人源性抗FGF-2中和性抗体的筛选，表达及鉴定

发布时间：2017-12-31 01:40

本文关键词：人源性抗FGF-2中和性抗体的筛选，表达及鉴定　出处：《南方医科大学》2010年博士论文　论文类型：学位论文

【摘要】： 目的和背景 FGF-2又称为碱性成纤维细胞生长因子(basic fibroblast growth factor,FGF-2),是成纤维生长因子家族的一员,最早由Gospodarowicz (1974)从牛脑垂体中提取的一种对BALB／C 3T3细胞等有明显促进生长作用的细胞因子。FGF-2有五种分子量,从18kD到34kD不等。所有的FGF-2异构体都缺乏信号肽,是通过一种现在还不十分清楚的机制分泌到细胞外的,其中18kD FGF-2在体内分布广泛,存在于中胚层及神经外胚层来源的细胞及多种肿瘤细胞中,对这些细胞有促增殖分化功能,参与了胚胎发育、血管生成、损伤修复、神经再生、肿瘤生长、组织纤维化等多项生理及病理过程。 FGF-2的生物学效应,除对成纤维细胞和血管内皮细胞具有显著的促增殖作用外,还参与肿瘤发生和发展,特别对肿瘤的浸润和转移具有重要作用。有相当部分的肿瘤(如乳癌、结肠癌、甲状腺癌、肺癌、膀胱癌、食道癌等)存在FGF-2的高表达(表2)。已知其作用环节主要有(1)FGF-2能促进表皮、内皮细胞再生,促进血管内皮细胞分裂,诱导其从基膜中分离出来,以刺激内皮细胞向肿瘤组织趋化运动,并形成管状结构,还提高组织中血纤维蛋白溶解酶原激活因子类及诱导内皮细胞产生其他蛋白酶,从而促进毛细血管的形成；(2)FGF-2能促进多种生长因子的分泌,如VEGF、HGF、FGF-1等,这些因子相互调节,共同促进肿瘤的生长；(3)肿瘤细胞存在FGF-2受体的高表达,通常比正常细胞上FGF-2R高10倍以上,许多肿瘤细胞(如神经胶质瘤、横纹肌肉瘤、白血病、肺瘤、黑色素瘤、肝癌等)既表达FGF2又表达FGFRs,因此FGF2可直接作用于肿瘤细胞,使肿瘤织的各种蛋白酶及胶原酸分泌增加,从而加速肿瘤细胞的转移过程；(4)FGF-2还与肿瘤的耐药性有关,增强肿瘤对药物诱导的细胞凋亡的抵抗能力[20]。故FGF-2与肿瘤生长、转移、预后有着非常密切的关系。 FGF-2与肿瘤肿瘤生长、转移、耐药有关,用抗体阻断FGF-2的活性被认为是一种治疗肿瘤的有效方法。已有多株抗FGF-2抗体体内外抑制肿瘤试验的报道。在多种动物体内,一株中和性的的单抗(GD2)能抑制FGF-2或肿瘤组织诱导的血管新生。在大鼠体内内GD2能抑制骨肉瘤的生长。另一株单抗,3H3,能抑制十二指肠溃疡的血管新生,从而延迟溃疡的愈合,同时它还能抑制一种转染的致瘤性细胞在裸鼠体内的生长。Aonuma等制备了两株中和性单抗,2G11和1E6,前者识别的是FGF-2的肝素结合位点,后者识别的是FGF-2与受体结合的位点,体内实验证实1E6能抑制肿瘤生长,而2G11不具备抗瘤作用。国内,谢庆祥等[7]通过建立裸鼠皮下移植膀胱癌动物模型进行研究,发现FGF-2抗体治疗组皮下瘤体积和重量比对照组均显著减少,瘤组织中增殖细胞核抗原(PCNA)指数和微血管密度(MVD)也显著降低,表明FGF-2抗体对膀胱癌的生长具有明显抑制作用,其主要通过抑制膀胱癌细胞增殖和减少肿瘤血管形成的途径而在荷瘤裸鼠体内发挥抗肿瘤作用。林卫等观察了FGF-2抗体对人卵巢癌细胞的增殖、卵巢癌腹腔移植瘤裸鼠的生存率和人卵巢癌移植瘤血管生成和肿瘤生长的影响。研究结果显示,FGF-2抗体能明显抑制卵巢癌细胞的增殖,卵巢癌的生长和血管生成,并呈浓度依赖性,提示FGF-2单抗有望成为卵巢癌生物治疗的新方法。本实验室前期制备了19株鼠抗FGF-2单抗,通过体外实验证实,其中三株抗体能够诱导B16细胞凋亡,近期的体内试验发现其中一株抗体能够明显抑制黑色素瘤的生长和转移,延长荷瘤小鼠的生存期,同时与化疗药物顺铂、紫杉醇等联合使用能够明显抑制黑色素癌的生长,而对于那些对化疗药物耐药的肿瘤抗FGF-2单抗能逆转其耐药性。这些报道证实在多种试验条件下,抗FGF-2抗体能阻断血管新生和抑制肿瘤生长。但这些抗体都是动物来源的抗体,用于人类疾病的治疗存在半衰期短、血清病发生率高、易在人体内产生抗体导致不能重复使用等弊。制备全人源性的抗FGF-2抗体,是克服上述弊端有效方法。