| 摘要: | (一)主旨與目標：目前仍有很多人類高致命率短存活期的惡性腫瘤，有待研發新療法，達到降低死亡率，延長存活期以及改善病人生活品質的目的。主持人近十多年來從事針對癌症之細胞(基因)免疫療法的研究，已完成之「惡性膠質腦瘤之自體樹突細胞腫瘤疫苗輔助療法」之第一相人體試驗，雖然在降低死亡率和延長存活期方面成果卓越（和美國相比），但也發現很多問題可做轉譯研究，以期提升第二、三相臨床醫學試驗的成效。此計劃的主要目的是以(1)惡性膠質腦癌（WHO grade Ⅳ）病人的腫瘤細胞和各種免疫細胞（包括腫瘤侵入淋巴球細胞(Tumor-infiltrating lymphocytes)）為研究平台，(2)探討此癌症特殊藥（如temozolomide）與放射線對身體抗癌免疫與免疫調控系統的影響。 (二)依據與背景：此二關鍵問題，目前正以衛生署資助十一個月之「樹突細胞腫瘤疫苗治療：臨床試驗後續之轉譯醫學研究(DOH95-TD-B-111- TM007)」開始獲得初步成果，必須繼續確定，並進一步針對抗癌免疫抑制的機制，研討對策(immuno-protection, immuno-enhancing modifiers)。此轉譯醫學之研究乃基於主持人四十年之癌症研究基礎，尤其是近十年對癌症免疫新療法之探討成果。又2006-2007年主持人將負責後續之第二相臨床試驗(已於2006年8月28日獲衛生署醫學臨床試驗委員會審查通過)，所以，如果此轉譯醫學研究計畫幸獲贊助，研究成果將很快可以應用於改進臨床試驗(經由IRB和DOH醫事審議委員會再次審查核准後執行)。 (三)實施方法及進行步驟 : 主要將繼續使用由惡性腦癌病人腫瘤組織培養出來的癌細胞以及病人自體之血液單核細胞：腫瘤侵入淋巴細胞(Tumor infiltrating lymphocytes)中CD4(+)CD25(+)CD45RO(+)FOXP3 RNA(+)之免疫調控T細胞以及具抗腫瘤特殊性之殺傷T細胞CD8(+) CTL。為達到第一目標，我們將大量培養上述之腫瘤與免疫細胞並利用[CFSE]生染術研發成研究平台。可使用於癌症化療放療或其他藥物機制之探討。為達第二目標，我們將以上述研究平台，篩檢而發現會影響CTL癌殺傷功能或Treg細胞免疫調控機能之藥物。此主要將以COX2酵素抑制藥與中草藥中具免疫調控作用的純化物如18--glycynhetinic acid 與其衍生化合物為研究模型，並提及其他的藥物。為達第三目標，我們將注重經過化療或放射線治療後消失而再發屬「癌幹細胞系」的惡性腫瘤細胞，因為此種再發的癌細胞常常對化療物或放射線已產生高度的抵抗性。我們將分離這些再發而具有｢癌幹細胞系｣性質的癌細胞，用以研究如何利用免疫療法來控制或消滅這些｢癌幹細胞系｣之惡性腫瘤。 (四)時程規劃：DOH96-TD-B-111TM007計劃正積極執行中。延續計劃擬申請二年，原因是主持人於公元2006-2009年期間正在執行「惡性膠質腦瘤之自體樹突細胞腫瘤疫苗輔助療法」第二相臨床試驗，將陸續有更多此類癌症病人的腫瘤細胞與腫瘤侵入免疫淋巴細胞，作完整性、足夠病例的研究，以確定Treg和CTL於抗癌免疫扮演的角色。又此計劃所建立的抗癌免疫細胞和自體腫瘤細胞的技術平台，勢將可應用於「新藥生科技國家型計畫」中其他計畫主持人所研發之抗癌新藥，測定這些新藥對人類免疫細胞的影響。這些研究目標至少需要兩年時間來完成。 (五)預期績效：所獲成果將很快應用於改進目前之腦惡性腫瘤之免疫疫苗療法，增進此致命癌之存活率。所研發之免疫機制與藥物調控知識與技術亦可應用於其他致命性人類癌症之治療。
I. Purpose and Specific Aims The present study is proposed to continue a currently DOH Translational Medicine Program-supported 11-month project (DOH95-TD-B-111-TM007 from January 19 to December 19, 2006) with the ultimate purpose of developing novel therapeutic approaches to decrease the mortality rate, to prolong the survival period and to improve the life quality of patients suffering from currently incurable human cancers of high mortality and short survival. Also, we have just completed a DOH-approved phase-I clinical trial “Autologous dendritic cell-tumor vaccine adjuvant therapy of glioblastoma multiforme (GBM)” and obtained highly promising results that warrant the following up with a multi-center phase-II clinical trial. However, from some non-responsive patients of the phase-I trial, we have also recognized a few important issues that require post-clinical trial translation research. For the past 6-7 months of the currently funded project, we have addressed two of the critical problems and produced significant experimental results for possible improvement of the GBM treatment. Thus, for the next one year, we will pursue the following specific aims:: 1. To develop a technology platform of human cancer cells and autologous immune T lymphocytes, particularly tumor-infiltrating T lymphocytes of CD4(+)CD25(+)CD45RO(+)foxp3RNA(+) T regulatory phenotypes, tumor-specific cytotoxic CD8(+) phenotype and CD8(+)CD56(+) NKT phenotypes, for characterization of various cancer therapeutic agents with the CFSE-labeling techniques we have developed for tracing the cell growth or inhibition kinetics in vitro, 2. To search for and characterize possible modifier biologicals and/or chemicals that can curtail or abrogate the negative effects of tumor-associated immuno-suppression of Treg cells while promoting the positive effects of anti-cancer innate and acquired immunity, including modifier drugs and purified compounds of Chinese traditional or herbal medicines believed to affect the immune system. As our phase-II clinical trial (planned for 80 new and recurrent glioblastoma patients) will be in progress during the next 3 years (2006-2009), any significant relevant results obtained from this translation research project will be rapidly utilized for the amendment or modification