单链抗体e23sFv的人源化抗体制备及其在HER2靶向肿瘤杀伤中的功能鉴定

发布时间：2018-06-04 12:39

  本文选题：HER2 + e23sFv　； 参考：《第四军医大学》2017年博士论文

【摘要】：HER2(p185~(erb B2/neu))是EGFR家族的成员,在乳腺癌、卵巢癌、胃癌、肺癌等多种肿瘤细胞中高表达,是肿瘤恶性行为以及不良预后的标志。靶向治疗是HER2阳性肿瘤的重要治疗手段,主要包括靶向HER2的单克隆抗体、单链抗体偶联效应分子等策略。HER2治疗性单克隆抗体能够有效抑制肿瘤细胞增殖、延长生存期,是HER2阳性进展期乳腺癌及胃癌的一线疗法。这些HER2靶向的治疗性抗体都由鼠源单克隆抗体经人源化改造而来,长期应用未观察到严重的免疫原性相关副作用。HER2单链抗体来源于单克隆抗体,常与生物效应分子融合表达或者表达于T细胞表面(CAR-T)靶向杀伤肿瘤细胞,具有广泛的应用价值。对鼠源单链抗体的人源化改造,是以其为基础的肿瘤靶向杀伤策略走向临床的重要一环。本研究以本组前期系列验证的、靶向效果明确的鼠源单链抗体e23sFv为模板,拟解决两个关键问题:一是在探索单链抗体人源化的技术改进;二是评价该人源化单链抗体用于肿瘤靶向治疗的有效性。针对第一个关键问题,本研究进行了两方面新的摸索。首先,在传统的鼠源互补决定区(CDR)—人源框架区(FR)移植策略的基础上,拓宽了人源化改造FR模板的选择范围,既选了与e23sFv同源性最高的全人抗体特异序列,也选了与e23sFv同源性最高的全人抗体亚类的通用序列,并将二者对比,为抗体人源化的FR模板选择提供线索。其次,针对所选的人源抗体亚类通用序列,将FR关键残基突变回鼠源亲本,以维持抗原构象、避免丧失亲和力。传统的突变策略有两种:一是定点突变,前提是抗体结构信息必须明确;二是随机突变,虽然可提高亲和力的多样性,但筛选工作量大。我们尝试将这二者相结合,选择13个关键氨基酸位点分别设计定点突变引物,同时又通过设计同位点的不突变对照、引物的随机组合分组等方法,增加这13个位点随机突变与组合的几率,最终结合噬菌体抗体展示技术,建立了库容为213的突变抗体库,经4轮亲和力淘选和噬菌体ELISA筛选,获得4株高亲和力的人源化单链抗体。基于上述构建和筛选,本研究得到2株特异序列来源的人源化单链抗体SGF1、SGF2,4株通用序列来源的人源化单链抗体P1h2、P1h3、P2h2、P2h5。通过同源建模的方法,模拟了e23sFv、SGF1、P1h2的结构并与HER2分子进行对接,预测人源化单链抗体的识别表位位于HER2胞外段第IV结构域,且SGF1主链碳原子构象比P1h2更接近e23sFv,而P1h2与HER2相互作用能比SGF1更强。经过原核蛋白表达纯化、包涵体复性,获得蛋白纯度大于90%,进行功能评价:(1)亲和力:通过表面等离子共振实验(SPR)观察人源化单链抗体对人重组HER2的亲和力,结果显示P1h2、P1h3、SGF1比e23sFv提高约10倍,P2h2、P2h5则与e23sFv相当;细胞ELISA观察它们对细胞表面天然表达HER2的亲和力,结果与SPR实验一致。(2)识别表位:荧光素标记人源化单链抗体,竞争性流式细胞术观察到,人源化单链抗体的HER2识别表位与e23sFv相同。(3)内化活性:激光共聚焦显微镜观察结果表明,荧光素标记的人源化单链抗体P1h2、P1h3、SGF1能够特异性结合并内化进入HER2阳性细胞;P2h2、P2h5、SGF2则不能内化。(4)免疫原性:ELISPOT及抗体偶联磁珠技术检测人源化单链抗体刺激细胞因子分泌的水平,结果表明,人源化单链抗体刺激PBMCs产生IFN-γ、TNF-α细胞因子的水平较鼠源单链抗体大幅下降,减少到鼠源单链抗体刺激水平的1/20。值得注意的是,特异序列来源的SGF1比通用序列来源的4株人源化单链抗体刺激细胞因子分泌的水平更高,提示其免疫原性相对较强。综上,我们从6株不同序列来源的人源化单链抗体中,优选出两株免疫原性低、亲和力高并且具有内化活性的单链抗体P1h2和P1h3,进行后续功能研究。针对第二个关键问题,我们探索了P1h2和P1h3用于两种靶向治疗策略的有效性:一是基于补体依赖的细胞毒作用(CDC)的间接肿瘤杀伤;二是基于本组前期建立成熟的免疫促凋亡策略的直接肿瘤杀伤。(1)CDC策略:在人源化单链抗体的C端融合表达人IgG1Fc,构建获得系列scFv-Fc融合蛋白,进行真核系统表达纯化。流式细胞术和体外CDC实验结果表明,P1h2-Fc、P1h3-Fc融合蛋白均能与HER2阳性肿瘤细胞特异性结合,间接杀伤肿瘤细胞。HER2高表达的乳腺癌细胞及中度表达的肺癌细胞中,P1h2-Fc、P1h3-Fc的杀伤作用均强于e23sFv-Fc。(2)免疫促凋亡策略:将人源化单链抗体构建替换入课题组前期建立的免疫促凋亡分子,获得scFv-Fdt-tBid,进行原核系统可溶表达纯化。流式细胞术和细胞杀伤实验观察到,P1h2-Fdt-tBid、P1h3-Fdt-tBid能够特异性结合HER2阳性肿瘤细胞,有效抑制细胞增殖;在HER2高表达的乳腺癌细胞、胃癌细胞、中度表达的肺癌细胞中,二者对肿瘤细胞的增殖抑制与e23sFv-Fdt-tBid相当。体内实验分别采用NOD-SCID鼠的三种不同荷瘤模型(乳腺癌原位瘤、胃癌原位瘤、肺癌皮下移植瘤),明确观察到人源化改构免疫促凋亡分子的体内抗肿瘤活性。在乳腺癌和胃癌模型中,P1h2-Fdt-t Bid、P1h3-Fdt-tBid的肿瘤抑制作用强于e23sFv-Fdt-tBid;而肺癌模型中,二者的抗肿瘤作用与e23sFv-Fdt-tBid相当。综上所述,本研究通过优化CDR移植策略,针对抗HER2鼠源单链抗体e23sFv进行人源化改构,成功筛选获得免疫原性降低、亲和力提高并具有内化活性的两株人源化单链抗体P1h2、P1h3,并通过体内外实验验证其用于CDC策略和免疫促凋亡策略的有效性,为单链抗体为导向的融合蛋白的人源化改造、安全性评价提供了实验依据。
[Abstract]:HER2 (p185~ (ERB B2/neu)) is a member of the EGFR family. It is highly expressed in a variety of tumor cells, such as breast cancer, ovarian cancer, gastric cancer, and lung cancer. It is a marker of malignant behavior and poor prognosis of the tumor. Target therapy is an important treatment for HER2 positive tumors, mainly including monoclonal antibodies targeting HER2 and coupling effect molecules of single chain antibody. The HER2 therapeutic monoclonal antibody can effectively inhibit the proliferation of tumor cells and prolong the survival time. It is a first-line therapy for breast cancer and gastric cancer in the HER2 positive progression. These HER2 targeted therapeutic antibodies are transformed by human monoclonal antibodies by humanization. Long term application has not observed the severe immunogenic associated side effects of.HER2 single strand resistance. It is derived from monoclonal antibodies and often fused with biological effector molecules to express or express on the surface of