本文選題：HER2 + e23sFv��； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：HER2(p185~(erb B2/neu))是EGFR家族的成員,在乳腺癌、卵巢癌、胃癌、肺癌等多種腫瘤細(xì)胞中高表達(dá),是腫瘤惡性行為以及不良預(yù)后的標(biāo)志。靶向治療是HER2陽性腫瘤的重要治療手段,主要包括靶向HER2的單克隆抗體、單鏈抗體偶聯(lián)效應(yīng)分子等策略。HER2治療性單克隆抗體能夠有效抑制腫瘤細(xì)胞增殖、延長生存期,是HER2陽性進(jìn)展期乳腺癌及胃癌的一線療法。這些HER2靶向的治療性抗體都由鼠源單克隆抗體經(jīng)人源化改造而來,長期應(yīng)用未觀察到嚴(yán)重的免疫原性相關(guān)副作用。HER2單鏈抗體來源于單克隆抗體,常與生物效應(yīng)分子融合表達(dá)或者表達(dá)于T細(xì)胞表面(CAR-T)靶向殺傷腫瘤細(xì)胞,具有廣泛的應(yīng)用價(jià)值。對(duì)鼠源單鏈抗體的人源化改造,是以其為基礎(chǔ)的腫瘤靶向殺傷策略走向臨床的重要一環(huán)。本研究以本組前期系列驗(yàn)證的、靶向效果明確的鼠源單鏈抗體e23sFv為模板,擬解決兩個(gè)關(guān)鍵問題:一是在探索單鏈抗體人源化的技術(shù)改進(jìn);二是評(píng)價(jià)該人源化單鏈抗體用于腫瘤靶向治療的有效性。針對(duì)第一個(gè)關(guān)鍵問題,本研究進(jìn)行了兩方面新的摸索。首先,在傳統(tǒng)的鼠源互補(bǔ)決定區(qū)(CDR)—人源框架區(qū)(FR)移植策略的基礎(chǔ)上,拓寬了人源化改造FR模板的選擇范圍,既選了與e23sFv同源性最高的全人抗體特異序列,也選了與e23sFv同源性最高的全人抗體亞類的通用序列,并將二者對(duì)比,為抗體人源化的FR模板選擇提供線索。其次,針對(duì)所選的人源抗體亞類通用序列,將FR關(guān)鍵殘基突變回鼠源親本,以維持抗原構(gòu)象、避免喪失親和力。傳統(tǒng)的突變策略有兩種:一是定點(diǎn)突變,前提是抗體結(jié)構(gòu)信息必須明確;二是隨機(jī)突變,雖然可提高親和力的多樣性,但篩選工作量大。我們嘗試將這二者相結(jié)合,選擇13個(gè)關(guān)鍵氨基酸位點(diǎn)分別設(shè)計(jì)定點(diǎn)突變引物,同時(shí)又通過設(shè)計(jì)同位點(diǎn)的不突變對(duì)照、引物的隨機(jī)組合分組等方法,增加這13個(gè)位點(diǎn)隨機(jī)突變與組合的幾率,最終結(jié)合噬菌體抗體展示技術(shù),建立了庫容為213的突變抗體庫,經(jīng)4輪親和力淘選和噬菌體ELISA篩選,獲得4株高親和力的人源化單鏈抗體�；谏鲜鰳�(gòu)建和篩選,本研究得到2株特異序列來源的人源化單鏈抗體SGF1、SGF2,4株通用序列來源的人源化單鏈抗體P1h2、P1h3、P2h2、P2h5。通過同源建模的方法,模擬了e23sFv、SGF1、P1h2的結(jié)構(gòu)并與HER2分子進(jìn)行對(duì)接,預(yù)測人源化單鏈抗體的識(shí)別表位位于HER2胞外段第IV結(jié)構(gòu)域,且SGF1主鏈碳原子構(gòu)象比P1h2更接近e23sFv,而P1h2與HER2相互作用能比SGF1更強(qiáng)。經(jīng)過原核蛋白表達(dá)純化、包涵體復(fù)性,獲得蛋白純度大于90%,進(jìn)行功能評(píng)價(jià):(1)親和力:通過表面等離子共振實(shí)驗(yàn)(SPR)觀察人源化單鏈抗體對(duì)人重組HER2的親和力,結(jié)果顯示P1h2、P1h3、SGF1比e23sFv提高約10倍,P2h2、P2h5則與e23sFv相當(dāng);細(xì)胞ELISA觀察它們對(duì)細(xì)胞表面天然表達(dá)HER2的親和力,結(jié)果與SPR實(shí)驗(yàn)一致。