摘要
肝细胞癌(HCC)是全世界范围内常见的恶性肿瘤之一，发病率高，预后较差，死亡率高，自然生存期短。手术彻底切除是最佳治疗，然而大多数肿瘤(75％以上)发现时已是中晚期，肿块太大或弥散，无法完全切除，传统的化疗和放疗对无法切除的原发性肝癌治疗效果不佳，放射免疫治疗是近年比较推崇的治疗晚期肝癌方法，其利用具有特异导向能力的单克隆抗体为载体，耦联放射性核素能有效的杀伤肿瘤细胞。国内外已有多种单克隆抗体做为载体应用于放射免疫实验研究，但由于单克隆抗体的自身特性、肿瘤抗原表达的异质性、抗原调变、肿瘤血管的特殊屏障作用、给药途径等多种因素，放射免疫治疗疗效受到很大影响。为增强放射免疫治疗的疗效，既往的研究主要是应用某些细胞因子、增强血管通透性等手段来加强标记抗体与肿瘤细胞抗原的结合能力。目前，运用两种或多种靶向性不同的抗体联合导向治疗、增强放射性核素在瘤体内浓聚和作用时间已经成为放射免疫治疗研究的新热点。
抗HBsAg Fab是一种以乙肝表面抗原为靶点的完全人源化Fab片段，可特异结合于细胞膜分泌的HBsAg，而流行病学表明我国肝癌的发生与HBsAg关系密切，某种意义上可称之为肝癌的“肿瘤相关抗原”；chTNT则是人／鼠嵌合的抗细胞核的单克隆抗体，能特异性结合肿瘤细胞坏死后暴露出来的细胞的核蛋白(DNA组蛋白H1复合体)，研究表明~(131)I标记的chTNT对多种实体瘤均具有抑制作用，已被中国药品食品管理局(SFDA)批准用于晚期肺癌的治疗。
索拉非尼(sorafenib)是一种小分子的多靶点口服抗癌新药，临床前研究和临床实验提示索拉非尼有广泛的抗肿瘤作用，既能抑制肿瘤血管的形成又能直接抑制肿瘤细胞的增殖，已被美国食品药品管理局(FDA)批准用于治疗晚期肾癌及肝癌，并且在其他实体瘤的Ⅱ／Ⅲ期研究中取得令人鼓舞的结果。我们的目的是研究~(131)I-chTNT联合~(131)I-抗HBsAg Fab片断对荷人肝癌HepG2．2．15移植瘤生长抑制的效果，以及应用sorafenib抑制肿瘤微血管生成对~(131)I-chTNT体内分布的影响，为临床提供实验数据，并为肝癌的多种抗体联合放射免疫治疗及与小分子药物的配合治疗提供理论基础。
第一部分抗乙肝表面抗原Fab片段联合抗细胞核单抗为载体的肝癌放射免疫治疗动物实验
一、方法
1．建立人肝癌细胞HepG2．2．15裸鼠皮下移植瘤模型：0．2ml(含活细胞1×10~7)人肝癌HepG2．2．15细胞(对数生长期)悬液接种裸鼠，注射部位为裸鼠右腋下部皮下。待裸鼠成瘤后剥取瘤体周围鱼肉状组织(活性好)为瘤源，分割成1-2cm~3小块，种植于其他裸鼠右侧腋窝下部皮下。
2．~(131)I标记抗HBsAg Fab、chTNT和无关抗体IgG(Idogen法)。测量标记物的标记率、放化纯度、稳定性以及免疫结合率。
3．实验分组：将25只荷HepG2．2．15移植瘤裸鼠按瘤体积随机分成~(131)I-抗HBsAg Fab组、~(131)I-chTNT组、联合用药组、~(131)I-无关抗体组(~(131)I-IgG)、空白对照组共五组，每组5只。
4．给药：①~(131)I-抗HBsAg Fab组：~(131)I-抗HBsAg Fab，14．8MBq／40ul，瘤内注射，(A组)；②~(131)I-chTNT组：~(131)I-chTNT，14．8MBq／40ul，瘤内注射，(B组)；③联合用药组：~(131)I-抗HBsAg Fab 7．4MBq／20μl+~(131)I-chTNT 7．4MBq／20μl，瘤内注射；(C组)；④阳性对照组：~(131)I-IgG，14．8MBq／40μl瘤内注射，(D组)；⑤阴性对照组：生理盐水40μl瘤内注射(E组)。
5．给药前测量每只裸鼠体重和肿瘤大小，给药后每天观察裸鼠的生长、生活情况，测量肿瘤的长径、短径，每周两次，按公式移植瘤体积=(长径×短径~2)／2计算瘤体积。
6．瘤内注射后第1、4、7天将荷瘤裸鼠麻醉后进行SPECT显像，计算瘤体放射性占全鼠放射性的比值和肿瘤与周围组织放射性比值(T／NT)，观察标记抗体在裸鼠体内的分布及代谢情况。第28天处死全部实验裸鼠，计算肿瘤抑制率并取移植瘤行病理形态学观察。
7．统计处理：实验数据均采用SPSS11．5软件进行统计处理，定量资料数据以(?)±S表示。标记抗体的免疫结合率比较采用两独立样本的t检验：抗体在裸鼠体内T／NT比值和用药前后对照组与各处理组移植瘤体积比较采用重复测量的方差分析，两两比较采用LSD法，方差不齐时采用Welch和Tambane's T2检验，P≤0．05为差异统计学意义。
二、结果
1．三氯醋酸法测得抗HBsAg Fab、chTNT、IgG平均标记率分别为60％、50％和58．3％，经纯化后测得放化纯度分别为100％、99％和99．3％；平均放射性比活度分别为22．2MBq／mg、30．8MBq／mg和19．5MBq／mg，结合分析法测定~(131)I-抗HBsAgFab与HepG2．2．15免疫结合率为68．3％，与HepG2免疫结合率为10．9％，两者之间有显著性差异(P<0．001)。
