Using Glycosyltransferases for Conjugation of Single-Chain Antibodies and Lipids

使用糖基转移酶缀合单链抗体和脂质

• 批准号: 8157471
• 负责人: Pradman K Qasba
• 金额: $ 25.49万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157471
• 关键词: Antibodies; Lipids; glycosyltransferase

Glycoconjugation of single chain antibodies (scFv) with various molecules for cancer diagnosis and treatment: To extend our bioconjugation work towards the immunoliposome formulation, we have taken up single chain antibodies (scFv) against CD22 and HER-2 proteins. The cDNAs of scFv against CD22 and HER-2 proteins were obtained from Dr. Dimitrovs group. These cDNAs were manipulated such that the single chain antibodies expressed in E. coli have a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. These scFv antibodies were expressed in E. coli mainly in inclusion bodies and very poorly as a soluble protein, and only micro gram quantities were obtained from the soluble fraction. Therefore an in vitro folding method has been developed that produced nearly 20 mgs of each from a one liter bacterial culture. Competitive ELISA assay indicated that the in vitro folded anti HER-2 scFv is correctly folded active protein. Furthermore, the C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins are used as the acceptor substrate for human polypeptide-R-nu-acetylgalactosaminyltransferase II (h-ppGalNAc- T2) that transfers either GalNAc or 2-keto-Gal, a modified galactose with a chemical handle, from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). Upon protease cleavage, the MALDI-TOF spectra of the glycosylated C-terminal fusion polypeptides showed that the glycosylated scFv fusion protein with a single Thr residue is fully glycosylated with a single 2-keto-Gal, whereas the glycosylated scFv fusion protein with 3 and 17 Thr residues is found as an equal mixture of 2-3 and 5-8 2-keto-Gal glycosylated fusion proteins, respectively. These fusion scFv proteins with the modified galactose are then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bind specifically to a human breast cancer cell line (SK-BR-3) that over expresses the HER2 receptor, indicating that the in Vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen.A large mucin protein like, muc6, has been expressed as a soluble protein in E. coli and has been in Vitro glycosylated with more than 50 sugars with GalNAc, using ppGalNAc-T1. Therefore, using our present site-specific and multiple site conjugating method, scFv proteins with a C-terminal muc6 fusion protein can be glycosylated with modified sugars and conjugated with bioactive molecules. Such complexes are expected to carry not just a few but several tens of bioactive molecules conjugated to scFv molecules. The methodology described here can generate site-specific and multiple site conjugated antibody-bioactive molecules that are in great need for the development of targeted MRI image contrast agents and a targeted drug delivery system.Synthesis of lipids carrying aminooxy or alkyne group for linking with a glycoprotein that has a sugar moiety linked with an orthogonal reactive group: The lipid molecule 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was converted into either DPPE-aminooxy or DPPE-alkyne derivatives for conjugation with scFV that carry sugar moiety with an orthogonal reactive group. The scFV molecules carrying lipid molecules will next used (in a collaborative project with Dr. Blumenthals and Dr. Dimitrovs groups), for the formulation of liposomes for the targeted drug delivery.

单链抗体（SCFV）的糖缀合具有各种用于癌症诊断和治疗的分子：为了将生物缀合功能扩展到免疫脂质体配方，我们采用了针对CD222和HER-2蛋白的单链抗体（SCFV）。 SCFV针对CD22和HER-2蛋白的CDNA从Dimitrovs Group博士获得。操纵这些cDNA，以使大肠杆菌中表达的单链抗体具有含有1、3或17苏氨酸（THR）残基的C末端融合多肽。这些SCFV抗体主要在大肠杆菌中表达，主要是在包含体中，并且作为可溶性蛋白非常差，并且仅从可溶性馏分中获得了微克量。因此，已经开发了一种体外折叠方法，该方法从一升细菌培养物中产生了近20 mg。竞争性ELISA分析表明，体外折叠抗HER-2 SCFV是正确折叠的活性蛋白。此外，这些重组SCFV融合蛋白的C末端扩展融合多肽被用作人多肽 - R-r-Nu-乙酰乳糖苷基氨基转移酶II（h-pppgalnac-t2）的受体底物，可以将其转移到galnac或2-酮的旋转中，并将其转换为a galnac或2-酮 - 固定型，并将其变形式融合液体供应。 THR残基的侧链羟基（S）。蛋白酶裂解后，糖基化的C末端融合多肽的MALDI-TOF光谱表明，具有单个THR残基的糖基化的SCFV融合蛋白被单个2-酮基完全糖基化了单个2-酮基因，而与3和17的糖基化的SCFV融合蛋白相等，是2-8-分别为2-酮基 - 糖基化融合蛋白。然后将这些与改性半乳糖的融合SCFV蛋白与荧光探针Alexa488结合，该荧光探针带有正交反应性基团。标记为SCFV蛋白的荧光特异性与人类乳腺癌细胞系（SK-BR-3）结合，该细胞系（SK-BR-3）表达HER2受体，表明体外折叠的SCFV融合蛋白具有生物活性，并且存在共轭的多个ALEXA488探针在其C-终端中的多个Anternical temential the Antection.Antection.Antection.Antection.ANTENTEN.AN.A.AA的MICIN.AAAA AA amen.aaa aa aa aa a，A。作为大肠杆菌中的可溶性蛋白质，使用PPGALNAC-T1与50多种糖一起在体外与50多种糖糖基化。因此，使用我们目前的位点特异性和多个位点共轭方法，具有C-末端MUC6融合蛋白的SCFV蛋白可以用修饰的糖糖基化并与生物活性分子共轭。这种复合物预计不仅携带少数，而且携带几十个与SCFV分子结合的生物活性分子。此处描述的方法可以生成位点特异性和多个位点共轭抗体生物活性分子，这些分子非常需要开发有针对性的MRI图像对比剂和有针对性的药物输送系统。脂质的含有脂质的含有氨基酸或碱基的脂质与与糖蛋白链接的脂肪蛋白链接的脂质相结合：将1,2-二氨木酰基-SN-甘油-3-磷酸乙醇胺（DPPE）转化为DPPE-AMINOOXY或DPPE-ALKYNE衍生物，以与SCFV结合，与SCFV结合，将糖与正交反应性基团带有糖部分。接下来将使用携带脂质分子的SCFV分子（在与Blumenthals博士和Dimitrovs组的协作项目中），以制定脂质体以用于靶向药物。