T cells (CAR-T) to target tumor cells. Humanization of mouse source single chain antibody is an important part of the tumor targeting killing strategy. It is proved that the targeted mouse source single chain antibody e23sFv is the template to solve two key problems: one is to explore the technical improvement of the humanization of single chain antibody and the two is to evaluate the effectiveness of the human derived single chain antibody for tumor targeting therapy. The first key problem is two new exploration. First, in the tradition On the basis of the mouse source complementary decision area (CDR) - human source framework area (FR) transplantation strategy, the selection range of the humanized transformation of FR template was widened, both the highest homologous specific sequence of human antibody with the e23sFv homology was selected, and the universal sequence of the highest homologous antibody subclass of e23sFv was selected, and the two were compared to the FR human derived FR The template selection provides clues. Secondly, the FR key residues are mutated back to the parent of the mouse to maintain the antigen conformation and avoid loss of affinity. There are two traditional mutation strategies: one is fixed point mutation, the premise is that the information of the antibody structure must be clear, and the two is a random mutation, although the affinity can be increased although the affinity can be raised. We try to combine the two and choose 13 key amino acid sites to design point mutation primers respectively. At the same time, the probability of random mutagenesis and combination of the 13 loci is increased by designing the non mutation control of the same point and the random combination of primers. Technology, a mutant antibody library with a capacity of 213 was established, and 4 high affinity human single chain antibodies were obtained by 4 rounds of affinity selection and phage ELISA screening. Based on the above construction and screening, this study obtained the human derived single chain antibody SGF1 from 2 specific sequence sources, and the human derived single chain antibody P1h2, P1h3, P2h2 of the SGF2,4 strain. P2h5., through homologous modeling, simulates the structure of e23sFv, SGF1, P1h2 and docking with HER2 molecules. It predicts that the identification epitopes of human derived single chain antibodies are located in the IV domain of the HER2 extracellular segment, and the carbon atom conformation of the SGF1 main chain is closer to e23sFv than P1h2, and the P1h2 and HER2 phase are stronger than those of the HER2 phase. Purified by prokaryotic expression, Inclusion body refolding, obtained protein purity greater than 90%, performed functional evaluation: (1) affinity: the affinity for human recombinant HER2 was observed by surface plasmon resonance test (SPR). The results showed that P1h2, P1h3, SGF1 were about 10 times higher than e23sFv, P2h2, P2h5 were equivalent to e23sFv; cell ELISA observed their natural expression of the cell surface. The affinity of ER2 was in accordance with the SPR experiment. (2) identification of epitopes: fluorescein labeled human single chain antibody, competitive flow cytometry showed that the HER2 recognition epitopes of human derived single chain antibodies were the same as e23sFv. (3) internalization activity: the results of laser confocal microscopy showed that fluorescein labeled human single chain antibody P1h2, P1h3, SGF1 energy Enough specifically binding and internalizing into HER2 positive cells; P2h2, P2h5, and SGF2 can not be internalized. (4) immunogenicity: ELISPOT and antibody coupled magnetic beads to detect the level of human derived single chain antibody stimulation of cytokine secretion. The results show that human derived single chain antibody stimulates PBMCs producing IFN- gamma, and the level of TNF- a cytokine is more than that of rat single chain antibody. 