2．瘤内注射标记抗体后第1天，各处理组T／NT比值均较高，组间无显著差异(P>0．05)。第7天，联合抗体组与~(131)I-抗HBsAg Fab组、~(131)I-chTNT组T／NT比值均有显著差异，P值分别为0．005和0．029，~(131)I-抗HBsAg Fab组与~(131)I-chTNT无显著差异(P=0．386)。阳性对照组第1天可见显影，第7天可见放射浓聚明显减低；阴性对照组肿瘤内始终未见放射性浓聚。
3．各处理组给药前后不同时间内裸鼠瘤体积有明显差异(F=477．615；P<0．001)，且各组间肿瘤大小差异显著(F=14．287、P<0．001)，终止观察时，~(131)I-chTNT组与联合组有显著差异(P=0．003)，~(131)I-HBsAg Fab组与联合组同样有显著性差异(P<0．001)，~(131)I-chTNT组与~(131)I-HBsAg Fab组无明显差异(P=0．325)，联合组的抑瘤率为84．32％，明显高于~(131)I-chTNT组的63．86％、~(131)I-抗HBsAg Fab组的51．49％及无关抗体组的14．87％。
4．肿瘤组织经HE染色，与对照组相比，三种用药处理组瘤体内坏死组织多，肿瘤细胞核明显固缩，结构破坏，部分区域肿瘤细胞全部坏死，联合抗体组最明显。
三、结论
1．~(131)I标记抗HBsAg Fab和chTNT具有良好的免疫活性和稳定性。
2．采用瘤内注射，核素标记抗体不需要血液循环，直接进入肿瘤组织，可明显增加标记抗体在肿瘤内的浓聚，提高导向治疗效果。
3．与单药相比，~(131)I-抗HBsAg Fab联合~(131)I-chTNT瘤内注射，放射性核素能更持久的存在于肿瘤组织，具有更强的肿瘤杀伤能力。
第二部分索拉非尼对~(131)I-chTNT裸鼠体内分布及抑制肿瘤生长能力的影响
一、方法
1．建立人肝癌细胞HepG2裸鼠皮下移植瘤模型：0．2ml(含活细胞2×10~6)人肝癌HepG2细胞(对数生长期)悬液接种裸鼠，注射部位为裸鼠右腋下部皮下。待裸鼠成瘤后剥取瘤体周围鱼肉状组织(活性好)为瘤源，分割成1-2cm~3小块，种植于裸鼠右侧腋窝下部皮下。
2．~(131)I标记chTNT(方法同第一章)。
3．索拉非尼对肿瘤微血管生成的作用及对~(131)I-chTNT裸鼠体内分布的影响：将33只荷瘤雄性BALB／c裸鼠随机分为3组，第一组12只，按40mg／kg给予sorafenib灌胃，第二组12只、第三组9只，均给予同等剂量生理盐水灌胃，持续14天。第15天于第一组和第二组各取3只裸鼠，处死，取出皮下瘤，免疫组织化学法行移植瘤微血管密度(MVD)检测。余下裸鼠均给予~(131)I-chTNT14．8 MBq／40ul，瘤内注射后，第二组按40mg／kg给予sorafenib灌胃，其他两组给予同等剂量生理盐水。瘤内注射~(131)I-chTNT后第1、4、7天每组各取三只裸鼠，行放射免疫显像后脱颈处死，取出每只裸鼠瘤体、心、肝、肾、血、肺和脾，分别称取重量(g)，并放入井型定标仪中测量放射性计数(D)，计算每克组织的放射性计数(D／g)，并计算瘤／非瘤(T／NT)比值。
4．索拉非尼联合~(131)I-chTNT对裸鼠移植瘤的抑制作用：25只荷瘤裸鼠分为5组，每组5只。①sorafenib组：sorafenib，40 mg／kg，0．1ml灌胃，持续14天+瘤内注射生理盐水40ul d1；②chTNT组：~(131)I-chTNT 14．8 MBq／40ul，瘤内注射d1+0．1ml生理盐水灌胃，持续14天；③序贯联合组：sorafenib，40 mg／kg，0．1ml，持续14天后瘤内注射~(131)I-chTNT 14．8 MBq／40ul；④同时联合组：sorafenib，40mg／kg，0．1ml持续14天+瘤内注射~(131)I-chTNT 14．8 MBq／40ul d1；⑤对照组：生理盐水0．1ml灌胃，持续14天+生理盐水40ul瘤内注射，d1。观察裸鼠移植瘤生长情况，4周后处死裸鼠，称重并测量瘤体积，计算肿瘤抑制率，并行病理形态学观察。
5．实验数据均采用SPSS 11．5软件进行统计处理，定量资料数据以(?)±S表示，两处理组MVD的比较采用两独立样本的t检验；抗体在裸鼠体内T／NT比值比较和用药前后对照组与各处理组移植瘤体积比较采用重复测量的方差分析，两两比较采用LSD法，方差不齐时采用Welch和Tambane's T2检验，P≤0．05为差异统计学意义。
二、结果
1．索拉非尼能明显抑制肿瘤微血管生成。经sorafenib灌胃14天后，裸鼠肿瘤组织MVD明显低于对照组(P=0．004)。
