抗独特型抗体及其在卵巢癌研究中的现状

随着近年来对独特型-抗独特型免疫调节网络的深入研究,抗独特型抗体作为肿瘤免疫治疗的新途径越来越受到人们重视。本文简述近年来抗独特型抗体在肿瘤研究中的进展,重点介绍有关卵巢癌方面抗独特型抗体研究现状,为妇科肿瘤免疫学研究提供新信息。     

卵巢癌抗独特型抗体的研究进展

卵巢癌抗独特型抗体的研究进展张香云崔恒钱和年抗独特型抗体（Ａｂ２）具有模拟抗原和免疫调节双重作用，同时能够克服机体免疫抑制或打破其免疫耐受状态。近年来，在国外已试用于结肠癌、恶性黑色素瘤、淋巴瘤等人类恶性肿瘤的治疗，并有成功的报道［１～４］。Ａｂ２在...     

应用基因工程方法制备卵巢癌抗OC125独特型抗体

目的；扩增和表达卵巢癌抗独特型鼠单克隆抗体变区基因，用于制备新型卵果癌疫苗。方法：自产生卵巢抗原内影象型抗ＯＣ１２５特特型抗体的小鼠杂交瘤细胞中提取ｍＲＮＡ，采用反转录聚合酶链，扩增抗体轻、重链可变区基因，连接成单链后重组至表面表达载体ｐＣＡＮＴＡＢ５Ｅ，转化大肠杆菌ＴＧ１，用辅助噬菌体拯救包装成完整噬菌体，用酶联免疫吸附法进行筛选鉴定。结果：获得抗ＯＣ１２５独特型抗体轻、重链可变区基因，长度约３     

模拟卵巢癌抗原的抗独特型抗体及其临床应用

肿瘤抗原在肿瘤基础研究及临床诊断和治疗中均占有极其重要的地位。但遗憾的是目前已明确的肿瘤抗原极少 ,且纯化困难 ,难以大量制备 ,加上肿瘤抗原为弱抗原 ,在体内缺乏第二信号系统的情况下 ,难以刺激机体产生有效的抗肿瘤细胞免疫反应。因此 ,为实现临床免疫诊断和免疫治疗 ,作为可行的替代方法之一是制备可模拟肿瘤抗原的抗独特型抗体。1　关于抗独特型抗体的基础理论1 1　免疫网络学说　免疫网络学说是著名诺贝尔奖获得者Ｊｅｒｎｅ于上个世纪 70年代提出的。该学说认为抗体、Ｂ细胞受体和Ｔ细胞受体的可变区均存在着具有淋巴细胞克隆…     

抗独特型抗体免疫治疗卵巢癌的动物实验动物实验研究

目的：研究抗独特型抗体对卵巢癌的免疫治疗作用，为临床试验提供动物实验依据。方法用人卵巢癌抗独特型抗体免疫杂交一代小鼠ＢＣＦ１，为实验组；用正常小鼠ＩｇＧ免疫小鼠ＢＣＦ１，为对照组。     

抗独特型抗体免疫治疗卵巢癌的动物实验研究

目的：研究抗独特型抗体对卵巢癌的免疫治疗作用，为临床试验提供动物实验依据。方法：用人卵巢癌抗独特型抗体（Ａｂ２）免疫杂交一代小鼠ＢＣＦ１，为实验组；用正常小鼠ＩｇＧ免疫小鼠ＢＣＦ１，为对照组。两组均免疫３次，于末次免疫后１周，将经裸鼠传代的人卵巢癌细胞系移植于ＢＣＦ１小鼠肾脏包膜下，分别于移植后第２、４、６、８和１０天处死小鼠，取血清进行抗独特型抗体（Ａｂ３）分析。移植瘤行组织学检查，观察宿主淋巴细胞浸润情况及瘤细胞可见率。结果：实验组小鼠肾脏包膜下人卵巢癌细胞系很快受到排斥，在移植后第６天，淋巴细胞浸润即达高峰，而对照组在第１０天才达高峰；瘤细胞可见率，在移植第６天后即明显降低，而对照组呈逐渐下降。结论：Ａｂ２作为抗原免疫小鼠后，能够排斥瘤细胞的生长。     