人源性抗体的制备技术主要有三种,第一种是通过表面展示技术从人源性抗体库中筛选人源性抗体,第二种技术是通过免疫转入人免疫球蛋白基因的转基因鼠来获得针对特异抗原的人源性抗体,第三种技术是通过细胞分选技术,从某些病人骨髓细胞或外周血细胞中筛选特异性针对某种抗原的B细胞或记忆性B细胞,通过某些技术如与人骨髓瘤融合或用EB病毒使其永生化,使得这些B细胞能在体外无限繁殖,从而分泌人源性抗体。这三种技术中第二种技术需要购买或构建转基因小鼠,需要耗费大量财力和物力。第三种技术需要特殊病人的血细胞,并且需要获得人源性杂交瘤或刺激B细胞的永生化,这些技术目前还不成熟。表面展示技术具有极强的筛选功能,将序列互不相同的一组多样化的抗体表达到细菌,酵母,病毒表面,得到抗体展示库因此,利用其表型与基因型的统一以及易于扩增的特性,可很容易的从库中筛选出针对某种特异抗原的抗体。其中以噬菌体展示技术应用最为广泛。从噬菌体抗体库中直接筛选获得高亲和力抗体,其主要影响因素是抗体库容量大小和抗体基因的成熟度,其多样性要能保证有这种抗体存,并且在经过扩增后具有被筛选出来所要求的拷贝数,因此增加噬菌体抗体库的库容量尤其重要。根据已有报道,从大于109的大容量天然抗体库中,往往可以得到高亲和力的抗体,无需进行抗体亲和力成熟。本研究所用的抗体库为经过单载体细胞内重组法(Lxop-cre定位重组系统)两次重组配对后的人源性大容量抗体库,重组后库容约为6×1010,沉淀浓缩后滴度约为1013cfu／ml,具有良好的多样性,可以充分满足筛选的需要,在获得高亲和力抗体以及进行抗体性能改造方面具有较强的优势。在抗体的进化过程中,基因突变并不是随机发生于可变区内的,而是倾向性的集中在CDR区的某些位置,即突变热点,有多种类型,其中研究较详细的热点类型有AGY／RGYW和TAY(R=A／G,Y=C／T,W=A／T)。这些位点也常常是位于和抗原直接结合的区域中。针对这类位点引入突变,相较于这类位点之外的区域将更可能得到亲和力提高的基因工程抗体,而且可以通过构建小型的突变库来实现。Ho等研究发现,CDR区的突变热点分为胚系热点和非胚系热点,只有突变胚系热点才能提高抗体的亲和力。因此对抗体胚系热点进行突变将是本文采取的方法。方法 1.利用固相筛选技术从大容量噬菌体抗体库(6×1010)中筛选能特异性结FGF-2的克隆,经过三到四轮的洗脱筛选,将得到的阳性克隆通过酶切鉴定,可变区多样性分析,基因测序和序列比对,确定能特异性结合FGF-2的序列正确的阳性克隆。 2.将测序正确的阳性克隆的基因插入到原核表达载体pComb3XSS中,在低温培养条件下,利用IPTG诱导scFv基因的可溶性表达,离心后收集菌体,PBS重悬后超声破碎,收集破碎上清,通过SDS-PAGE, Western-blot, ELISA鉴定上清中抗体的特异性,通过竞争ELISA检测者几株scFv能否抑制FGF-2与其高亲和力受体FGFR1的结合。 3.将能够抑制FGF-2与其高亲和力受体FGFR1的结合的44克隆通过重叠延伸PCR构建成全长的人源性抗FGF-2抗体,插入到真核表达载体pIGG中,构建成真和表达载体pIGG-44,利用转染试剂FuGene HD转染HEK293T细胞,进行真核可溶性表达,收集细胞培养上清,通过SDS-PAGE, Western-blot,ELISA鉴定上清中抗体的特异性,通过硫酸铵沉淀和蛋白G纯化抗体,通过夹心ELISA测定抗体的浓度。 4.表达的全长的人源性抗体通过体外细胞实验验证抗体的中和FGF-2的活性,通过血管内皮细胞促增值实验,迁移实验和成管实验验证抗体对血管内皮细胞的作用,通过肿瘤细胞增殖抑制试验验证抗体对肿瘤细胞的增值抑制作用,通过Hoechst 33258染色观察抗体作用后肿瘤细胞细胞核的变化情况,来观察细胞是否发上了凋亡 5.对具有中和活性的44号克隆进行亲和力成熟,利用基因比对从Genebank中搜索出与44号克隆序列最接近的抗体胚系基因序列,然后通过IMTG中的抗体可变区基因在线分析工具V-QUEST,找出重链胚系基因中CDR区的突变热点,并与44号克隆进行比较,确定这些热点在44号克隆重链CDR区中的位置,随后我们通过定点突变对重链可变区CDR1区中的三个胚系热点进行定点随机突变,构建噬菌体抗体突变库,通过三轮洗脱筛选,每轮逐渐增加洗脱次数和减少包被的FGF-2的量来得到亲和力提高的抗体突变株,将高亲和力突变株进行可溶性表达后,通过异硫氰酸铵洗脱法测定抗体的相对亲和力。