2．肿瘤微血管密度减少能明显提高~(131)I-chTNT在瘤内的浓聚程度，延长瘤内浓聚时间。经过sorafenib灌胃14天后的序贯给药组T／NT比值明显高于给予生理盐水灌胃的同时给药组和chTNT组，有显著性差异，均为P<0．01，从瘤内注射~(131)I-chTNT后第1天到第7天均如此，同时给药和chTNT组间无显著差异，第1天和第7天P值分别为0．187和0．933。
3．序贯联合给药组与同时联合给药组两者抑制肿瘤能力无显著差异，但是两组分别与其他组肿瘤大小比较均出现显著性差异，均为P<0．05。序贯联合组的抑瘤率为85．02％，同时联合给药组为89．05％，明显高于~(131)I-chTNT组的57．64％和sorafenib组的56％，
4．与对照组比较，其他处理组可见大片细胞凝固性坏死，细胞与细胞间质无明显分界，细胞壁遭到破坏，细胞核固缩、碎裂，序贯联合组和同时联合组最明显。
三、结论
1．索拉非尼能明显抑制肿瘤的新生血管生成。
2．肿瘤的微血管密度减少能够延长瘤内注射~(131)I-chTNT的代谢时间，提高肿瘤组织的~(131)I-chTNT浓度，减少其他器官的~(131)I-chTNT浓度。
3．联合运用sorafenib和~(131)I-chTNT比单用sorafenib或~(131)I-chTN具有更强的肿瘤杀伤效果。
Hepatocellular carcinoma (HCC) is one of the common malignant tumor in world-wide. High incidence, poor prognosis, high mortality, and short survival time are its features. Surgical excision is the best treatment, However, the majority of tumors (75%) are in intermediate or advanced stage when found. The volume of tumor is so big that it can not be completely resected. The traditional chemotherapy and radiotherapy are ineffective in the unresectable primary liver cancer. Radioimmunoassay treatment is respected as a better treatment of advanced hepatocellular carcinoma in recent years. It uses monoclonal antibodies as carrier which has specific oriented ability, and it can kill tumor cell when coupling with radionuclide. a variety of monoclonal antibodies are applied to radioimmunoassay experimental study as carrier at home and abroad. But as for some factors such as characteristics of Monoclonal Antibody, the expressive heterogeneity of tumor antigen,the antigen modulation, the special barriers function of tumor angiogenesis, the route of administration, the effects of radioimmunotherapy has been greatly affected. Some means such as applying cytokines, increasing vascular permeability are used to strengthen the binding capacity of labeled antibody with tumor cell antigen, then the effect of radioimmunotherapy can be enhanced. Recently, some methods such as combining two or more different targeting antibodies, enhancing the concentration of radionuclide in the tumor tissue has become the new hot spots of RIA treatment.
