；P ＜0.001)。當與T細胞共培養時，BMDC相比於IMDC顯著提高T細胞增殖水準(平均值±SEM，89.5±4.5×10² 個細胞相對於8.7±0.2×10² 個細胞；P =0.0031)(圖5D)。總體而言，此等結果指示吾人製備之BMDC具有最佳成熟及功能。實例 4  BMDC 及 PD-1/PD-L1 抗體之組合治療使得總存活期比原位 HCC 小鼠中之任一單獨治療長 Mature DC can synthesize high levels of IL-12, which mediate T cell activation and proliferation during the contact between DC and T cells. ¹¹ Later, we evaluated the ability of BMDC to secrete IL-12 and stimulate T cell proliferation. As shown in Figure 5C, compared with IMDC, the IL-12 concentration in the culture supernatant of BMDC was significantly increased (mean ± SEM, 5078.0 ± 73.7 pg/mL vs. 166.3 ± 25.7 pg/mL; P < 0.001). When co-cultured with T cells, BMDC significantly increased the proliferation level of T cells compared to IMDC (mean±SEM, 89.5±4.5×10 ² cells vs. 8.7±0.2×10 ² cells; P =0.0031) (Figure 5D). Overall, these results indicate that the BMDC prepared by us has the best maturity and function. Example 4 The combined treatment of BMDC and PD-1/PD-L1 antibody makes the overall survival longer than any single treatment in orthotopic HCC mice

為了評估BMDC與PD-1/PD-L1抗體之組合用於治療HCC之功效，以1天間隔向原位HCC小鼠(6隻小鼠/組)投與總共三個劑量之BMDC (1×10⁶ 個細胞/劑量)及/或PD-1/PD-L1抗體(100或200微克/劑量)且追蹤存活(圖6A)。如圖6B中所示，相比於以下各組，用BMDC或抗PD-1/PD-L1治療之小鼠組具有顯著提高之總存活期(天數，平均值±SEM(中值，範圍)：小鼠對照組(BMDC (1×10⁶ 個細胞/劑量)：44.33±0.95 (44.0，42至48)；抗PD-L1 (100微克/劑量)：43.80±1.93 (45.0，38至49)；抗PD-1 (200微克/劑量)：43.50±4.50 (43.5，38至48)；抗PD-L1 (100微克/劑量)：42.00±2.85 (42.5，35至48)；抗PD-L1 (200微克/劑量)：45.80±0.58 (45.0，45至48)；對照組：36.00±1.00 (36.5，32至38)。在治療之後的38天時，對照組中之所有小鼠均已死亡，而幾乎所有用BMDC或抗PD-1/PD-L1治療之小鼠仍存活且具有約48至49天之最長存活時間。然而，對於抗PD-1/PD-L1治療，未觀測到對小鼠之總存活期之明顯劑量依賴性影響。In order to evaluate the efficacy of the combination of BMDC and PD-1/PD-L1 antibody for the treatment of HCC, a total of three doses of BMDC (1×1) were administered to orthotopic HCC mice (6 mice/group) at 1 day intervals. 10 ⁶ cells / dose) and / or PD-1 / PD-L1 antibody (100 or 200 ug / dose) and tracking survival (FIG. 6A). As shown in Figure 6B, compared with the following groups, the mouse group treated with BMDC or anti-PD-1/PD-L1 had significantly improved overall survival (days, mean±SEM (median, range) : Mouse control group (BMDC (1×10 ⁶ cells/dose): 44.33±0.95 (44.0, 42 to 48); anti-PD-L1 (100 μg/dose): 43.80±1.93 (45.0, 38 to 49) ; Anti-PD-1 (200μg/dose): 43.50±4.50 (43.5, 38 to 48); Anti-PD-L1 (100μg/dose): 42.00±2.85 (42.5, 35 to 48); Anti-PD-L1 ( 200μg/dose): 45.80±0.58 (45.0, 45 to 48); control group: 36.00±1.00 (36.5, 32 to 38). At 38 days after treatment, all mice in the control group had died. Almost all mice treated with BMDC or anti-PD-1/PD-L1 survived and had the longest survival time of about 48 to 49 days. However, for anti-PD-1/PD-L1 treatment, no pair of mice was observed Obvious dose-dependent effect on the overall survival of mice.

