联合应用MALDI-TOFMS抗原表位确定技术与分子对接进行抗体人源化改造

发布时间：2018-06-17 12:20

本文选题：表位 + 质谱　；参考：《第四军医大学》2010年硕士论文

【摘要】： 众所周知,肿瘤是最致命的疾病之一。自20世纪70年代以来,我国癌症患者的人数逐年升高,约有百分之八十的癌症患者死于肺癌、肝癌、胃癌等常见肿瘤。通常的治疗方法包括外科手术、放射治疗、化疗、生物治疗等,这些治疗方法有时单独使用,有时联合使用。抗体治疗是生物治疗的一种,它的重要性越发明显。抗体治疗的前景是光明的,目前已批准20余种抗体药物用于治疗各种疾病,在临床上已取得预期效果。抗体药物是利用以细胞工程技术和基因工程技术为主体的抗体工程技术制备的药物,但由于其伴随的人抗鼠抗体反应(HAMA),抗体药物的应用往往受到了一定程度的限制。为了降低抗体药物的HAMA反应,基于蛋白质结构信息发展了一系列的改造抗体的方法。随着分子生物学的发展,嵌合抗体和人源化抗体的出现,成为了解决抗体应用中降低HAMA反应的有效途径,但改造的抗体往往伴随着亲和力的大幅下降。本研究建立了一种新型的抗体人源化方法,首次联合基于质谱的表位确定技术和分子对接对抗体进行人源化改造。通过鉴定出HAb18G/CD147的抗原表位进行HAb18抗体的人源化设计,这样就在保证了抗体HAb18亲和力的基础上进行人源化改造,之后通过基因重组技术合成新的抗体片段并检测该抗体片段的生物学活性,下面将从以下三部分分别说明。第一部分:MALDI-TOF MS确定HAb18G/CD147的抗原表位目的:确定HAb18G/CD147的表位序列方法:利用配套的磁架洗涤新的磁珠,洗去磁珠上的防腐剂。加入适量的HAb18抗体,在室温的条件下与磁珠结合1~2小时并轻微摇晃,使抗体与磁珠充分结合。之后用PBS(0.1% BSA,pH=7.4)洗涤磁珠4~5次,以封闭未结合抗体磁珠的活性位点。将适量的HAb18G/CD147加入到制备好的抗体磁珠混悬液中,室温下结合1~2小时,使抗原与抗体充分结合。加入胰酶溶液酶解HAb18G/CD147,用PBS洗涤掉未结合的抗原肽段,并用0.1㳠TFA(pH=2.5)洗脱HAb18G/CD147抗原表位肽并迅速冻干,用MALDI-TOF MS检测制备的样品。结果:制备了HAb18G/CD147抗原表位肽,说明了利用免疫磁珠酶解抗原的办法是可行的,并且制备出的样品符合MALDI-TOF MS检测的标准。对制备的HAb18G/CD147抗原表位肽进行MALDI-TOF MS分析,并与PeptideCutter软件预测的胰酶酶切HAb18G/CD147各肽段分子量进行对比,得到了HAb18G/CD147抗原表位肽的序列信息。第二部分:应用确定的表位与分子对接对抗体HAb18人源化设计目的:确定突变的氨基酸位点方法:根据已有的HAb18抗体模型和得到的HAb18G/CD147抗原表位序列,应用DOCKING软件建立HAb18G/CD147与HAb18的分子对接模型,并预测表位的关键氨基酸残基。在模拟出HAb18G/CD147同其抗体HAb18的对接模型后,用表面重塑的方法对HAb18可变区进行人源化设计。利用序列分析和结构分析确定鼠源抗体外露的差异残基,选定将要突变的氨基酸残基位点,以便能够合成低免疫原性又具有抗原结合活性的人源化抗体。结果:建立了一套新的人源化抗体的方法,首次将基于MALDI-TOF MS确定抗原表位技术引入到分子对接中,建立了HAb18G/CD147和HAb18的分子对接模型,完成了HAb18可变区的人源化设计,确定突变的氨基酸位点为H42E,H90A和L43S。第三部分:人源化抗体片段HAb18-huscFv的表达与鉴定目的:验证该人源化方法方法:根据设计的人源化方案,用overlapping PCR的方法合成人源化形式HAb18-huscFv的基因,克隆至表达载体pCANTAB-6His-HAb18-huscFv中,转化至E. coli中诱导表达。经Ni2+柱纯化表达的蛋白后,采用SDS-PAGE、Western blot、细胞免疫荧光等方法对表达产物进行检测、鉴定,同步构建表达其亲代鼠源形式抗体为对照。采用间接竞争ELISA法分析抗体HAb18-huscFv与HAb18G/CD147的结合活性, SPR法测定抗体HAb18-huscFv与HAb18G/CD147的亲和力,并用同步表达的鼠源形式抗体作为对照,分析两种抗体有无差异。最后采用HAMA阳性血清,对比了HAb18-huscFv和鼠源HAb18-scFv的免疫原性差异。结果:根据已选择的突变位点,用overlapping PCR的方法合成了新的基因并克隆至表达载体,测序验证正确后转化到E. coli中诱导表达,经Ni2+柱纯化后获得了较纯的HAb18-huscFv。SDS-PAGE和Western blot检测目标蛋白分子量约为27kDa。经SPR法测定,HAb18-huscFv与HAb18-scFv相比亲和力无明显差异。用细胞免疫荧光法检测HAb18-huscFv与肝癌细胞SMMC-7721具有一定结合能力。间接竞争ELISA法结果显示:抗体HAb18-huscFv与HAb18-scFv相比具有较低的免疫原性。结论:基于利用新方法建立的抗体人源化方案,改造后的抗体HAb18-huscFv与HAb18-scFv相比,在降低了免疫原性的同时,又保留了抗体的抗原结合能力,证明了该方法是有效可行的,为抗体人源化设计提供了新的思路。
