脂质体异体瘤苗对肿瘤的治疗效应研究

应用瘤苗诱导肿瘤宿主产生抗肿瘤免疫反应以治疗肿瘤的主动免疫治疗肿瘤是目前肿瘤生物治疗有效的方法之一 ,受到国内外的广泛重视[1,2 ] 。我们已开展自体瘤苗治疗肿瘤的研究并已取得一定进展[3] 。进一步提高瘤苗免疫治疗肿瘤的疗效 ,寻求制备高效、安全的异体瘤苗的途径极为重要。据文献报道脂质体 (ｌｉ ｐｏｓｏｍｅ)包裹瘤苗抗原性强 ,又可作异体瘤苗的载体 ,其诱导机体产生的抗体比单纯性瘤苗高 10 0～ 4 0 0倍 ,且具有作用持久、毒副反应轻等优点[4 ,5] 。为此本研究观察了脂质体异体瘤苗对肿瘤的抑制作用 ,以期为临床应用提供实验…     

β-榄香烯综合瘤苗主动免疫治疗临床试验研究-40例癌症患者的疗效分析

在过去瘤苗主动免疫治疗实验研究和初步临床试验的基础上，进一步扩大临床试验，进行应用评价。用本室申报的发明专利——以β-榄香烯为主的复合因素制备的自体瘤苗或异体瘤苗主动免疫疗法，治疗40例直肠癌，结肠癌及胃癌等术后患者,制备瘤苗的肿瘤组织取自癌症患者术后切下之肿瘤标本，在无菌条件下选取新鲜肿瘤组织，     

琐阳花色甙复合瘤苗免疫治疗肺肿瘤的实验研究

目的:研究琐阳花色甙复合瘤苗免疫治疗肺肿瘤的综合效应。方法:对肺肿瘤细胞系LCC,用琐阳花色甙对其进行系列生物修饰,构建琐阳花色甙复合瘤苗,用以治疗肺肿瘤。结果:经琐阳花色甙修饰构建的复合瘤苗,明显阻止LCC细胞的肺转移;显著抑制皮下肿瘤结节形成;明显延长荷瘤鼠的生存时间;治疗组小鼠脾细胞对小鼠肺癌的细胞毒活性显著增强。结论:琐阳花色甙修饰构建的LCC复合瘤苗,促进脾细胞的杀瘤活性,对肺癌有较好的实验治疗作用。     

肿瘤疫苗的研究进展

近年来随着分子生物学的发展和基因工程技术的出现,肿瘤疫苗的研究有很大飞跃,成为肿瘤主动性免疫治疗的重要手段之一。本文从转基因瘤苗、树突状细胞（ＤＣ）与瘤苗、抗独特型瘤苗、瘤苗制备和修饰的其它手段等几个方面综述肿瘤疫苗的部分研究进展。     

DC肿瘤融合瘤苗抗肿瘤效应的实验研究

目的:观察DC与肿瘤细胞融合后的瘤苗体内诱导的抗肿瘤免疫应答以及对荷瘤小鼠的治疗作用.方法:应用免疫磁珠分选和贴壁培养方法收集融合细胞,应用3H-TdR掺入法、4h51Cr释放法观察T细胞增殖反应的能力和CTL活性,并观察瘤苗对荷瘤小鼠保护性免疫反应和免疫治疗作用.结果:DC肿瘤融合瘤苗具有强烈的激活T细胞增殖和抗原提呈的能力,在体外、体内诱导出更强的特异CTL细胞毒活性,使免疫小鼠产生一定的免疫保护作用,抵抗Hepa1-6肝癌细胞的再次攻击,使治疗的小鼠肿瘤的生长明显缓慢,具有更明显的治疗作用.结论:DC与肿瘤细胞融合后进行体内免疫和治疗,能诱导出显著的抗肿瘤免疫反应,为DC介导的肿瘤免疫治疗开辟了新的途径.     

