抗卵泡刺激素受体纳米抗体的制备及其在肿瘤分子显像和抗血管治疗中的作用

Follicle-stimulating hormone receptor(FSHR) is expressed only in the human granulosa cells of the ovary and the sertoli cells of the testis. It is minimally expressed by the endothelial cells of gonadal blood vessels. For tumors, most researches believed that FSHR is only expressed in ovarian surface epithelium and epithelial ovarian cancer. The relationship of FSHR with most common cancer especially the hormone independent cancer was not established , the role of follicle-stimulating hormone receptor（FSHR）in tumorgenesis was not clear.Recent research discovered FSHR is selectively expressed on the surface of the blood vessels of a wide range of huamn tumors，including early T1 tumors.This suggested that FSHR might play an important role in tumor angiogenesis. Taking into consideration the specific espression of FSHR in tumor vasculature, and the potential to distingush from normal tissue and nonmalignant inflammatory lesion, FSHR is a promisimg tumor molecular imaging and potential antiangiogenic target.For developing efficent tumor imaging and antiangiogenic agents, we exploied the property of camel derived single domain antibody fragments (VHHs,also desigend nanobody),which are naturally occurring antibodies devoid of light chains.Nanobodies posess several advantages compared with heterotetrameric conventional antibody, such as small molecular size,easy cloning, high expression and easy purified from bacterial, fast clearance from the blood, recognition of hidden antigenic sites. Particularly appealing is their ability to simultaneously inhibit various crucial growth factor receptors or their ligands with a single molecule and the rapird clearance from blood. These properties make nanobody a promising and proficient tools in diagnostics and blocking especially in tumor imaging and antiangiogenic therapy.In this project, we firstly plan to investigate the potential role of FSHR in proliferation, immigration and tube formation of tumor with the transfection of FSHR in HUVEC, Caov-3 and CT26 colon cancer cells. Secondly, we develoment FSHR specific VHH nanobody（namely αFSHR）by screening the VHH phage display library, and examnation its blocking property of FSH with FSHR in vitro.Thirdly, the αFSHR was labelled with radio nuclide 99mTc，the in vitro specificity of 99mTc-labeled nanobody with FSHR and ex vivo biodistribution and blood clearance were measured on an automated gamma counter.Fourthly，the Babl/C mice bearing CT26 was used for in vivo targeting of tumor xenografts with 99mTc-αFSHR nanobody. Finally，the antitumor and antiangiogenic effects of αFSHR nanobody alone and combined with bevacizumab were evaluated on the CT26 tumor xenografts mice model, and the angiogenesis was evaluated by measuring the density of blood vessels before and after antibody therapy. This project will provied evidences for the role of FSHR in the tumor angiogensis and the diagnositc and therauptic potentials of camei nanobody based approach.

卵泡刺激素受体FSHR在肿瘤中的作用一直未能明确。最近研究发现 FSHR 广泛表达于各种进程的多种肿瘤内皮细胞表面，并且可以有效的区分肿瘤和正常组织，是一个理想的体内显像靶抗原和治疗靶点。基于此，本研究拟初步探讨 FSHR 在肿瘤新生血管形成中的作用，并利用骆驼纳米抗体具有的分子小、容易获得、可优先结合受体配体结合部位以及原核表达量大并具有完全活性的优点，制备抗 FSHR 纳米抗体（简称为αFSHR），作为潜在的肿瘤体内显像剂和靶向血管治疗药物。本研究主要通过（1）FSHR基因过表达对肿瘤细胞和血管内皮细胞的生理功能的影响；（2）FSHR纳米抗体的制备和特性分析；（3）99mTc-FSHR纳米抗体的标记和抗原结合和分布研究；（4）99mTc-FSHR纳米抗体肿瘤体内显像以及（5）FSHR纳米抗体抗血管治疗和机制分析等方面，初步阐明FSHR在肿瘤中的作用并探讨FSHR抗体的应用前景。

该项目通过对HUVEC、Caov-3、Scov-3和CT26细胞FSHR表达和成管、迁移及增殖的研究，确定了FSHR基因在Caov-3高表达，而在HUVEC低表达。确定和验证了FSH在血管形成中的作用，初步明确了FSHR基因过表达对肿瘤血管形成的促进作用。采用噬菌体展示技术和高通量测序技术及亲和力转移技术获得了5个具有一定FSHR结合活性的纳米抗体。并探讨了采用CDR3突变和与多聚化载体蛋白偶联进行亲和力成熟的方法。其中VHH-FSHR-06和VHH-FSHR-3F9对Caov-3与FSHR表达阳性的Caov-3细胞具有特异结合作用，VHH-FSHR-3F9对FSH与其受体结合具有阻断作用。建立了人卵巢癌Caov-3和Scov-3细胞裸鼠和CT26小鼠肿瘤皮下移植瘤动物模型。采用p-SCN-Bn-NOTA试剂与纳米抗体VHH-FSHR-06反应，利用64Cu核素对纳米抗体进行标记，建立了以放射性标记纳米抗体的方法，获得了核素标记的特异结合Caov-3细胞的纳米抗体示踪剂64Cu-NOTA- VHHFSHR-06。利用该纳米抗体注射Caov-3荷瘤小鼠，在microPET/microCT仪器中可以显示体内肿瘤的大小和位置。利用CT26小鼠肿瘤动物模型研究了FSHR纳米抗体VHH-FSHR-06和多抗在体内抗肿瘤作用及初步机制，初步结果表明，抗FSHR多克隆抗体和VHH-FSHR-06纳米抗体在治疗21天时对小鼠CT26肿瘤的生长具有明显抑制作用（p<0.05），但纳米抗体在进一步治疗中没有明显抑制作用（p>0.05）。另外，对治疗前后的小鼠肿瘤组织的免疫组化分析显示，抗FSHR抗体治疗可减少CT26肿瘤组织中的血管密度。提示抗肿瘤作用可能是通过抑制新生血管形成发挥作用。除此，采用大鼠卵巢切除骨质疏松症动物模型初步研究提示抗FSHR抗体对实验大鼠骨质疏松形成具有一定的抑制作用。