1/20., which is significantly reduced to the level of mouse single chain antibody stimulation, is noteworthy that the specific sequence source of SGF1 is higher than the 4 human derived single chain antibody stimulated cytokine secretion from the common sequence source, suggesting that the immunogenicity is relatively strong. In the summary, we choose from the human derived single chain antibody from 6 different sequence sources. Two single chain antibodies, P1h2 and P1h3, with low immunogenicity, high affinity and internalized activity, were used for subsequent functional studies. For the second key problems, we explored the effectiveness of P1h2 and P1h3 for two target therapy strategies: one is the indirect tumor killing based on complement dependent cytotoxic action (CDC); two is based on this group. (1) CDC strategy: fusion expression of human IgG1Fc at the C end of human single chain antibody and construction of a series of scFv-Fc fusion proteins for expression and purification of eukaryotic system. Flow cytometry and in vitro CDC test showed that P1h2-Fc, P1h3-Fc fusion protein could be associated with HER2 positive tumor. Cell specific binding,.HER2 high expression of breast cancer cells and moderately expressed lung cancer cells, P1h2-Fc and P1h3-Fc are more lethal than e23sFv-Fc. (2) immunization promotion strategy: the construction of human derived single chain antibody was replaced by the prophase immuno apoptotic molecules established by the project group, and scFv-Fdt-tBid was obtained. The prokaryotic system can be expressed and purified. Flow cytometry and cell killing experiments have observed that P1h2-Fdt-tBid and P1h3-Fdt-tBid can specifically bind HER2 positive tumor cells to inhibit cell proliferation effectively; in the breast cancer cells with high expression of HER2, gastric cancer cells, and moderately expressed lung cancer cells, the two of the tumor cells inhibit the proliferation of tumor cells and e23sFv-F Dt-tBid is equivalent. In vivo, three different tumor bearing models of NOD-SCID mice (in situ breast tumor, gastric carcinoma in situ tumor, subcutaneous tumor of lung cancer) were used to observe the antitumor activity in vivo of human derived modified apoptosis inducing molecules in vivo. In breast cancer and gastric cancer model, P1h2-Fdt-t Bid, P1h3-Fdt-tBid was stronger than E2 3sFv-Fdt-tBid; in the lung cancer model, the antitumor effect of the two is equivalent to that of e23sFv-Fdt-tBid. To sum up, this study, by optimizing the CDR transplantation strategy, against the human derived single chain antibody e23sFv of the HER2 mouse, successfully screened the human derived single chain antibody, P1h2, with the reduction of immunogenicity, the enhancement of affinity and the internalization activity. P1h3, and through the experiments in vivo and in vitro, verified the effectiveness of its application to the strategy of CDC and immunization of apoptosis. It provides an experimental basis for the humanized transformation of single chain antibody oriented fusion protein and the evaluation of safety.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.51
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本文编号：1977385