计算抗体亲和力提高倍数。同时通过竞争ELISA法检测突变株是否还能抑制FGF-2与其高亲和力受体FGFR1的结合。结果 1.经过3到4轮筛选从104个随机挑选的克隆中,筛得39株抗FGF-2的抗体,通过对抗体可变区基因多样性分析,发现有8种不同抗体可变区基因型,挑选其中8株phage ELISA显色最高的克隆进行DNA酶切和测序鉴定,发现其中7株为正确的抗体基因序列,通过大肠杆菌HB2151成功的进行了scFv的可溶性表达,通过竞争ELISA发现其中三株scFv能够抑制FGF-2与其高亲和力受体FGFR1βⅢC的结合,其中44号克隆的抑制效果最好。 2.通过对转染试剂,转染方法,培养温度,转染试剂与细胞的孵育时间,组蛋白抑制剂的加入等条件进行优化使人源性抗FGF-2抗体在HEK293T细胞中的表达量从1.5mg／L提高到了15.1mg／L。表达产物经硫酸铵沉淀,透析后上蛋白G亲和层析柱纯化,纯化后的产物经超滤管超滤除盐,最后经SDS-PAGE鉴定,获得纯度为90%的电泳纯抗体,双抗夹心法测定抗体含量,显示产量可以达到每升细胞培养上清12mg抗体。 3.通过一系列体外实验证实44号克隆能够抑制人脐静脉血管内皮细胞的增殖,迁移和成管,同时还能抑制神经胶质瘤细胞株U87MG的增殖,Hoechst 33258染色后发现抗FGF-2抗体作用能够诱导U87MG的凋亡 4.通过热点随机突变构建44号克隆的单链抗体噬菌体突变库,通过三轮洗脱筛选,筛选到了4株亲和力提高的抗体突变株,经过可溶性表达后,通过异硫氰酸铵洗脱法测定抗体的相对亲和力,显示其中最高的一株亲和力比44号克隆的亲和力提高了4倍。结论 1.通过对人源性噬菌体抗体库的筛选得到了七株不同的人源性抗FGF-2抗体,经过体外实验证实其中一株抗体能够中和FGF-2的多种生物学作用,如促血管内皮增殖作用,促细胞迁移作用,内皮细胞成管作用等,更为重要的此株抗体还能诱导神经胶质瘤细胞株U87MG的凋亡。 2.通过胚系热点突变使此株中和性抗体的亲和力提高了4倍,与此同时也提高了抗体的中和活性,从而证实了胚系热点突变提高抗体亲和力的可行性。本研究的创新处： 1.成功筛选到三株人源性中和性抗FGF-2抗体,其中一株的抑制FGF-2与其高亲和力受体FGFR1结合的效果最好,通过细胞实验证实此株抗体能够中和FGF-2的多种生物学作用,为FGF-2抗体药物研究奠定基础。 2.成功利用CDR区胚系热点突变提高了一株中和性抗体的亲和力,同时也使得抗体的中和活性增强,证实了此方法可在不改变抗原识别位点的情况下提高抗体的亲和力。
[Abstract]:Purpose and background
FGF-2 is also called the basic fibroblast growth factor (basic fibroblast, growth factor, FGF-2), is a member of the fibroblast growth factor family, first by Gospodarowicz (1974) extracted from bovine pituitary in a cell growth factor.FGF-2 had significant promoting effect of the five kinds of molecular weight of BALB / C 3T3 the cell, from 18kD to 34kD FGF-2 range. All of the isoforms are lack of signal peptide, is through a now is not clear the mechanism of secretion to the extracellular 18kD FGF-2, which is widely distributed in the body, cells and many tumor cells exist in the mesoderm and neuroectoderm source, can promote the proliferation and differentiation of function these cells are involved in embryonic development, angiogenesis, wound repair, nerve regeneration, tumor growth, tissue fibrosis and other physiological and pathological processes.