of the clinical trial protocol (following the IRB and DOH reviews and approvals). II. Rationale and Background Although cancer chemotherapy, radiotherapy and other anti-cancer measures have improved considerably, many human cancer diseases are still incurable and fatal. Recent cancer therapeutic research has therefore placed emphasis on novel approaches, particularly gene therapy and cell therapy. Hundreds of cancer gene therapy and immunotherapy protocols have been put to clinical trials, mostly in the United States and European countries and also several in Taiwan. The results reported of these clinical trials have so far not been impressive, suggesting a lack of sufficient translational research before the clinical trial (as pointed out by some recent expert reviews). For more than a decade, the major goal of this laboratory has been to develop safe and effective, if not life-saving, combination therapy measures for currently incurable and fatal human cancer diseases. Because of his more than two decades of gene transfer research experiences, this P.I was initially interested in the gene therapy approaches. But most of the promising therapeutic genes, discovered by animal model studies, are associated with the proliferation, differentiation and functions of dendritic cells. This laboratory has therefore shifted from gene therapy to dendritic cell-based immunotherapeutic approaches. With continuous DOH funding supports through the “Genes in Health Sciences” Frontier Science and Technology Program and the National Program of Genomic Medicine, the P.I. has performed translational research for more than six years and finally been able to come up with a protocol of dendritic cell-based tumor vaccine therapy for WHO grade IV glioblastoma multiforme (GBM) and grade III anaplastic astrocytoma. Despite current standard therapeutic measures of surgery and radiotherapy, GBM remains an essentially 100% fatal disease sowing survival period of 8-14 months after diagnosis. It is therefore possible to tell whether this dendritic cell-based tumor vaccine therapy is useful or not in 3 years with a relatively small number of tested patients. From this clinical trial we have obtained results that are highly significant and much better than those reported from US, Europe and Japan. We are now in the process of publishing these results into three papers, while probably filing a patent application. However, this clinical trial has also revealed specific points for further translational research that may not only serve to improve the present protocol but also to collect important data for cancer therapy in general. Therefore, we proposed the research project entitled “Dendrtic Cell-based Tumor Vaccine Therapy: Post Clinical Trial Translational Research” (DOH95-TD-B-111-TM007) and were fortunately supported by the DOH Translational Medicine Program. For the past 7 months, significant progress has been made by finding a possible important role played by T regulatory lymphocytes (Treg) in immuno-suppressive effects of GBM and also of emergence of tumor-specific cytotoxic T lymphocytes (CTL) as a key benefit of dendritic cell-based tumor vaccine therapy. These results are to be firmly established by using tumor-infiltrating lymphocytes (TILs) of more numbers of vaccinated GBM patients, while the possible effects of radiotherapy and chemotherapy on anti-tumor immune cells are being investigated. A research platform of the GBM TILs and autologous GBM tumor cells will be developed for the discovery of therapeutic agents (biologicals, chemicals and purified essential components of Chinese traditional medicines and herbs) that may enhance the immune potency and negate the immunosuppression in cancer therapy. III. Methods of Procedures To achieve the specific aims, we will continue