肿瘤免疫治疗疗效评价的新标准

目的:肿瘤免疫治疗近年来发展迅猛,已被越来越多的临床医生所认可,并广泛应用于多种恶性肿瘤的治疗。与传统的化疗不同,免疫治疗发生严重毒性反应的概率更低,患者耐受性更好,尤其对患者生活质量的改善作用更明显。但遗憾的是,目前临床上采用传统的肿瘤治疗评价体系对肿瘤免疫治疗疗效评价时往往不能令人满意,导致部分进入Ⅲ期临床试验的免疫治疗项目最后以失败告终。鉴于现有WHO或RECIST(response evaluation criteria in solid tumor)标准很难对肿瘤免疫治疗的临床疗效进行准确的解读和确切的评价,因此在2009年第23期的Clinical Cancer Research上专门刊载了由纽约Me-morial Sloan-Kettering癌症中心Wolchok教授等撰写的论文——《针对实体瘤免疫治疗疗效评价指南:免疫相关疗效评价标准》,深入探讨了肿瘤免疫治疗疗效评价新标准的意义和应用前景。本文以该论文为主,结合其他相关文献,对"肿瘤免疫疗效评价的新标准"这一肿瘤学界的热点问题作一介绍。     

TNFα基因转染的肿瘤细胞抗肿瘤转移作用的探讨

肿瘤瘤苗进行的主动性免疫治疗是一条较为理想的治疗途径，近几年发展起来的细胞因子基因转染的肿瘤细胞瘤苗，开创了肿瘤主动免疫治疗的新时代。肿瘤细胞靶向的TNFα基因治疗开展得较早，TNFα基因转染的肿瘤细胞的体内致瘤性显著下降，但是对于此肿瘤细胞制备成瘤苗对已发生的肿瘤能否具有抗肿瘤转移作用尚未见报道。     

白血病肿瘤疫苗联合1-甲基色氨酸免疫治疗荷瘤小鼠疗效观察

 目的　探讨白血病肿瘤疫苗（简称瘤苗）主动免疫治疗及联合吲哚2,3双加氧酶（IDO）的抑制剂1-甲基色氨酸（1-MT）,在白血病荷瘤小鼠治疗中的作用。方法　采用FBL-3细胞皮下注射建立荷瘤白血病小鼠模型;实验分为5组：正常对照组、PBS对照组、环磷酰胺（CTX）化疗组、单用瘤苗治疗组和瘤苗联合1-MT治疗组;观察各组小鼠的一般状况、肿瘤缓解率、肿瘤大小、转移情况及生存期。结果　PBS对照组小鼠活动迟缓,体质量（含瘤结节质量）比其余各组均高;单用瘤苗组和瘤苗联合1-MT组小鼠活动、进食正常,体质量与正常小鼠差异不大;化疗组体质量明显减轻,出现脱毛、弓背、活动减少等,差异有统计学意义（F＝57.71,P＝000）;单用瘤苗组和瘤苗联合1-MT组治疗相关死亡率明显低于化疗组（0,0,40 %）。瘤苗联合1-MT组完全缓解率与单用瘤苗组（61.1 %、70.0 %）比较,差异无统计学意义（χ2＝0.221,P＞0.05）,但瘤苗联合1-MT组的复发率低于单用瘤苗组（0,36.36 %）;复发小鼠再应用1-MT,能明显抑制瘤结节的生长。单用瘤苗组和瘤苗联合1-MT组小鼠中位存活期明显高于化疗组和PBS对照组（χ2＝52.13,P＜0.01）。各组小鼠整体瘤结节的变化比较差异有统计学意义（F＝89.966,P＝0.000）。结论　白血病瘤苗在动物实验具有肯定的疗效,能明显抑制肿瘤的生长,延长小鼠生存时间,且副作用小。免疫治疗联合1-MT对白血病进行治疗,可以显著减少肿瘤的复发率;而免疫治疗有效后复发时应用1-MT,可以显著抑制肿瘤的生长。     