应用卵巢癌抗独特型单克隆抗体6B11进行血清中卵巢癌抗体检测

探讨应用卵巢癌抗独型单克隆抗体６Ｂ１１进行血清中相关抗体检测的可行性。方法；采用间接免疫酶法测定妇科良性与恶性肿瘤患者各３０与８８例，子宫内膜异位症患者９例，正常女性献血员４５例共１７２份血清，１５例卵巢癌患者年内１３３份血清卵巢部抗体水平，并与病情作比较。     

6B11抗独特型微抗体负载树突状细胞对卵巢癌细胞系细胞杀伤作用的体外研究

目的　用 6B1 1抗独特型微抗体体外负载树突状细胞 (DC) ,以期诱导出抗原特异性的抗肿瘤免疫反应。方法　分离培养HLA -A2 健康人外周血树突状细胞 ,培养过程中用 6B1 1微抗体负载 ,无关抗原F (ab)’2负载及未负载组为对照。光镜、电镜下观察树突的形态 ;流式细胞仪检测DC表面分子表达 ;3 H -TdR掺入法测DC刺激自体T细胞增殖 ;51 Cr 6h释放试验测激活的T细胞对卵巢癌细胞系的杀伤。结果　培养的树突细胞有典型形态 ,CD80、CD86、HLA -DR等表面分子呈高表达 (分别为 6 5 %、 86 2 %、 93 7% ) ;6B1 1微抗体负载的DC不仅能刺激自体T细胞的增殖 ,而且其诱导的CTL细胞对HLA -A2 、OC1 6 6 - 9 卵巢癌细胞系HOC1A有特异性的杀伤 ,结论　 6B1 1抗独特型微抗体模拟卵巢癌抗原OC1 6 6 - 9负载树突状细胞在体外可以诱导出抗原特异性的细胞毒T细胞 ,为进一步研究 6B1 1微抗体对卵巢癌免疫治疗作用提供了依据     

卵巢癌抗独特型抗体诱导特异性迟发过敏反应的实验研究

本研究应用卵巢癌抗原内影像型单克隆抗独特型抗体６Ｂ１１和１Ｈ１２代替肿瘤抗原，诱导同系小鼠产生卵巢癌特异性细胞免疫反应－迟发性过敏反应（ＤＴＨ），从而证明了６Ｂ１１和１Ｈ１２具有抗原模拟作用。经过６Ｂ１１或１Ｈ１２免疫的小鼠，用卵巢癌细胞系ＳＫＯＶ３进行脚掌注射攻击。而后，测量脚掌水肿厚度作为ＤＴＨ反应强度。６Ｂ１１诱导的ＤＴＨ平均水肿厚度为０．９３ｍｍ，卵巢癌组织抗原ＯＣ１６６－９诱导的阳性对照     

6811抗独特型微抗体负载树突状细胞对卵巢癌细胞系细胞杀伤作用的体外研究

目的 用6811抗独特型微抗体体外负载树突状细胞(DC)，以期诱导出抗原特异性的抗肿瘤免疫反应。方法 分离培养HLA-A2 健康人外周血树突状细胞，培养过程中用6811微抗体负载，无关抗原F(ab)2负载及未负载组为对照。光镜、电镜下观察树突的形态；流式细胞仪检测DC表面分子表达；^3H-TdR掺入法测DC刺激自体T细胞增殖；^51Cr6h释放试验测激活的T细胞对卵巢癌细胞系的杀伤。结果 培养的树突细胞有典型形态，CD80、CD86、HLA-DR等表面分子呈高表达(分别为65％、86．2％、93．7％)；6811微抗体负载的DC不仅能刺激自体T细胞的增殖，而且其诱导的CTL细胞对HLA-A2 、OC166-9 卵巢癌细胞系HOC1A有特异性的杀伤，结论 6811抗独特型微抗体模拟卵巢癌抗原OC166-9负载树突状细胞在体外可以诱导出抗原特异性的细胞毒T细胞，为进一步研究6811微抗体对卵巢癌免疫治疗作用提供了依据。     

A novel human monoclonal antibody against cervical cancer: its immunoreactivity with normal tube and ovary and with ovarian tumor tissue