值得注意的是，與任一單獨治療(BMDC+抗PD-1 (200微克/劑量)：49.75±2.92 (49.5，43至57)；BMDC+抗PD-L1 (100微克/劑量)：50.20±2.13 (51.0，44至56)；BMDC+抗PD-L1 (200微克/劑量)：55.25±4.13 (52.5，49至67)相比，BMDC及抗PD-1/PD-L1之組合治療可進一步延長小鼠之總存活期；除了BMDC+抗PD-1(100微克/劑量)治療之程度較低(44.00±1.00 (44.0，41至47)(圖6B)。小鼠之最長存活時間自藉由BMDC (1×10⁶ 個細胞/劑量)、抗PD-1(200微克/劑量)或抗PD-L1 (100或200微克/劑量)單一治療之48天，延長至藉由BMDC+抗PD-1 (200微克/劑量)之57天、藉由BMDC+抗PD-L1 (100微克/劑量)之56天及藉由BMDC+抗PD-L1 (200微克/劑量)組合治療之67天。此外，BMDC與抗PD-1/PD-L1之組合治療以劑量依賴性方式延長小鼠之總存活期。用BMDC及抗PD-L1治療之小鼠組比用BMDC及抗PD-1治療之彼等小鼠展現更佳總存活期。實例 5 臨床試驗研究 It is worth noting that with either single treatment (BMDC+anti-PD-1 (200μg/dose): 49.75±2.92 (49.5, 43 to 57); BMDC+anti-PD-L1 (100μg/dose): 50.20±2.13 ( 51.0, 44 to 56); BMDC + anti-PD-L1 (200 μg/dose): 55.25±4.13 (52.5, 49 to 67) Compared with the combination treatment of BMDC and anti-PD-1/PD-L1, the mice can be further prolonged The overall survival time of mice; except for BMDC+anti-PD-1 (100 micrograms/dose), the treatment degree is low (44.00±1.00 (44.0, 41 to 47) (Figure 6B). The longest survival time of mice is free from BMDC (1 ×10 ⁶ cells/dose), anti-PD-1 (200 micrograms/dose) or anti-PD-L1 (100 or 200 micrograms/dose) for 48 days of monotherapy, extended to BMDC + anti-PD-1 (200 micrograms) /Dose) 57 days, 56 days with BMDC+anti-PD-L1 (100μg/dose) and 67 days with BMDC+anti-PD-L1 (200μg/dose) combination treatment. In addition, BMDC and anti-PD- The 1/PD-L1 combination treatment prolongs the overall survival of mice in a dose-dependent manner. The group of mice treated with BMDC and anti-PD-L1 performed better than those treated with BMDC and anti-PD-1 Overall survival. Example 5 Clinical trial study

進行臨床試驗研究以評估BMDC及抗PD-1/PD-L1之組合治療之功效。在組合治療中，以約1×10⁵ 個細胞/劑量/天至約1×10⁸ 個細胞/劑量/天範圍內之劑量向HCC患者投與BMDC，隨後以約50微克/劑量/天至約400微克/劑量/天範圍內之劑量的抗PD-L1或抗PD-1進行治療。在第一天，個體接受初打疫苗劑量；隨後將在6週後接受兩個追打疫苗劑量。每週將獲取末梢血液以藉由四聚體分析監測針對各肽之免疫反應。抗PD-1療法在個體之最後一次樹突細胞疫苗之後5至8週開始。初步評估顯示接受組合治療之患者展現顯著腫瘤萎縮。A clinical trial study was conducted to evaluate the efficacy of the combination therapy of BMDC and anti-PD-1/PD-L1. In combination therapy, BMDC is administered to HCC patients at a dose ranging from about 1×10 ⁵ cells/dose/day to about 1×10 ⁸ cells/dose/day, and then about 50 μg/dose/day to A dose of anti-PD-L1 or anti-PD-1 within the range of about 400 micrograms/dose/day is used for treatment. On the first day, the individual receives the first dose of vaccine; thereafter will receive two follow-up doses of vaccine 6 weeks later. Peripheral blood will be obtained every week to monitor the immune response to each peptide by tetramer analysis. Anti-PD-1 therapy starts 5 to 8 weeks after the individual's last dendritic cell vaccine. Preliminary evaluation showed that patients receiving the combination therapy showed significant tumor shrinkage.

圖1顯示原位HCC小鼠模型之建立。(A)為建立原位HCC小鼠模型，將小鼠肝腫瘤Hep-55.1C細胞直接注射至經受中線剖腹術之小鼠之左肝葉中。用綠色虛線指示之區域在下圖中放大。細胞注射位點用綠色圓圈指示。(B)當小鼠死亡時，觀測到腫瘤在中線剖腹術之後在小鼠肝臟中原位地形成。用綠色虛線指示之區域在下圖中放大。原位腫瘤用綠色箭頭指示。Figure 1 shows the establishment of an orthotopic HCC mouse model. (A) To establish an orthotopic HCC mouse model, mouse liver tumor Hep-55.1C cells were directly injected into the left liver lobe of mice undergoing midline laparotomy. The area indicated by the green dashed line is enlarged in the figure below. The cell injection site is indicated by a green circle. (B) When the mouse died, tumors were observed to form in situ in the mouse liver after midline laparotomy. The area indicated by the green dashed line is enlarged in the figure below. Tumors in situ are indicated by green arrows.