Anti HBsAg Fab is a complete humorized Fab fragment which targets for hepatitis B surface antigen(HBsAg), and it can bind with the HBsAg excreted from cell membrane specificly. The epidemiology shows that HBsAg has a close correlation with the occurrence of liver cancer in China, and HBsAg is called "HCC associated antigen" in a sense. Chimeric Tumor Necrosis Treatment (chTNT) is a human/mouse chimeric monoclonal antibody which can bind with nucleoprotein that exposes when tumor cell necrosis (DNA histone H1 complex). Study shows that chTNT labeled with ~(131)I has a suppressive function to a variety of solid tumors, and it is licensed to treat advanced lung cancer by Chinese Food and Drug Administration (SFDA).
Sorafenib is a multi-target small molecule oral anticancer drug. Preclinical study and clinical trial predicts that sorafenib has a wide anti-tumor function, for it can not only inhibit the formation of vessel but also suppress proliferation of tumor cell directly, and it is licensed to treat advanced renal carcinoma by Food and Drug Administration (FDA). It also achieves encouraging results in II /III stage reseaches on other solid tumor. Our aim is to study the inhibitory effect of combining ~(131)I-chTNT with ~(131)I- anti-HBsAg Fab to the subcutaneous humor liver caner xenografts of HepG2.2.15 and the effect of distribution in vivo of ~(131)I-chTNT by applying sorafenib to suppress the Tumor micrangiogenesis, to provide experiment statistics for clinic, and to lay the foundation for combining multi-antibodies target therapy and Small molecule drugs.
Chapter 1. Animal experiment studies on the radioimmunotherapy of the
hepatoma with intratumoral injection combining ~(131)I-humanrized anti-HBsAgFab with ~(131)I-chTNT
Methods:
1. A model of human liver cancer was establishes by subcutaneous transplantation of HepG2.2.15 cells in nude mice: 0.2ml suspension containing 1×10~7 HepG2.2.15 hepatoma cells is inject into the hypo of armpit. Then divided the tumor tissue into small pieces of l-2cm3 when the tumor has big enough, and implantate them into the hypo of armpit of other nude mice.
2. Measure the marking rate, constancy and the immunobinding rate of the anti-HBsAg Fab, chTNT and IgG was labeled by ~(131)I.
3. 25 mice are randomly divided into 5 groups: ~(131)I-anti-HBsAg Fab group; ~(131)I-chTNT group; combination routine treatment group; positive control group; negative control group.
4. Treatments is designed as follows: the mice in 131I-anti-HBsAg Fab group (A group) receives 14.8MBq/40ul ~(131)I-anti-HBsAg Fab; the mice in ~(131)I-chTNT group(B group) receives 14.8MBq/40ul ~(131)I-chTNT; the combination routine treatment group(C group) receives 7.4MBq/20ul ~(131)I-chTNT + 7.4MBq/20ul ~(131)I- anti-HBsAg Fab; the mice in positive control group(D group) receives 14.8MBq/40ul ~(131)I-IgG; the mice in negative control group(E group) receives 40ul normal saline. Treatment is given by intratumorally injection.