1-1-2D, a novel human monoclonal antibody (MAb) raised against cervical cancer, was examined for its immunohistochemical reactivity with ovarian cancer. Six of 10 ovarian cancer cell lines showed positive staining, while 3 of 5 cervical cancer cell lines were positive. Among tumor tissues, 15 of 18 (83%) ovarian serous cystadenocarcinomas and 10 of 12 (83%) ovarian clear cell adenocarcinomas were positive. We also performed immunohistochemical staining of the same cancer specimens with OC 125 and compared their reactivity. The frequency of positivity was similar, but the reactivity of the two MAbs was different. 1-1-2D stained the apical surface of the glandular epithelial cells and secretory products of the gland. On the other hand, OC 125 stained the cytoplasm as well as the plasma membrane of the glandular epithelial cells. These results suggest that 1-1-2D MAb recognizes a different antigen from that recognized by OC 125.     

卵巢癌独特型疫苗免疫接种方案的探讨

目的：探讨卵巢癌抗独特型抗体（６Ｂ１１ ）疫苗免疫接种的理想方案。方法：用６Ｂ１１ 免疫ＢｃＦ１ 小鼠,观察基础免疫和加强免疫诱导的免疫应答情况,探讨加强免疫间隔时间、剂量及免疫途径对诱导免疫应答的影响,基础免疫为６Ｂ１１ １００μｇ—ＫＬＨ＋ ＣＦＡ,加强免疫为６Ｂ１１—ＫＬＨ＋ ＩＦＡ（或ＰＢＳ）,免疫部位为小鼠尾根及足底皮下或腹腔。观察指标：体液免疫为免疫鼠血清中抗原特异结合性抗独特型抗体（Ａｂ３ ）滴度,细胞免疫为免疫鼠脾脏淋巴细胞ＣＤ４＋ 、ＣＤ８＋ 亚群的变化。结果：（１）６Ｂ１１ 基础免疫后,Ａｂ３ 阳性率为８０．００％ ,Ａｂ３ 滴度高峰在第１４～２１天（１：３２０）,ＣＤ４＋ 亚群增加,ＣＤ＋８ 亚群变化不明显;（２）间隔４ 周加强免疫诱导的Ａｂ３ 滴度较２ 周者高,ＣＤ４＋ 亚群增加明显;６Ｂ１１５０μｇ 加强免疫诱导的免疫应答强于３０、１５μｇ;皮下途径加强免疫诱导的免疫应答略强于腹腔注射;（３）６Ｂ１１３ 次免疫后,Ａｂ３ 产生快（第３天１：３２０）,滴度高（最高１：６４０）,持续时间长（第４９天为１：４０）,ＣＤ＋４ ／ＣＤ８＋ 比值明显增加。结论：卵巢癌抗独特型抗体６Ｂ１１可作为肿瘤疫苗诱导ＢｃＦ１小鼠?     

卵巢上皮性癌单克隆抗体183B2相关抗原的纯化和鉴定

目的纯化卵巢上皮性癌单克隆抗体（单抗）183B2相关抗原,并对该抗原及其理化特性进行鉴定和分析。方法用间接酶联免疫吸附试验（ELISA）方法筛选出与单抗183B2阳性反应较强的卵巢上皮性癌腹水标本,继而用免疫亲和层析技术纯化得到相应抗原。纯化后的抗原分别经高碘酸钠、神经氨酸酶、去糖脂混合液及胰蛋白酶、链酶蛋白酶破坏糖链和肽链后,用间接ELISA方法检测其活性,并用十二烷基磺酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）及蛋白印迹（western blot）法进行鉴定。再进一步用双向电泳结合western blot对卵巢上皮性癌细胞株SKOV3细胞的相应抗原进行验证。结果纯化后的抗原经破坏糖链处理后活性不变,而经破坏肽链及加热处理后活性消失。SDS—PAGE显示,该抗原两个不同亚单位的相对分子质量分别为56000和25000,而前者能与单抗183B2反应。纯化后的抗原N端氨基酸测序后进行同源序列分析发现,此序列与人Ig重链N端的15个氨基酸相同。确定了双向电泳凝胶上3个目标点的位置,并初步确定该蛋白点的相对分子质量约为56000,等电点为5．3～5．8。结论卵巢上皮性癌单抗183B2相关抗原决定簇位于糖蛋白的肽链上,为Ig超家族成员,它可能是一种新的肿瘤相关抗原。     