The biological effects of FGF-2, besides of fibroblasts and vascular endothelial cells proliferation significantly, is also involved in the occurrence and development of malignant tumor, especially in tumor invasion and metastasis plays an important role. There is a considerable part of the tumor (such as breast cancer, colon cancer, thyroid cancer, lung cancer, bladder cancer, esophageal cancer, etc.) the high expression of FGF-2 (Table 2). The known part of its role mainly (1) FGF-2 can promote the regeneration of epidermis, endothelial cells, promote vascular endothelial cell division and induce the separated from the basement, to stimulate endothelial cells to tumor tissue chemotaxis, and the formation of tubular structure, but also improve the blood of fibrous tissue protein plasminogen activators and other proteases induced by endothelial cells, thereby promote capillary formation; (2) FGF-2 can promote the secretion of various growth factors, such as VEGF, HGF, FGF-1 and so on, these factors regulate each other and jointly promote Progressive tumor growth; (3) the high expression of FGF-2 receptor in tumor cells than in normal cells, usually FGF-2R 10 times higher than that of many tumor cells (such as glioma, rhabdomyosarcoma, leukemia, lung, melanoma, liver cancer) expressed both FGF2 and FGFRs, so the FGF2 can be directly applied to the tumor the tumor cells, proteases and collagen fabric acid secretion increased, thus speeding up the transfer process of tumor cells; (4) FGF-2 is associated with tumor resistance, enhance the ability to resist [20]. on the apoptosis of the tumor FGF-2 and tumor growth, metastasis, prognosis has a very close relationship.