圖2顯示原位HCC小鼠模型之驗證。(A)顯示原位HCC小鼠(n=6)之存活時間及肝臟重量、腫瘤重量及腫瘤體積與體重之比的圖表。水平線表示平均值。平均值±SEM及中值(範圍)顯示於各圖表下方。(B)所有六隻小鼠之肝臟中之腫瘤生長(表示為#1至#6) 用黑色箭頭指示腫瘤。(C)藉由H&E染色之腫瘤組織病理學。黑色虛線界定肝臟組織中之腫瘤及非腫瘤部分之區域。比例尺顯示於影像之右下角。初始放大倍數，×20。Figure 2 shows the validation of the orthotopic HCC mouse model. (A) A graph showing the survival time, liver weight, tumor weight, and tumor volume to body weight ratio of orthotopic HCC mice (n=6). The horizontal line represents the average value. The mean±SEM and median (range) are shown below each graph. (B) Tumor growth in the livers of all six mice (indicated as #1 to #6) The tumors are indicated by black arrows. (C) Tumor histopathology by H&E staining. The black dashed line delimits the tumor and non-tumor parts of the liver tissue. The scale is displayed in the lower right corner of the image. The initial magnification, ×20.

圖3顯示BMDC之生成及形態表徵。(A)說明自小鼠骨髓生成BMDC之示意圖。與表現高含量之此四種分子之BMDC相比，IMDC表現高含量之CD11c，但表現低含量之CD40、CD80及CD86。(B)藉由反相對比顯微法檢驗之細胞形態。黑色箭頭指示懸浮細胞之樹突突起。比例尺顯示於各影像之右下角。初始放大倍數，×20 (第1天及第3天)；×40 (第6天及第7天)。Figure 3 shows the generation and morphological characterization of BMDC. (A) A schematic diagram illustrating the generation of BMDC from mouse bone marrow. Compared with BMDC, which exhibits high content of these four molecules, IMDC exhibits high content of CD11c but low content of CD40, CD80, and CD86. (B) Cell morphology examined by reverse phase contrast microscopy. The black arrow indicates the dendritic protrusion of the suspended cell. The scale is displayed in the lower right corner of each image. Initial magnification, ×20 (day 1 and 3); ×40 (day 6 and 7).

圖4顯示BMDC之表現型表徵。對IMDC及BMDC上之DC表面標記物(包括CD11c、CD40、CD80及CD86)之表現的流動式細胞測量術分析。為了偵測各標記物，將IMDC及BMDC用各標記物之抗體(分別為橙色及紅色實曲線)或同型匹配對照抗體(青色實曲線)染色或保持未染色(黑色實曲線)。將FITC強度高於用同型匹配對照抗體染色之細胞之FITC強度的染色細胞閘控且視為對指定標記物呈陽性之細胞。表現指定標記物之細胞之頻率計算為所有分析細胞之百分比且顯示於各圖表之右上角。Figure 4 shows the phenotypic characterization of BMDC. Flow cytometry analysis of the performance of DC surface markers (including CD11c, CD40, CD80 and CD86) on IMDC and BMDC. In order to detect each marker, IMDC and BMDC were stained with each marker antibody (orange and red solid curves respectively) or isotype matching control antibody (cyan solid curve) or kept unstained (black solid curve). Stained cells whose FITC intensity is higher than the FITC intensity of cells stained with an isotype-matched control antibody are gated and regarded as cells positive for the designated marker. The frequency of cells expressing the designated marker is calculated as a percentage of all analyzed cells and displayed in the upper right corner of each graph.