卵巢癌单抗183B2相应抗原的纯化及其特性分析

目的　纯化卵巢癌单克隆抗体 1 83B2相应抗原 ,并鉴定该抗原的理化特性。方法　将STRE AMLINErProteinA纯化的单抗 1 83B2与CNBr活化的sepharose 4B相偶联 ,制备亲和层析柱 ,再将用饱和硫酸铵粗提的病人腹水上柱纯化得到 1 83B2相应抗原。提纯抗原经高碘酸钠、胰蛋白酶、链酶蛋白酶、神经氨酸酶、去糖脂混合液及加热处理后进行间接ELISA检测。结果　经亲和层析纯化得到活性好的 1 83B2相应抗原。经高碘酸钠、神经氨酸酶、去糖脂混合液处理后抗原活性不变 ,而胰蛋白酶、链酶蛋白酶、及加热处理后抗原活性消失。结论　单抗 1 83B2相应的抗原决定簇位于肽链上 ,它可能是一种新的卵巢癌肿瘤相关抗原     

模拟卵巢癌抗原的人源化抗独特型抗体的构建与表达

目的 对模拟人卵巢癌抗原的鼠源性抗独特型单链抗体6B11scFv进行人源化改造和表达。方法采用重叠PCR和基因工程技术,将6811scFv基因与人IgG1铰链区和CH3区的基因进行融合,构建人源化抗独特型抗体6811VHVLhC基因。用IPTG诱导大肠杆菌进行表达,并采用酶切法、DNA测序、SDS—PAGE电泳、ELISA和免疫印迹技术等进行鉴定。结果酶切和DNA测序表明插入片段正确;SDS—PAGE凝胶电泳显示融合蛋白分子量50 000 Da处有一诱导蛋白带;而不连续非变性凝胶电泳表达产物分子量为100 000 Da;ELISA和免疫印迹结果显示,表达出的6811VHVLhC人源化抗体仍具有6B11scFv和人IgG1CH3的活性。结论 已成功构建并表达出双价的可模拟卵巢癌抗原的人源化抗独特型抗体。     

Imaging of ovarian cancer with radiolabelled monoclonal antibodies

Summary This article presents the state of the art of immunoscintigraphy (IS) of ovarian cancer. We will review the monoclonal antibodies (MoAbs) used in clinical trials: (HMFG1/2, OC125, H317, H17E2, NDOG2 and 791T/36). We conclude that none of the afore mentioned MoAbs are clearly superior and that IS cannot yet replace laparotomy for the diagnosis of overian cancer but may have a role in the follow-up of ovarian cancer, in timing second-look surgery and assessing the response/TD treatment.     

Anti-tumor activity of an anti-DR5 monoclonal antibody, TRA-8, in combination with taxane/platinum-based chemotherapy in an ovarian cancer model

Objective

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis via binding to death receptors and enhances the anti-tumor effect of conventional cancer therapies. We evaluated the efficacy of TRA-8, an agonistic antibody to DR5, combined with docetaxel and carboplatin in vitro in an intraperitoneal (IP) ovarian cancer model.

Methods

Luciferase positive ES2 cells (ES2H) were treated in 96 well plates with TRA-8, carboplatin, docetaxel, and combination therapy. Cell viability was assessed using ATP-lite assay. Apoptosis was confirmed via Western blot analysis. ES2H cells were injected IP into female athymic nude mice. Animals were sorted based on bioluminescent signal with the following treatments: 1) untreated; 2) TRA-8 alone; 3) docetaxel + carboplatin; and 4) docetaxel + carboplatin + TRA-8. Animals receiving TRA-8 antibody were injected IP with 200 μg of TRA-8 twice weekly until death. Animals receiving docetaxel + carboplatin were injected IP with 5 mg/kg and 15 mg/kg respectively every 3 weeks until death. Animals were assessed for tumor burden using bioluminescence imaging and overall survival.

Results

Combination therapy reduced viability of ES2H cells in vitro over single agent therapy. Tumor burden was lowest in the chemotherapy + TRA-8 group at days 23 (p < 0.001) and 30 (p = 0.04). Mean survival was greatest in the chemotherapy + TRA-8 group (41 days) compared to the chemotherapy only group (34 days) and control group (27 days) as determined by Kaplan-Meier analysis (p < 0.001).