实体瘤外周血循环肿瘤细胞与转移相关性的研究进展

目的:总结实体瘤外周血中循环肿瘤细胞(CTC)和转移相关性的研究进展。方法:以"循环肿瘤细胞、实体瘤、转移"为关键词,检索2000-01-2010-10 PubMed、Science Direct、Ovid、Springer、CNKI和维普等数据库的相关文献。纳入标准:关于实体瘤CTC与转移密切相关的分子机制、临床相关性的文献。共纳入分析42篇文献。结果:随着分子生物学和材料技术的发展,越来越多的方法有效地富集和鉴定不同类型实体瘤外周血CTC。细胞基因水平证实CTC具有恶性生物学特性,CTC自身基因和转移相关蛋白谱的表达、肿瘤微环境、免疫系统等因素影响着CTC远处器官转移灶的形成。CTC数目、特定基因、蛋白的表达与治疗疗效、预后等具有相关性。结论:研究CTC参与血液播散转移的机制,为全面、准确地阐明恶性实体瘤转移的机制、个体化的治疗提供新的工具。     

雷公藤内酯醇对人宫颈癌细胞的凋亡诱导效应

目的:探讨腺病毒介导AFP基因修饰的DC(AFP-DC)瘤苗经不同途径免疫后机体抗肿瘤免疫应答反应.方法:采用皮下注射、静脉注射和瘤体注射三种途径回输AFP-DC瘤苗,比较观察AFP-DC瘤苗对荷瘤小鼠免疫治疗作用,应用4h51cr释放杀伤实验、T细胞与NK体内剔除实验等方法,观察AFP-DC瘤苗对荷瘤小鼠免疫治疗作用及保护性免疫反应.结果:皮下注射AFP-DC瘤苗治疗效果在抑制肿瘤生长、延长小鼠存活期方面都明显优于瘤体内注射或尾静脉注射(P＜0.05),AFP-DC瘤苗体内能更有效地诱导特异CTL细胞毒活性,能使免疫动物产生一定的免疫保护作用,抵抗肿瘤细胞的再攻击.在AFP-DC瘤苗诱导抗肿瘤免疫排斥反应过程中,必需有CD4 T和CD8 T细胞的参与;而在其效应阶段,则依赖于CD8 T细胞的参与,CD4 T细胞为非必需;在免疫诱导及效应阶段剔除NK细胞对抗肿瘤免疫应答无明显影响.结论:皮下注射AFP-DC瘤苗能有效诱导机体产生抗肿瘤免疫反应,为DC介导的肝癌免疫治疗开辟了新的途径.     

Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer

Tumor vaccines composed of autologous tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) (GVAX) have demonstrated clinical activity in advanced-stage non-small-cell lung cancer (NSCLC). In an effort to remove the requirement for genetic transduction of individual tumors, we developed a 'bystander' GVAX platform composed of autologous tumor cells mixed with an allogeneic GM-CSF-secreting cell line. We conducted a phase I/II trial of this vaccine (3-12 biweekly vaccinations) in advanced-stage NSCLC. Tumors were harvested from 86 patients, tumor cell processing was successful in 76, and 49 proceeded to vaccination. The most common toxicity was local vaccine injection site reactions. Serum GM-CSF pharmacokinetics were consistent with secretion of GM-CSF from vaccine cells for up to 4 days with associated transient leukocytosis confirming the bioactivity of vaccine-secreted GM-CSF. Evidence of vaccine-induced immune activation was demonstrated; however, objective tumor responses were not seen. Compared with autologous GVAX vaccines prepared by transduction of individual tumors with an adenoviral GM-CSF vector, vaccine GM-CSF secretion was approximately 25-fold higher with the bystander GVAX vaccine used in this trial. However, the frequency of vaccine site reactions, tumor response, time to disease progression, and survival were all less favorable in the current study.     

TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner

Our neoadjuvant clinical trial of a GM-CSF secreting allogeneic pancreas tumor vaccine (GVAX) revealed the development of tertiary lymphoid aggregates (TLAs) within the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment 2 weeks after GVAX treatment. Microarray studies revealed that multiple components of the TGF-β pathway were suppressed in TLAs from patients who survived greater than 3 years and who demonstrated vaccine-enhanced mesothelin-specific T cell responses. We tested the hypothesis that combining GVAX with TGF-β inhibitors will improve the anti-tumor immune response of vaccine therapy. In a metastatic murine model of pancreatic cancer, combination therapy with GVAX vaccine and a TGF-β blocking antibody improved the cure rate of PDA-bearing mice. TGF-β blockade in combination with GVAX significantly increased the infiltration of effector CD8⁺ T lymphocytes, specifically anti-tumor-specific IFN-γ producing CD8⁺ T cells, when compared to monotherapy controls (all p < 0.05). TGF-β blockade alone did not deplete T regulatory cells (Tregs), but when give in combination with GVAX, GVAX induced intratumoral Tregs were depleted. Therefore, our PDA preclinical model demonstrates a survival advantage in mice treated with an anti-TGF-β antibody combined with GVAX therapy and provides strong rational for testing this combinational therapy in clinical trials.     