圖5顯示BMDC之功能表徵。(A)為了抗原攝取分析，將IMDC及BMDC與FITC-聚葡萄糖在37℃下(紅色實曲線)或在冰上(青色實曲線)或保持未經處理(黑色實曲線)下一起培育，隨後進行流動式細胞測量術分析。將37℃下之FITC強度高於冰上之FITC強度的聚葡萄糖處理之細胞閘控且視為具有攝取聚葡萄糖之能力的細胞。所示為三個獨立實驗之代表性結果。(B)對FITC-聚葡萄糖呈陽性之細胞的頻率計算為所有經分析細胞之百分比。資料表示三個獨立實驗之平均值±SEM誤差槓。**P ＜ 0.01。(C)由IMDC及BMDC產生之IL-12。IMDC及BMDC之培養物上清液中之IL-12之濃度係藉由ELISA量測且表示為三個獨立實驗之平均值±SEM誤差槓。***P ＜ 0.001。(D)由IMDC及BMDC誘導之T細胞增殖。在細胞培養孔中共培養T細胞與IMDC或BMDC之後，對細胞培養***物中之T細胞數目計數且顯示為三個獨立實驗之平均值±SEM誤差槓。**P ＜ 0.01。Figure 5 shows the functional characterization of BMDC. (A) For antigen uptake analysis, IMDC and BMDC were incubated with FITC-polydextrose at 37°C (red solid curve) or on ice (cyan solid curve) or kept untreated (black solid curve), and then Perform flow cytometry analysis. Cells treated with polydextrose whose FITC intensity at 37°C is higher than the FITC intensity on ice are gated and regarded as cells with the ability to take up polydextrose. Shown are representative results of three independent experiments. (B) The frequency of cells positive for FITC-polydextrose is calculated as a percentage of all analyzed cells. The data represents the mean ± SEM error bars of three independent experiments. ** P <0.01. (C) IL-12 produced by IMDC and BMDC. The concentration of IL-12 in the culture supernatant of IMDC and BMDC was measured by ELISA and expressed as the average of three independent experiments ± SEM error bars. *** P <0.001. (D) T cell proliferation induced by IMDC and BMDC. After co-culturing T cells with IMDC or BMDC in the cell culture wells, the number of T cells in the cell culture insert was counted and displayed as the mean ± SEM error bars of three independent experiments. ** P <0.01.

圖6顯示在原位HCC小鼠中單獨或與PD-1/PD-L1抗體治療組合之BMDC評估。(A)原位HCC小鼠中之BMDC及/或抗PD-1/PD-L1治療時程之示意性時刻表。在第0天向8週齡C57BL/6雄性小鼠注射Hep-55.1C腫瘤細胞。在腫瘤細胞注射後第7天開始治療且以1天時間間隔進行總計三個劑量。在治療之後，追蹤所有小鼠直至死亡以確定存活時間。(B)用BMDC (1×10⁶ 個細胞/劑量)及/或抗PD-1/PD-L1 (100或200微克/劑量)治療後之原位HCC小鼠之卡普蘭-邁爾(Kaplan-Meier)存活曲線。累積存活率係針對腫瘤細胞注射之後的天數標繪。各組小鼠(n=6)中之總存活率顯示為以天數計的平均值±SEM及中值(範圍)。分析不同小鼠治療組之間的總存活率差異之顯著性且與小鼠對照組比較。將小於0.05之P 值視為顯著的。Figure 6 shows BMDC evaluation in orthotopic HCC mice alone or in combination with PD-1/PD-L1 antibody treatment. (A) Schematic timetable of BMDC and/or anti-PD-1/PD-L1 treatment schedule in orthotopic HCC mice. On day 0, 8-week-old C57BL/6 male mice were injected with Hep-55.1C tumor cells. The treatment was started on the 7th day after tumor cell injection and a total of three doses were given at 1-day intervals. After treatment, all mice were tracked until death to determine survival time. (B) Kaplan-Meier (Kaplan) of orthotopic HCC mice treated with BMDC (1×10 ⁶ cells/dose) and/or anti-PD-1/PD-L1 (100 or 200 μg/dose) -Meier) survival curve. The cumulative survival rate is plotted against the number of days after tumor cell injection. The overall survival rate of mice in each group (n=6) is shown as the mean±SEM and median (range) in days. Analyze the significance of the difference in overall survival rate between different mouse treatment groups and compare it with the mouse control group. A P value of less than 0.05 is considered significant.

Claims (16)