Conclusion

Conventional chemotherapy combined with TRA-8 reduced cell-viability via activation of apoptotic pathways, reduced tumor burden and improved survival in this ovarian cancer model.     

A pilot phase 2 study of oregovomab murine monoclonal antibody to CA125 as an immunotherapeutic agent for recurrent ovarian cancer

This prospective, open-label, pilot phase 2 study examined the clinical and immunologic effects of oregovomab (OvaRex) in heavily pretreated patients with recurrent ovarian cancer (OC). Thirteen women were administered intravenous oregovomab (2 mg) at weeks 0, 2, 4, 8, and 12, followed by quarterly doses for up to 2 years or disease progression. Concomitant chemotherapy was not permitted. Eligibility criteria included recurrence after one or more platinum-based chemotherapy regimens, CA125 >35 U/mL, evaluable or measurable disease. Tumor burden was evaluated by physical or radiologic methods pretreatment, weeks 12, 24, and every 24 weeks thereafter. Immune responses, including antibodies and T cells to oregovomab and CA125, were demonstrated in over half the patients. Stabilization of disease and survival >2 years was observed in 3 of 13 patients and coincided with robust immune responses. Shrinkage of marker lesions was not observed; however, four patients showed decreases in CA125 levels. Treatment was well tolerated without serious adverse events or discontinuations due to therapy. This pilot study supports immunologic activity and safety of oregovomab in recurrent OC. Further study of this agent in the consolidation and adjuvant setting is ongoing to establish its clinical utility.     

bFGF单克隆抗体对卵巢癌移植瘤血管生成的抑制作用

目的:探讨碱性成纤维细胞生长因子(bFGF)在卵巢上皮性肿瘤中的表达强度与肿瘤血管生成之间的关系,及其单克隆抗体bFGFMAb对人卵巢癌移植瘤血管生成和肿瘤生长的影响。方法:(1)应用免疫组化法和图像分析系统研究bFGF在正常卵巢组织,良性、交界性和恶性卵巢上皮性肿瘤中的表达部位和强度;以及bFGF的表达强度与肿瘤新生血管的关系;(2)利用卵巢癌裸鼠皮下移植瘤模型,每周2次将不同浓度的bFGFMAb注射于瘤体周围,连续8周,每周测量肿瘤体积,绘制移植瘤生长曲线;对移植瘤组织行Ⅷ因子的免疫组化染色,测定肿瘤内微血管密度(MVD)。结果:(1)bFGF主要在卵巢上皮性肿瘤细胞浆内表达,在交界性和恶性肿瘤中,部分细胞同时存在核表达;bFGF在正常卵巢组织,良性、交界性和恶性卵巢上皮性肿瘤中的表达强度逐步增强,同时MVD逐步增高,二者呈正相关(P<0.001);(2)30、20、10μgbFGFMAb组移植瘤体积分别是对照组的31.34%、49.20%和71.25%,MVD分别是对照组的27.80%、52.37%和81.86%(P<0.05),其作用呈浓度依赖性。结论:bFGFMAb能明显抑制卵巢癌的血管生成和肿瘤生长,有望成为卵巢癌生物治疗的新方法。     

罗勒多糖对卵巢癌生物学行为的影响及其机制研究

目的:观察中药罗勒多糖对卵巢癌生物学行为的影响,探讨其抗卵巢癌作用的可能机制。方法:建立荷NuTu-19卵巢癌大鼠模型,观察罗勒多糖对荷瘤鼠肿瘤生物学行为的影响;抽提肿瘤组织总RNA,逆转录标记cDNA探针,用Cy3-dUTP标记对照组,Cy5-dUTP标记实验组,与表达谱芯片进行杂交,ImaGene3.0软件分析统计。同时取肿瘤组织,进行常规病理学检测。结果:与对照组相比,罗勒多糖治疗组肿瘤的生长及转移行为都受到明显的抑制:肿瘤数量减少,体积减小,腹水量显著减少(P<0.05);腹腔脏器转移程度积分显著降低(P<0.01)。罗勒多糖作用后,卵巢癌的基因表达谱发生了明显变化,应用表达谱芯片共筛选出168条差异表达基因。结论:罗勒多糖通过多个药效靶点抗肿瘤增殖及转移。