蛋白质和抗体的分子修饰技术是指用化学修饰的方法将生物方法所产生的药用蛋白和抗体进行分子改性的技术，可以用来解决或缓解蛋白和抗体在药用过程中存在的诸多问题。其中，蛋白质的聚乙二醇修饰技术和抗体的偶联药物技术是分子修饰技术的两个重要组成部分。在前人研究的基础上，本文对这两个方向进行了新的探索，创新性的发展了聚乙二醇酰肼氮末端修饰技术和抗体片段偶联药物技术，用于构建长效蛋白药物和靶向型抗体偶联药物。蛋白质的聚乙二醇修饰是一种广泛应用的改善蛋白质性质的方法，经过聚乙二醇修饰的蛋白药物其稳定性提高，体内保留时间延长，免疫原性降低，体内药代药效学性质得到明显改善。聚乙二醇修饰方法分为随机修饰和定点修饰两种。由于定点修饰的明显优势，现在聚乙二醇修饰领域越来越朝着定点修饰的方向发展。其中，蛋白药物的氮末端醛基修饰方法使用最广，这一修饰方法在一定程度上可以减少多修饰产物及单修饰产物位置异构体。然而，氮末端醛基修饰的方法仍然会产物多修饰产物，这样，产物间的分离纯化和定量定性过程会变得复杂，影响了这一方法的广泛使用。在本论文的研究中，我们开发了一种氮末端的修饰策略。首先我们将干扰素β1b的氮末端丝氨酸使用高碘酸钠定点氧化生成醛基，然后与聚乙二醇酰肼修饰剂发生偶联。修饰以后的干扰素β1b其半衰期明显延长，从原蛋白的2 h延长的8 h以上，其体内免疫原性基本消失，应用药效学性质得到改善。随后，我们对酰肼修饰方法和传统醛基修饰方法从修饰选择性、反应速率、产率以及药代动力学性质进行了比较。结果发现，酰肼修饰方法是一种高度特异性的修饰反应，聚乙二醇专一性的修饰在干扰素β1b的氮末端，而醛基修饰过程中出现了多修饰产物。并且酰肼修饰的单修饰产率要显著高于醛基修饰过程，显示出了更高的修饰效率。这些结果表明，蛋白质的酰肼修饰方法是一种实用性强的氮末端修饰方法，可以用于氮末端是丝氨酸或者苏氨酸蛋白质的定点修饰，所得的单修饰产物，其体内药代动力学性质也得到明显改善。 文献已有的抗体偶联药物制备方法一般是氨基偶联或者二硫键全还原后进行偶联，不可避免的会产生混合产物。由于产物的种类及分子结构各不相同，导致后续的分离纯化及产物评价过程异常复杂，在一定程度上制约了相应抗体偶联药物的应用。在本论文的研究中，我们开发了一种偶联策略用于制备定点均一的抗体片段偶联药物（AFDC）。研究中，作为模型药物的抗肿瘤药物阿霉素通过聚乙二醇偶联剂的定点偶联，特异性的连接于抗CD20抗体的Fab’片段铰链区，获得一种载药量确定的抗体片段偶联药物。抗CD20抗体首先使用胃蛋白酶酶解获得F(ab’)2片段，然后使用β巯基乙胺定点还原F(ab’)2片段铰链区获得Fab’片段，Fab’片段铰链区存在两个可供定点偶联的游离巯基。与此同时，通过化学合成手段将阿霉素和PEG偶联剂进行反应，获得MAL-PEG-DOX中间产物。这一产物与Fab’片段发生特异性偶联，生成最终的抗体片段偶联药物。由于两端反应的特异性，最终得到的产物中，两分子阿霉素连接于Fab’片段的铰链区，其药物/抗体偶联比为2。激光共聚焦结果和细胞ELISA结果表明，所得抗体偶联药物能够进入并且聚集于抗CD20抗体阳性细胞中，并且表现出肿瘤细胞杀伤作用。在体内实验中，Fab’-PEG-DOX偶联物表现出了肿瘤部位靶向作用以及良好的肿瘤治疗效果，Fab’-PEG-DOX治疗组的小鼠肿瘤抑制率更高，存活时间延长，毒副作用降低。这一抗体偶联药物制备策略可以广泛应用于其他种类抗体及细胞毒素的偶联过程中，具有良好的应用前景。

Molecule modification technology is a method of chemical conjugation of therapeutic proteins and antibodies produced by biological methods to solve or alleviate the problems in application process. Wherein, the protein PEGylation and antibody drug conjugates technology are two important aspects of molecular modification technology. On the basis of previous studies, we have explored innovative development of N-terminal hydrazide PEGylation and antibody fragments drug conjugates (AFDC) strategy, constructing long-term / targeting pharmaceuticals.PEGylation, including non-specific and site-directed ways, is a well-established and validated strategy to increase the stability, in vivo plasma retention time, and efficacy of protein pharmaceutics come together with the reduction of immunogenicity and hydrophobicity. Site-directed conjugation by PEG-aldehyde is the most widely used method for N-terminal modification, however, the generation of multi-modified products is inevitable due to lysine chemistry which always leads to difficulties in purification and quantification procedure. In this study, we developed a specific PEGylation strategy through the periodation of N-terminus of interferon beta-1b （IFN-β-1b） followed by the coupling of PEG-hydrazide. The prolonged elimination half-life and significantly diminished immunogenicity of PEG hydrazide-modified protein indicated an effective process in improving the properties of pharmacology and immunogenicity of IFN-β-1b. We further conducted comparisons on selectivity, velocity, yield and pharmacokinetics of the two methods. Results demonstrated that the hydrazide-based conjugation was a highly specific coupling reaction that only produced homogeneous N-terminal mono-PEGylated conjugate, while heterogeneous multi-modified products were generated in aldehyde-based process. In addition, fairly higher PEGylation yield was presented in the hydrazide conjugation compared with that of aldehyde strategy. These investigations supplied a practical approach for site specific modification of proteins with N-terminal serine or threonine to achieve improved homogeneity of conjugates as well as enhanced pharmacological properties.Conventional preparation strategies for antibody-drug conjugates （ADCs） result in heterogeneous products with various molecular size and species. In this study, we developed a homogenous preparation strategy by site-specific conjugation of the anticancer drug with antibody fragment. The model drug doxorubicin （DOX） was coupled to the Fab’ fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate （AFDC）. Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate Fab’ fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol （NHS-PEG-MAL） to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab’ fragment to form a Fab’-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and the introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment with two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab’-PEG-DOX retained appreciable targeting ability and improved anti-tumor activity, demonstrating excellent therapeutic effect on lymphoma mice model for better cure rate and significantly reduced side effects. The results in this study show that site-speci?c reduction of IgG and conjugation to the resulting Fab’ fragment is an applicable and e?cient strategy for preparing antibody drug conjugates with de?nite composition to achieve improved antitumor activity.