一種用於治療肝細胞癌(HCC)之方法，該方法包含向患者共投與基於樹突細胞之疫苗與免疫檢查點抑制劑之組合。A method for treating hepatocellular carcinoma (HCC), the method comprising co-administering a combination of a dendritic cell-based vaccine and an immune checkpoint inhibitor to the patient. 如請求項1之方法，其中該基於樹突細胞之疫苗係以約1×10⁵ 個細胞/劑量/天至約1×10⁸ 個細胞/劑量/天範圍內之劑量投與。The method of claim 1, wherein the dendritic cell-based vaccine is administered at a dose ranging from about 1×10 ⁵ cells/dose/day to about 1×10 ⁸ cells/dose/day. 如請求項1之方法，其中該基於樹突細胞之疫苗係以約1×10⁶ 個細胞/劑量/天之劑量投與。The method of claim 1, wherein the dendritic cell-based vaccine is administered at a dose of about 1×10 ⁶ cells/dose/day. 如請求項1之方法，其中該基於樹突細胞之疫苗為不成熟樹突細胞、成熟樹突細胞、骨髓樹突細胞(cDC)、漿細胞樣樹突細胞(pDC)或骨髓源性樹突細胞。The method of claim 1, wherein the vaccine based on dendritic cells is immature dendritic cells, mature dendritic cells, bone marrow dendritic cells (cDC), plasmacytoid dendritic cells (pDC) or bone marrow-derived dendrites cell. 如請求項1之方法，其中該免疫檢查點抑制劑為針對以下之抗體：細胞毒性T-淋巴細胞抗原4 (CTLA-4或CD152)或計劃性細胞死亡配位體-1 (PDL-1)或計劃性細胞死亡蛋白1 (PD-1)。The method of claim 1, wherein the immune checkpoint inhibitor is an antibody against: cytotoxic T-lymphocyte antigen 4 (CTLA-4 or CD152) or planned cell death ligand-1 (PDL-1) Or planned cell death protein 1 (PD-1). 如請求項1之方法，其中該免疫檢查點抑制劑係以約50微克/劑量/天至約400微克/劑量/天範圍內之劑量投與。The method of claim 1, wherein the immune checkpoint inhibitor is administered at a dose ranging from about 50 micrograms/dose/day to about 400 micrograms/dose/day. 如請求項1之方法，其中，該免疫檢查點抑制劑係以約100微克/劑量/天或約200微克/劑量/天之劑量投與。The method of claim 1, wherein the immune checkpoint inhibitor is administered at a dose of about 100 micrograms/dose/day or about 200 micrograms/dose/day. 如請求項1之方法，其中該基於樹突細胞之疫苗與免疫檢查點抑制劑之組合係藉由輸注或注射投與。The method of claim 1, wherein the combination of the dendritic cell-based vaccine and the immune checkpoint inhibitor is administered by infusion or injection. 如請求項1之方法，共投與係經由靜脈內、腹膜內、肌肉內、鞘內或皮下之途徑。As in the method of claim 1, co-administration is via intravenous, intraperitoneal, intramuscular, intrathecal, or subcutaneous routes. 如請求項1之方法，其中該基於樹突細胞之疫苗及該免疫檢查點抑制劑可提供為用於相同時間或在不同時間投與之獨立藥劑。The method of claim 1, wherein the dendritic cell-based vaccine and the immune checkpoint inhibitor can be provided for the same time or at different times to be administered with independent agents. 如請求項1之方法，其中該共投與係週期性地重複。Such as the method of claim 1, wherein the co-investment is repeated periodically. 如請求項11之方法，其中投與週期包含每隔一天投與該基於樹突細胞之疫苗及該免疫檢查點抑制劑，持續總共三個劑量。The method of claim 11, wherein the administration cycle comprises administering the dendritic cell-based vaccine and the immune checkpoint inhibitor every other day for a total of three doses. 如請求項11之方法，其中該共投與包含同時投與或分開投與該基於樹突細胞之疫苗及該免疫檢查點抑制劑。The method of claim 11, wherein the co-administration comprises simultaneous administration or separate administration of the dendritic cell-based vaccine and the immune checkpoint inhibitor. 如請求項13之方法，其中該基於樹突細胞之疫苗及該免疫檢查點抑制劑經調配用於分開投與且係同時或依序地投與。The method of claim 13, wherein the dendritic cell-based vaccine and the immune checkpoint inhibitor are formulated for separate administration and are administered simultaneously or sequentially. 如請求項14之方法，其中可首先投與該基於樹突細胞之疫苗，隨後投與該免疫檢查點抑制劑。The method of claim 14, wherein the dendritic cell-based vaccine can be administered first, and then the immune checkpoint inhibitor can be administered. 一種用於投與基於樹突細胞之疫苗與免疫檢查點抑制劑之組合之醫藥套組，其包含關於投與基於樹突細胞之疫苗及免疫檢查點抑制劑的印刷說明書，以及呈用於至少一個週期之劑量單位的基於樹突細胞之疫苗及免疫檢查點抑制劑之組合。A medical kit for administering a combination of a dendritic cell-based vaccine and an immune checkpoint inhibitor, which includes printed instructions for administering a dendritic cell-based vaccine and immune checkpoint inhibitor, and is presented for at least A combination of a dendritic cell-based vaccine and immune checkpoint inhibitor in one cycle of dosage unit.

Images