5.After treatment, the situation of growth and living of nude mice is observed everyday. Measure the long-diameter (a) and the short diameter (b). Calculate the volume of transplanted tumor using the formula: volume =0.5*a*b*b.
6.On the first, forth and seventh day, anesthetize the nude mice for SPECT imaging, and calculate the ratio of radioactivity in tumor/non-tumor tissue, then observe the distribution and metabolism of antibody labeled with ~(131)I in vivo. On the 28~(th) day, all mice are killed by anesthetic overdose. The tumors are obtained from xenografts, then calculate the inhibition ratio of transplanted tumor and observe the pathological situation in microscope.
7. The sample data are analyzed statistically by using SPSS 11.5 software. The quantity data are represented by the way of "Mean±SD"; As for the immunobinding rate of antibody labeled with radionuclide, Independent-Sample T Test is used ; As the ratio of T/NT of antibody in nude mice and the comparison of the transplant tumor volume among groups before and after taking the medicine, Repeated Measure ANOVA is used. among it, the comparison between two groups use LSD Test while Welch or Tambane's T2 Test with equal variances not assumed.
Results:
1.The average marking ratio of anti-HBsAg Fab, chTNT and IgG measured by TCA is 60%, 50% and 58.3% separately. The radiochemical purity of ~(131)I-anti-HBsAg Fab,~(131)I-chTNT and ~(131)I-IgG after purification is 100%、99 %和99.3% separately, and the mean activity ratio of them is 22.2MBq/mg、30.8MBq/mg和19.5MBq/mg individually. The immunobinding rate of ~(131)I-anti-HBsAg Fab with HepG2.2.15 cell is 68.3% while that of ~(131)I-anti-HBsAg Fab with HepG2 cell is 10.9% ( P<0.001).
2. The ratios of T/NT in ~(131)I-chTNT group (B group), ~(131)I-anti-HBsAg Fab (A group) group and combination group (C group) arise gradually, Form the point of view of time, there is no significant difference in T / NT ratio among groups on the first day after the treatment (P >0.05).On the seventh day, there is significantly difference in ratio of T/NT between C group and A or B group, the P value is 0.005 and 0.029 separately, nor is the ratio between A group and B group(P=0.386). image is visible in positive control group on the first day, but on the seventh day, the concentration of radiation decreased significantly. Radioactivity can not be found in negative control group all the time.
3. As for the volume of tumor, there is significant difference among groups at the different time(F= 14.287、P<0.001), so is the difference in the volume of tumor among groups (F= 14.287、P<0.001). In the forth week, there is significantly difference in volume of tumor between C group and B group(P<0.003), so is the difference between C group and A group(P<0.001), and nor is the difference between A group and B group(P=0.325). The growth inhibitory rates of C group is 84.32% when the observation is over, which is significant higher than 63.86% of B group, 51.49% of A group and 14.87% of D group.
4. After staining with HE, the necrosis tissue in the there groups of drug treatment are more than that in control group, the nucleus of tumor cells are pycnosis significantly, and the combination group is most obviously.
Conclusion:
1. The ~(131)I-anti-HBsAg Fab and ~(131)I-chTNT have good constancy and immunocompetence.
2. Intratumoral injection can increase the concentration of antibody labeled with radionuclide in the tumor tissue to improve the effect of targeted therapy, because it can induce the antibody to enter into tumor tissue directly and need not circulate through the blood.
3. Combining ~(131)I-anti-HBsAg Fab with ~(131)I-chTNT, radionuclides can be more durable in the tumor tissue and have a stronger capacity of killing tumor cells than using ~(131)I-anti-HBsAg Fab or ~(131)I-chTNT alone.
Chapter 2 The study of how sorafenib effect the distribution of ~(131)I-chTNT innude mice and the ability to inhibit tumor growth of ~(131)I-chTNT
Methods
1. A model of human liver cancer was establishes by subcutaneous transplantation of HepG2 cells in nude mice: 0.2ml suspension containing 2×10~6 HepG2 hepatoma cells is inject into the hypo of armpit. Then divided the tumor tissue into small pieces of 1-2cm~3 when the tumor has big enough, and implantate them into the hypo of armpit of other nude mice.