GM-CSF修饰肿瘤细胞疫苗治疗前后肺癌患者Treg的变化及其临床意义

目的:观察GM-CSF基因修饰的肿瘤细胞疫苗(GM-CSF modified tumor cell vaccine,GVAX)治疗前后肺癌患者外周血中CD4+CD25+调节性T细胞(regulatory T cell,Treg)比例的变化,探讨其与肺癌临床病理特征的关系及对患者生存的影响。方法:选择2007年3月至2010年7月天津医科大学附属肿瘤医院接受GVAX治疗的85例肺癌患者,其中鳞癌28例、腺癌47例、小细胞癌10例,早期(Ⅰ～ⅢA期)23例、晚期(ⅢB～Ⅳ期)62例。流式细胞术检测患者GVAX治疗前后外周血Treg的比例,分析治疗前后Treg比例变化与肺癌临床病理特征和患者生存期的关系。结果:接受GVAX治疗的85例肺癌患者1、3年的生存率分别为69.3%、48.2%,治疗后外周血Treg比例明显下降[(4.86±2.52)%vs(5.52±2.68)%,P<0.05]。患者血清LDH水平正常组治疗后Treg比例明显下降[(4.50±2.23)%vs(5.59±2.76)%,P<0.05],而LDH增高组治疗后Treg比例上升[(6.04±3.07)%vs(5.31±2.48)%,P<0.05];GVAX治疗超过1个疗程者较治疗1个疗程者治疗后Treg比例下降更明显[(-0.39±2.39)%vs(-2.11±1.62)%,P<0.05]。治疗前后外周血Treg比例的变化与患者预后相关,治疗后Treg比例下降组患者中位总生存期(overall survival,OS)明显长于升高组(21个月vs 10个月,P<0.05),晚期肺癌患者中Treg比例下降者的OS延长更明显(18个月vs 8个月,P<0.05)。结论:肺癌患者GVAX治疗前后外周血Treg比例变化可能成为评价GVAX疗效及判断患者预后的免疫指标,GVAX治疗后患者Treg比例的下降提示其疗效和预后都较好。     

Development of thyroglobulin antibodies after GVAX immunotherapy is associated with prolonged survival

Cancer immunotherapy induces a variety of autoinflammatory responses, including those against the thyroid gland, which can be exploited to predict clinical outcomes. Considering the paucity of information about thyroid autoimmunity in patients receiving cancer vaccines, we designed our study to assess the development of thyroglobulin antibodies (TgAbs) in patients treated with GVAX (vaccine made of a tumor cell type transfected with GM‐CSF) and/or ipilimumab and correlated seroconversion with survival. Using both in house and commercial ELISA assays, we measured TgAbs in patients with pancreatic (No. = 53), prostate (No. = 35) or colon (No. = 8) cancer, before and after treatment with GVAX only (No. = 34), GVAX plus ipilimumab (No. = 42) or ipilimumab (No. = 20), and correlated their levels with patient's survival, disease status and T‐cell surface markers. Antibodies to thyroperoxidase, myeloperoxidase, proteinase 3, insulin and actin were also measured. TgAbs specifically developed after GVAX, independent of the underlying cancer (81% in prostate, 75% colon cancer and 76% pancreatic cancer) and co‐administration of ipilimumab (75% in GVAX only and 78% in GVAX plus ipilimumab). This TgAbs seroconversion could be detected mainly by the in house assay, suggesting that the thyroglobulin epitopes recognized by the antibodies induced by GVAX are different from the epitopes seen in the classic form of Hashimoto thyroiditis. Notably, TgAbs seroconversion was associated with significantly prolonged survival (p = 0.01 for pancreas and p = 0.005 for prostate cancer). In conclusion, GVAX immunotherapy induces the appearance of TgAbs that recognize a unique antigenic repertoire and associate with prolonged survival.     