(2)識(shí)別表位:熒光素標(biāo)記人源化單鏈抗體,競爭性流式細(xì)胞術(shù)觀察到,人源化單鏈抗體的HER2識(shí)別表位與e23sFv相同。(3)內(nèi)化活性:激光共聚焦顯微鏡觀察結(jié)果表明,熒光素標(biāo)記的人源化單鏈抗體P1h2、P1h3、SGF1能夠特異性結(jié)合并內(nèi)化進(jìn)入HER2陽性細(xì)胞;P2h2、P2h5、SGF2則不能內(nèi)化。(4)免疫原性:ELISPOT及抗體偶聯(lián)磁珠技術(shù)檢測人源化單鏈抗體刺激細(xì)胞因子分泌的水平,結(jié)果表明,人源化單鏈抗體刺激PBMCs產(chǎn)生IFN-γ、TNF-α細(xì)胞因子的水平較鼠源單鏈抗體大幅下降,減少到鼠源單鏈抗體刺激水平的1/20。值得注意的是,特異序列來源的SGF1比通用序列來源的4株人源化單鏈抗體刺激細(xì)胞因子分泌的水平更高,提示其免疫原性相對(duì)較強(qiáng)。綜上,我們從6株不同序列來源的人源化單鏈抗體中,優(yōu)選出兩株免疫原性低、親和力高并且具有內(nèi)化活性的單鏈抗體P1h2和P1h3,進(jìn)行后續(xù)功能研究。針對(duì)第二個(gè)關(guān)鍵問題,我們探索了P1h2和P1h3用于兩種靶向治療策略的有效性:一是基于補(bǔ)體依賴的細(xì)胞毒作用(CDC)的間接腫瘤殺傷;二是基于本組前期建立成熟的免疫促凋亡策略的直接腫瘤殺傷。(1)CDC策略:在人源化單鏈抗體的C端融合表達(dá)人IgG1Fc,構(gòu)建獲得系列scFv-Fc融合蛋白,進(jìn)行真核系統(tǒng)表達(dá)純化。流式細(xì)胞術(shù)和體外CDC實(shí)驗(yàn)結(jié)果表明,P1h2-Fc、P1h3-Fc融合蛋白均能與HER2陽性腫瘤細(xì)胞特異性結(jié)合,間接殺傷腫瘤細(xì)胞。HER2高表達(dá)的乳腺癌細(xì)胞及中度表達(dá)的肺癌細(xì)胞中,P1h2-Fc、P1h3-Fc的殺傷作用均強(qiáng)于e23sFv-Fc。(2)免疫促凋亡策略:將人源化單鏈抗體構(gòu)建替換入課題組前期建立的免疫促凋亡分子,獲得scFv-Fdt-tBid,進(jìn)行原核系統(tǒng)可溶表達(dá)純化。流式細(xì)胞術(shù)和細(xì)胞殺傷實(shí)驗(yàn)觀察到,P1h2-Fdt-tBid、P1h3-Fdt-tBid能夠特異性結(jié)合HER2陽性腫瘤細(xì)胞,有效抑制細(xì)胞增殖;在HER2高表達(dá)的乳腺癌細(xì)胞、胃癌細(xì)胞、中度表達(dá)的肺癌細(xì)胞中,二者對(duì)腫瘤細(xì)胞的增殖抑制與e23sFv-Fdt-tBid相當(dāng)。體內(nèi)實(shí)驗(yàn)分別采用NOD-SCID鼠的三種不同荷瘤模型(乳腺癌原位瘤、胃癌原位瘤、肺癌皮下移植瘤),明確觀察到人源化改構(gòu)免疫促凋亡分子的體內(nèi)抗腫瘤活性。在乳腺癌和胃癌模型中,P1h2-Fdt-t Bid、P1h3-Fdt-tBid的腫瘤抑制作用強(qiáng)于e23sFv-Fdt-tBid;而肺癌模型中,二者的抗腫瘤作用與e23sFv-Fdt-tBid相當(dāng)。綜上所述,本研究通過優(yōu)化CDR移植策略,針對(duì)抗HER2鼠源單鏈抗體e23sFv進(jìn)行人源化改構(gòu),成功篩選獲得免疫原性降低、親和力提高并具有內(nèi)化活性的兩株人源化單鏈抗體P1h2、P1h3,并通過體內(nèi)外實(shí)驗(yàn)驗(yàn)證其用于CDC策略和免疫促凋亡策略的有效性,為單鏈抗體為導(dǎo)向的融合蛋白的人源化改造、安全性評(píng)價(jià)提供了實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.51
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本文編號(hào)：1977385