FGF-2 and tumor growth, metastasis, drug resistance, antibody blocking FGF-2 activity was found to be an effective method for treating tumors. Tumor inhibition test has been reported in several strains of anti FGF-2 antibodies in vitro and in vivo. In a variety of animal, a neutralizing monoclonal antibody (GD2) could inhibit FGF-2 induced tumor angiogenesis can inhibit osteosarcoma. GD2 in vivo in rat growth. Another monoclonal antibody, 3H3, can inhibit the angiogenesis of duodenal ulcer, and delayed healing of the ulcer, and it can inhibit a transfection of tumorigenic cells by two strains of neutralizing monoclonal antibody in nude mice and the growth of.Aonuma so, 2G11 and 1E6, the former is the identification of the heparin binding site of FGF-2, which is a combination of FGF-2 and receptor recognition sites in vivo experiments confirmed that 1E6 can inhibit the growth of tumor, and 2G11 does not have antitumor effect. At home, Xie Qingxiang [7]. Study to establish the animal model of bladder carcinoma transplanted subcutaneously in nude mice, found FGF-2 antibody treatment than in the control group were significantly reduced in group subcutaneous tumor volume and weight of tumor tissues, proliferating cell nuclear antigen (PCNA) index and microvessel density (MVD) were significantly decreased, indicating that the growth of FGF-2 antibody on bladder cancer has obvious inhibitory effect, the mainly through the inhibition of tumor angiogenesis and ways to reduce the proliferation of bladder cancer cells and the antitumor effect in nude mice. Lin Wei observed the proliferation of FGF-2 antibody against human ovarian cancer cells, and affect the survival of human ovarian cancer xenograft tumor angiogenesis and tumor growth in nude mice to study. The results showed that FGF-2 antibody can inhibit ovarian cancer cell proliferation, growth and angiogenesis of ovarian cancer, and in a concentration dependent manner, suggesting that FGF-2 is expected to become the biological therapy of ovarian carcinoma monoclonal antibody The new method. The laboratory has prepared 19 strains of mouse anti FGF-2 monoclonal antibody, in vitro experiments confirmed that the three antibodies can induce B16 cell apoptosis in vivo, recently found that one strain antibody can significantly inhibit melanoma growth and metastasis, prolong the survival time of the mice, at the same time with the chemotherapy drug the combined use of cisplatin, paclitaxel can inhibit the growth of melanoma, and to those of the chemotherapy drug resistance tumor anti FGF-2 monoclonal antibody can reverse the drug resistance.
These reports have confirmed that in a variety of experimental conditions, the anti FGF-2 antibody can inhibit angiogenesis and tumor growth inhibition. But these antibodies are antibodies of animal origin, for the treatment of human disease has short half-life, high rate of occurrence of serum sickness, easy to produce antibody can not cause repeatition harm in the human body. Preparation of the whole people the source of anti FGF-2 antibody, is the effective method to overcome these disadvantages.
There are three main types of human antibody preparation technology, the first is through the surface screening of human antibodies from human antibody library, second transgenic mice by immune technology is transferred to Human Immunoglobulin gene to obtain human antibodies against specific antigen, third kinds of technology through cell sorting technique screening, specific to antigens of B cells and memory B cells in some patients with bone marrow cells or peripheral blood cells, and by some techniques such as human myeloma or fusion with EB virus immortalized B cells can proliferate, the infinite in vitro, and the secretion of human antibodies. Three second kinds of technology technology need to purchase or construction of transgenic mice, requires a lot of financial and material resources. Third techniques require special blood cells, and need to get human hybridoma or stimulation of B cells The immortal, the technology is not mature. The surface display technology has the function of screening, a group of diverse expression of antibody to bacteria, yeast, virus surface sequences are different from each other, so the obtained antibody display library, using the unified phenotype and genotype and easy amplification characteristics, can be very easy screening from the library for a specific antigen antibody. The phage display technology is most widely used.
Screening from phage antibody library directly to obtain high affinity antibodies, the main factor is the antibody library size and antibody gene maturity, its diversity to ensure the antibody, and after amplification has been screened out copy number required, thus increasing the storage capacity of particular phage antibody library important. According to the existing reports, from more than 109 large capacity natural antibody library, often can get high affinity antibody, without antibody affinity maturation. Antibody library used in this study is through a single cell carrier recombination method (Lxop-cre recombination system) of human large phage antibody library two recombinant pairing after the reorganization, the volume is 6 x 1010, precipitation concentration was approximately 1013cfu / ml, with good diversity, can fully meet the need in the screening, to obtain high affinity antibodies and anti The improvement of body performance has strong advantages.