Granulocyte-macrophage colony-stimulating factor gene-transfected autologous tumor cell vaccine: focus[correction to fcous] on non-small-cell lung cancer

Traditionally, non-small-cell lung cancer (NSCLC) is not thought of as an immunosensitive malignancy. However, recent clinical results with GVAX, a granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced autologous tumor vaccine, may suggest otherwise. This review summarizes immune-induced activity caused by GM-CSF protein and GM-CSF gene-transfected vaccines. Initial indication of use for GM-CSF protein (sargramostim) was to improve neutrophil recovery following cytotoxic chemotherapy. However, several trials involving patients with hematologic malignancy demonstrated improvement in survival related to delayed disease progression in patients receiving sargramostim in combination with chemotherapy. Subsequently, others explored potential antitumor activity with sargramostim in a variety of trials. Results did not consistently demonstrate sufficient antitumor activity to justify routine use of sargramostim as an anticancer agent. Preclinical work with GM-CSF gene-transfected vaccines, however, did demonstrate significant activity, thereby justifying clinical investigation. Patients with metastatic NSCLC who had previously failed chemotherapy demonstrated response to GVAX (3 of 33 complete responses) and dose-related improvement in survival (471 days vs. 174 days).     

74例肺癌患者GVAX治疗前后外周血树突状细胞变化及其临床意义

  目的   探讨粒-巨噬细胞集落刺激因子（granulocyte-macrophage colony stimulating factor,GM-CSF）基因修饰的肿瘤细胞疫苗治疗前后肺癌患者外周血中DC两个不同亚群（DC1,DC2）的比例、治疗前后比例变化与临床病理特征的相关性及对生存的影响。   方法   对74例接受GM-CSF基因修饰的肿瘤细胞疫苗（GM- CSF modified tumor cell vaccine,GVAX）治疗的肺癌患者,采用流式细胞技术检测患者治疗前后外周血DC及淋巴细胞亚群的比例,分析治疗前后DC比例的变化、治疗前后DC比例与治疗前血清标志物及免疫细胞的关系、治疗前后DC比例对肺癌患者生存的影响。   结果   接受GVAX疫苗治疗后外周血DC1与DC2比例无明显变化（P_DC1=0.786,P_DC2=0.779）;神经烯醇化酶（NSE）水平升高组中的DC亚群比例高于NSE水平正常组;治疗后DC2比例与治疗前Treg呈负相关;对于早期肺癌患者,治疗后DC2比例低于均值者的生存时间比高于均值者的生存时间明显延长。   结论   治疗后DC2比例可作为早期患者GVAX疫苗疗效及判断预后的免疫指标,其对预后的影响可能与患者外周血Treg比例有一定的相关性。      

Phase II Trial of Neoadjuvant Docetaxel and CG1940/CG8711 Followed by Radical Prostatectomy in Patients With High‐Risk Clinically Localized Prostate Cancer

Background.

Prostate cancer (PC) is the most commonly diagnosed noncutaneous malignancy in American men. PC, which exhibits a slow growth rate and multiple potential target epitopes, is an ideal candidate for immunotherapy. GVAX for prostate cancer is a cellular immunotherapy, composed of PC-3 cells (CG1940) and LNCaP cells (CG8711). Each of the components is a prostate adenocarcinoma cell line that has been genetically modified to secrete granulocyte-macrophage colony-stimulating factor. Hypothesizing that GVAX for prostate cancer could be effective in a neoadjuvant setting in patients with locally advanced disease, we initiated a phase II trial of neoadjuvant docetaxel and GVAX. For the trial, the clinical effects of GVAX were assessed in patients undergoing radical prostatectomy (RP).

Methods.

Patients received docetaxel administered at a dose of 75 mg/m² every 3 weeks for 4 cycles. GVAX was administered 2–3 days after chemotherapy preoperatively for four courses of immunotherapy. The first dose of GVAX was a prime immunotherapy of 5×10⁸ cells. The subsequent boost immunotherapies consisted of 3×10⁸ cells. After RP, patients received an additional six courses of immunotherapy. Pathologic complete response, toxicity, and clinical response were assessed. The primary endpoint of the trial was a pathologic state of pT0, which is defined as no evidence of cancer in the prostate.