[Abstract]:As we all know, cancer is one of the most deadly diseases. Since 1970s, the number of cancer patients in our country has increased year by year, and about eighty percent of cancer patients die from common tumors such as lung, liver and stomach cancer. The common treatment methods include surgery, radiation therapy, chemotherapy, biological treatment and so on. These treatments are sometimes alone. Use, sometimes combined.
Antibody therapy is a kind of biological therapy, its importance is more and more obvious. The prospect of antibody treatment is bright. At present, more than 20 kinds of antibody drugs have been approved for the treatment of various diseases, and the expected effect has been achieved. Antibody drugs are made by using antibody engineering technology based on cell engineering and genetic engineering technology. But because of its anti mouse antibody response (HAMA), the application of antibody drugs is often limited to a certain extent. In order to reduce the HAMA reaction of antibody drugs, a series of methods for transforming antibodies have been developed based on protein structure information. With the development of molecular biology, the emergence of chimeric and humanized antibodies has become the result of the development of molecular biology. The effective way to reduce the HAMA reaction in the application of antibodies is to solve the problem, but the modified antibody is often accompanied by a sharp decrease in affinity.
In this study, a new method of humanization of antibody was established. It was first combined with mass spectrometry based epitope determination technique and molecular docking for humanization of antibody. By identifying the epitopes of HAb18G/CD147, the humanized design of HAb18 antibody was carried out, so that the humanized transformation was made on the basis of guaranteeing the affinity of antibody HAb18. After that, the new antibody fragment was synthesized by gene recombination technology and the biological activity of the antibody fragment was detected. The following three parts will be explained below.
Part I: MALDI-TOF MS determines the epitope of HAb18G/CD147.