to employ the GBM patient-derived cancer cells, autologous peripheral blood mononuclear cells, tumor-infiltrating CD4(+)CD25(+)CD45RO(+)foxp3RNA(+) Treg cells and tumor-infiltrating tumor-specific CD8(+) cytotoxic T lymphocytes for in vitro studies.. For the first specific aim, we will expand the in vitro growth of glioblastoma tumor cells and corresponding autologous immune lymphocytes including Treg cells and tumor-specific cytotoxic T lymphocytes of a few GBM patients and, with the use of the fluorescent [CFSE]-labeling assay that we have developed, we will establish a technological platform for “drug screening” or “novel drug discovery”. To achieve the second specific aim, we will examine various candidate drugs for possible modification of the immuno-suppressive property of GBM glioma cells by circumventing the Treg cell immuno-suppressive function and/or by boosting the anti-tumor CTL potency. For example, we will study some COX-2 inhibitors and also examine pure glycyrrhizin as well as its in vivo active metabolite, 18-beta-glycyrrhetinic acid, for their versatile properties of anti-oxidant, gap-junction closure and Th1 lymphocyte stimulation. To achieve the third specific aim, we will particularly exploit the presumed “cancer stem line” GBM cells that survive intensive radiotherapy and chemotherapy and become refractory to further radiotherapy and chemotherapy. These radio-resistant and chemo-resistant “cancer stem lines” will be examined for possible sensitivity to immune attack or serve for designing combined radiotherapy, chemotherapy and immunotherapy approaches to treat post-surgical GBM patients. IV. Time scheme Although a two-year renewal of this translational research project was proposed, the review committee recommended approval for only one year with marked curtailment of the requested budget to NT$1,000,000. We therefore can only focus on the “essential” part of the proposed research. This would definitely affect the multi-center phase II trial of autologous dendritic cell-based tumor vaccine adjuvant therapy planned for the next three-year period. (V). Resources and Research Environment We will perform all the experimental works within the GLP barrier facility newly built for clinical trial purposes in China Medical University Hospital. To carry out this post-clinical trial translation research project, we will continue active collaboration mainly with neurosurgeon teams of Der-Yang Chow (China Medical Univ Hospital Department of Neurosurgery) and Chen-nen Chang (Chang-gun Memorial Medical Center Department of Neurosurgery).on GBM. The principal investigator (W.K. Yang) is an invited member of the 40-member International Immunotherapy Task Force of the American Neurosurgeon Society Neuro-oncology Section and thus is well-informed with respect to novel immunotherapeutic approaches to glioblastomas and other brain malignant tumors. He has also collaborated with colorectal surgeons, K.W. Chen (CMUH) and W.S. Chen (VGH-Taipei), on colorectal cancer; and with Y.M. Chen (VGH-Taipei Chest Medicine) on lung non-small cell ling cancer. Furthermore, in collaboration with C.T. Peng’s clinical team (CMUH Pediatric Oncology), the P.I. has initiated a collaborative research project to develop adjuvant immunotherapy of childhood neuroblastoma, which will be actively pursued in the present study. (VI). Expected Results We will determine how tumor infiltrating Treg cells cause failures of DC-tumor vaccine therapy in some GBM patients and find out what drugs can abrogate or circumvent the Treg effect. These drugs (especially already in clinical use but for other purposes) will be incorporated to improve the DC-tumor vaccine immunotherapy of glioblastoma multiforme and thus further increase the survival rates and periods of the treated GBM patients. |