In the evolutionary process of antibody, gene mutation and random variable is not occurred in the area, but the tendency of certain locations concentrated in the CDR area, namely hot spot mutation, there are many types, including detailed research hotspot types are AGY / RGYW and TAY (R=A / G, Y=C / T, W=A / T). These sites are often located and direct antigen binding region. For this kind of site mutation, compared to the outside of this kind of site will be more likely to improve the affinity of gene engineering antibody, but also through the construction of mutant library to achieve small.Ho research found that mutation hotspot region CDR points for germline hot and non germline mutations in the germ line hot hot, only to improve the affinity of the antibody. So the mutation will be the approach of germline antibody hotspot.
Method
1. using the solid-phase screening technology from a large phage antibody library (6 * 1010) in screening the specific binding of FGF-2 clones, after three to four rounds of panning, the positive clones were obtained by enzyme digestion, diversity analysis of variable region gene sequencing and sequence alignment, can determine the sequence of positive clone FGF-2 the correct binding specificity.
2. sequencing positive clones the gene was inserted into prokaryotic expression vector pComb3XSS, in low temperature conditions, the soluble expression of scFv gene induced by IPTG, after centrifugation, collecting thalli, ultrasonic suspended in PBS, collect the broken supernatant, through SDS-PAGE, Western-blot, ELISA and identification of specific antibodies in the supernatant by. With the competitive ELISA detection of several strains of scFv can inhibit FGF-2 and its high affinity receptor FGFR1.
3. will be able to inhibit the binding of FGF-2 to its high affinity receptor FGFR1 44 cloned by overlap extension PCR to construct a full-length humanized anti FGF-2 antibody, inserted into eukaryotic expression vector pIGG, construct and true expression vector pIGG-44 using transfection reagent FuGene HD transfected HEK293T cells, eukaryotic expression of soluble, collecting cells the culture supernatant by SDS-PAGE, Western-blot, ELISA and identification of specific antibodies in the supernatant, purified by ammonium sulfate precipitation and protein G, the antibody concentration was determined by sandwich ELISA.
4. activity and FGF-2 expression of human antibodies against full-length antibody by in vitro experiments, the endothelial cell proliferation experiment, migration and tube formation experiment experimental verification antibody on vascular endothelial cells, the inhibition of tumor cell proliferation test antibody inhibition value of tumor cells, changes by Hoechst 33258 antibody staining of tumor cells after the role of the nucleus, to observe the hair on the cell apoptosis
5. of the 44 clones with neutralizing activity of affinity maturation, using gene ratio from Genebank to search the closest and clone 44 sequence antibody germline gene sequence, then the antibody variable genes in IMTG online analysis tool V-QUEST, find out the heavy chain germline gene mutation hotspot in CDR region, and compared with the 44 clones, determine the location of these hot spots in clone 44 heavy chain in the CDR area, then we by point mutation in CDR1 region of heavy chain variable region in three germline hot point mutation, mutant phage antibody library, through three rounds of panning, each round gradually increased and washing times reduce the amount of FGF-2 package is to improve the affinity of antibody mutant, the high affinity mutant soluble expression, by ISO thiocyanate elution method for the determination of the relative affinity of antibody antibody affinity calculation. At the same time, a competitive ELISA method was used to detect whether the mutant strain could inhibit the combination of FGF-2 and its high affinity receptor FGFR1.
Result
1. to 4 after 3 rounds of screening from 104 clones randomly selected, screened anti FGF-2 antibody of 39 strains, the gene of antibody variable region diversity analysis, found that 8 genes of different antibody variable type, selected among 8 strains of phage ELISA were cloned by DNA color of the highest enzyme digestion and DNA sequencing. 7 of them were found, antibodies to the correct gene sequence of Escherichia coli HB2151, through the success of the soluble expression of scFv, ELISA found through competition among the three scFv strains could inhibit FGF-2 to its high affinity receptor FGFR1 beta III binding to C, one of the best inhibitory effect of clone 44.
2. through the transfection reagent, transfection method, transfection reagent and cell culture temperature, incubation time, adding conditions such as inhibitors of histone deacetylase optimize expression of anti FGF-2 antibody in HEK293T cells from 1.5mg / L to 15.1mg / L. expression products were purified by ammonium sulfate precipitation, dialysis protein G affinity chromatography, purified by ultrafiltration ultrafiltration

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【参考文献】