Results.

Six patients completed neoadjuvant docetaxel and GVAX therapy. No serious drug-related adverse events were observed. Median change in prostate-specific antigen (PSA) following neoadjuvant therapy was 1.47 ng/ml. One patient did not undergo RP due to the discovery of positive lymph nodes during exploration. Of the five patients completing RP, four had a downstaging of their Gleason score. Undetectable PSA was achieved in three patients at 2 months after RP and in two patients at 3 years after RP.

Conclusions.

Neoadjuvant docetaxel/GVAX is safe and well tolerated in patients with high-risk locally advanced PC. No evidence of increased intraoperative hemorrhage or increased length of hospital stay postoperatively was noted. These results justify further study of neoadjuvant immunotherapy.     

新城疫病毒处理瘤细胞的免疫实验研究

目的：观察新城疫病毒（简称ＮＤＶ）对体外培养的肿瘤细胞免疫原性的影响。方法：将经新城疫病毒处理的小鼠肝癌细胞Ｈ２２为活瘤进行免疫接种。结果：ＮＤＶ处理瘤细胞制备的瘤苗可完全排斥再次接种未经病毒处理瘤细胞的生长,瘤苗能增强小鼠体内细胞免疫。结论：ＮＤＶ处理后的瘤细胞能使肿瘤生长减慢、荷瘤生存期明显延长。     

红细胞肿瘤疫苗的制备及对肿瘤复发转移作用的观察

目的：制备一种新型肿瘤疫苗，观察其对肿瘤复发的转移的作用。方法：用PEG将血红细胞（RBC）与小鼠于宫颈癌（U14）细胞融合，制图瘤苗。MTT法检测新型瘤苗诱发的CTL活性。用复发瘤抑制率及肿瘤转移凶制率来评价瘤苗的疗效。结果：融合细胞携带了RBC的血型抗原。在小鼠体内瘤苗可激发特异的CTL活性，对U14细胞的杀伤率为37．8％；而对照小鼠前胃癌（MFC）细胞的杀伤率为22．0％。瘤苗治疗组复发瘤抑制率为39．9％，肺转移的抑制率为69．9％。结论：RBC与肿瘤细胞融合制备的细胞可作为瘤苗。该瘤苗可有效抑制肿瘤复发和转移。     

米托蒽醌处理B16F10-ESAT-6-gpi/IL-21瘤苗特征及其诱导的抗肿瘤免疫反应初探

背景与目的:蒽环类药物处理可使肿瘤细胞免疫原性增加。本文旨在分析米托蒽醌(mitoxantrone,MIT)处理的B16F10-ESAT-6-gpi/IL-21瘤苗特征,初步探讨该瘤苗诱导的抗肿瘤免疫反应。方法:MIT处理B16F10-ESAT-6-gpi/IL-21瘤苗后,用吖啶橙/嗅化乙啶(AO/EB)染色法观察瘤苗细胞形态,流式细胞仪(FCM)检测其粒度及凋亡比例,荧光显微镜观察瘤苗凋亡后细胞膜表面结核杆菌早期分泌靶抗原6KD(ESAT-6)的表达情况,蛋白质印迹法(Western blot)检测瘤苗经MIT处理后IL-21的表达。瘤苗免疫小鼠后,FCM检测了补体依赖的细胞毒性(complement dependent cytotoxicity,CDC)及细胞毒T细胞(cytotoxicT lymphocyte,CTL)活性。结果:经MIT处理后,瘤苗停止分裂,细胞逐渐增大,数日内可保持生物活力,并表达IL-21。瘤苗细胞凋亡后,ESAT-6成点簇状分布于胞膜表面。MIT处理的瘤苗能诱导小鼠产生抗肿瘤免疫应答,免疫鼠血清和CD8+T细胞可分别通过CDC和CTL杀伤野生型B16F10细胞。结论:MIT处理的B16F10-ESAT-6-gpi/IL-21瘤苗失去增殖能力,但仍能表达IL-21且具有免疫原性,能诱导小鼠产生抗肿瘤免疫反应。