2. Labeling chTNT by 131I.
3. 33 mice are randomly divided into 3 groups. Treatments is designed as follows: twelve mice in the first group receive sorafenib ,40mg.kg~(-1); twelve mice in the second group and nine mice in the third group receive normal saline at the same dose, Treatment is given by intragastric administration and lasts for 14 days; In the 15~(th) day, take 3 mice from the first and the second group separately, kill them by anesthetic overdose and dislodge the transplanted tumor, and tumor microvessel density(MVD) is measured by inmunohistochemistry. The remaining mice are given intratumorally injection with 14.8MBq/20ul ~(131)I-chTNT, then the mice in second group receives sorafenib, 40mg.kg~(-1). The other mice receives normal saline at the same dose. On the first, forth and seventh day after being treated with ~(131)I-chTNT, take 3 mice separately in every group to make SPECT imaging, Then kill the mice, take out and weigh(g) the tumor, heart, blood, liver, lung, kidney, spleen of each mouse. Next measure the radiocounting (D) by well-type calibrating machine. Calculate the proportionality of D/g and the ratio of tumor/non-tumor tissue (T/NT).
4. 25 mice and divide them into 5 groups stochastically. Treatments is designed as follows: the mice in sorafenib group receives sorafenib 40mg.kg~(-1)+ normal saline 40ul by intratumorally injection; the mice in ~(131)I-chTNT group receives 14.8MBq/40ul ~(131)I-chTNT + normal saline 1ml, with intragastric administration lasting for 14 days; the sequent combination group receives 14.8MBq/40ul ~(131)I-chTNT after sorafenib 40mg.kg~(-1) is given 14 days; the mice in simultaneous combined group receives 14.8MBq/40ul ~(131)I-chTNT + sorafenib 40mg.kg~(-1) at the same time; the mice in control group receives 40ul normal saline intratumoral injection + normal saline 1ml intragastric administration, lasting 14 days. sorafenib is given by intragastric administration and continues for 14 days, and ~(131)I-chTNT is given by intratumoral injection. After treatment, observe the situation of growth and living of nude mice. All mice are killed after 4 weeks, then calculate the volume of transplanted tumor and the inhibition ratio, then observe the pathological situation of transplanted tumor in microscope.
5. The sample data are analyzed statistically by using SPSS 11.5 software. The quantity data are represented by the way of "Mean±SD"; As for MVD in tumor tissue of two groups, Independent-Sample T Test is used ; As the ratio of T/NT of antibody in nude mice and the comparison of the transplant tumor volume among groups before and after taking the medicine,, the Repeated Measure ANOVA is used, among it,, and the comparison between two groups use LSD Test while Welch or Tambane's T2 Test with equal variances not assumed.
Results
1. The MVD of tumor tissue in sorafenib group is significantly lower than that of in control group (P=0.004).
2. The radionuclide concentration level of tumor tissue in sequent combination group is higher than that of in other groups obviously (F=74.258, P<0.001). From the first day to the seventh day after intratumoral injection, comparing with the simultaneous combination group and the ~(131)I-chTNT group, the ratio of T/NT in sequent combination group is obviously higher than that of in them after intratumorally injecting ~(131)I-chTNT, and it has statistical difference among there groups , the value of P is all less than 0.01. At the same time, there is no statistical difference between simultaneous combination group with the ~(131)I-chTNT group, and the P value in the first and seventh day is 0.187 and 0.933 respectively.
3. In the transplantat tumor suppressive experiment, tumor growth in sequent combination group and simultaneous combination group is suppressed significantly, and there is no statistical difference between them, but there is statistical difference between groups mentioned above and other groups, the P values are less than 0.05. At the end of experimental period, the growth inhibitory rates in sequent combination group and simultaneous combination group is 85.02% and 89.05%, while that of 131I-chTNT group and sorafenib group is 57.64% and 56%.
4.the necrosis tissue in the groups of drug treatment are more than that in control group, the nucleus of tumor cells are pycnosis significantly, and the simultaneous combination group and sequent combination group is most obviously.
Conclusion:
1. Sorafenib can significantly inhibit the angiogenesis of transplant tumor.
2. The decrease MVD transplant tumor can extend the metabolism time of radionuclides and enhance concentration of ~(131)I-chTNT in tumor tissue.
3. Combining sorafenib with ~(131)I-chTNT have a stronger anti-tumor effect than using sorafenib or ~(131)I-chTNT alone.
引文
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