Objective: to determine the epitope sequence of HAb18G/CD147
Methods: wash the new magnetic beads and remove the antiseptic on the magnetic beads. Add appropriate HAb18 antibody, combine the magnetic beads with the magnetic beads for 1~2 hours at room temperature and gently shake them, so that the antibody and magnetic beads are fully combined. Then PBS (0.1% BSA, pH=7.4) is used to wash the magnetic beads 4~5 times to close the active site of unconjugated magnetic beads. HAb18G/CD147 was added to the prepared antibody magnetic bead suspension. The antigen was combined with the antibody at room temperature for 1~2 hours at room temperature. The trypsin solution HAb18G/CD147 was added to the enzyme solution and the unbound antigen peptide was washed out with PBS, and the HAb18G/CD147 antigen epitopes were eluted with 0.1 TFA (pH=2.5) and freeze-dried quickly, and the samples were prepared by MALDI-TOF MS Goods.
Results: the HAb18G/CD147 epitope peptide was prepared, indicating that the method of using immunomagnetic beads to solve the antigen was feasible, and the prepared samples were in conformity with the standard of MALDI-TOF MS detection. The MALDI-TOF MS analysis of the prepared HAb18G/CD147 epitopes was carried out and the peptide segments of the pancreatin were predicted by the PeptideCutter software. The sequence information of HAb18G/CD147 epitope peptide was obtained by comparing the subunits.
The second part: the application of designated epitope and molecular docking to the humanization design of antibody HAb18.
Objective: to determine the amino acid site of the mutant
Methods: Based on the existing HAb18 antibody model and the HAb18G/CD147 epitope sequence obtained, the molecular docking model of HAb18G/CD147 and HAb18 was established by DOCKING software, and the key amino acid residues in the epitopes were predicted. After simulating the docking model of HAb18G/CD147 with its antibody HAb18, the HAb18 variable region was carried out by surface remodeling. Source design. Using sequence analysis and structural analysis to determine the differential residues of mouse source antibody exposure, select the amino acid residues that will be mutated, in order to be able to synthesize human derived antibodies with low immunogenicity and antigen binding activity.
Results: a new method of humanized antibody was established. The MALDI-TOF MS based epitope technique was first introduced into the molecular docking, and the molecular docking model of HAb18G/CD147 and HAb18 was established. The humanized design of the HAb18 variable region was completed, and the mutant amino acid sites were H42E, H90A and L43S..
The third part: expression and identification of humanized antibody fragment HAb18-huscFv.
Objective: to verify the humanization method
Methods: according to the designed humanization scheme, the gene of human derived form HAb18-huscFv was synthesized by overlapping PCR, and was cloned into the expression vector pCANTAB-6His-HAb18-huscFv and transformed into E. coli to induce expression. After purification of the expressed protein by Ni2+ column, the expression products were introduced by SDS-PAGE, Western blot, cell immunofluorescence and so on. An indirect competitive ELISA method was used to analyze the binding activity of antibody HAb18-huscFv and HAb18G/CD147, and the affinity between the antibody HAb18-huscFv and HAb18G/CD147 was measured by SPR method, and the antibody of the mouse source was used as the control, and the two kinds of antibodies were analyzed. HAMA positive serum was used to compare the immunogenicity of HAb18-huscFv and mouse HAb18-scFv.
Results: according to the selected mutation sites, a new gene was synthesized and cloned to the expression vector by overlapping PCR. After the sequencing verification was correct, the expression was transformed into E. coli. After the purification of the Ni2+ column, the molecular weight of the pure HAb18-huscFv.SDS-PAGE and Western blot detection target protein was about 27kDa. by SPR method and HAb18-h. There was no significant difference in affinity between uscFv and HAb18-scFv. The binding ability of HAb18-huscFv to SMMC-7721 cells was detected by cell immunofluorescence. The result of indirect competitive ELISA showed that the antibody HAb18-huscFv had a lower immunogenicity compared with HAb18-scFv.
Conclusion: Based on the antibody humanization scheme established by the new method, the modified antibody HAb18-huscFv is compared with HAb18-scFv, while reducing the immunogenicity and retaining the antigen binding ability of the antibody, it is proved that this method is effective and feasible, and provides a new idea for the antibody humanization design.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】