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
基本信息
- 批准号:8157471
- 负责人:
- 金额:$ 25.49万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:
项目摘要
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)与多种分子的糖偶联物用于癌症诊断和治疗:为了将我们的生物偶联工作扩展到免疫脂质体配方,我们已经获得了针对CD22和HER-2蛋白的单链抗体(ScFv)。抗CD22和HER-2单链抗体的cDNAs来源于Dr.Dimitrovs团队。对这些cDNA进行了操作,使在大肠杆菌中表达的单链抗体具有含有1、3或17个苏氨酸(Thr)残基的C末端融合多肽。这些ScFv抗体在大肠杆菌中主要以包涵体形式表达,以可溶性蛋白的形式表达很少,从可溶性部分中只能获得微量的ScFv抗体。因此,已经开发出一种体外折叠方法,从一升细菌培养物中产生每种近20毫克的蛋白质。竞争酶联免疫吸附试验表明,体外折叠的抗HER-2单链抗体是正确折叠的活性蛋白。此外,这些重组单链抗体融合蛋白的C末端延伸融合多肽被用作人polypeptide-R-nu-acetylgalactosaminyltransferase II(h-ppGalNAc-T2)的受体底物,h-ppGalNAc-T2将GalNAc或2-keto-Gal(一种带有化学手柄的修饰的半乳糖)从各自的UDP-糖转移到Thr残基的侧链羟基(S)。酶切后,糖基化的C端融合多肽的MALDI-TOF谱表明,含有单一Thr残基的糖基化ScFv融合蛋白与单一的2-keto-Gal完全糖基化,而含有3个和17个Thr残基的糖基化ScFv融合蛋白分别由2-3和5-8个2-keto-Gal糖基化的融合蛋白组成。然后,将这些与修饰的半乳糖融合的ScFv蛋白与带有正交反应基团的荧光探针Alexa488连接。荧光标记的单链抗体蛋白与高表达HER2受体的人乳腺癌细胞株(SK-BR-3)特异性结合,表明体外折叠的单链抗体融合蛋白具有生物学活性,其C末端的多个共轭Alexa488探针的存在不会干扰其与抗原的结合。像MUX6这样的大粘蛋白已在大肠杆菌中以可溶性蛋白的形式表达,并通过ppGalNAc-T1与50多个含GalNAc的糖体外糖基化。因此,利用我们目前的位点特异性和多位点偶联方法,带有C端MUX 6融合蛋白的ScFv蛋白可以被修饰的糖糖基化并与生物活性分子偶联。这类复合体预计将携带几十个生物活性分子与scFv分子结合。制备靶向磁共振成像对比剂和靶向给药系统所需要的靶点特异性和多位点结合的抗体生物活性分子。携带氨氧基或炔基的脂类的合成,用于与糖蛋白连接:脂类分子1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(DPPE)被转化为DPPE-氨氧基或DPPE-炔衍生物,用于与单链抗体偶联,后者携带糖基和正交基。携带脂质分子的单链抗体分子接下来将用于(在与Blumenthals博士和Dimitrovs博士团队的合作项目中),为靶向药物输送配制脂质体。
项目成果
期刊论文数量(0)
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Pradman K Qasba其他文献
Pradman K Qasba的其他文献
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{{ truncateString('Pradman K Qasba', 18)}}的其他基金
Structural Studies and 3D Structure Determination of Recombinant <FONT FACE=symb
重组体的结构研究和 3D 结构测定 <FONT FACE=symb
- 批准号:
6433157 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
Oligosaccharide substrate interactions with beta-1,4-Ga
寡糖底物与 beta-1,4-Ga 的相互作用
- 批准号:
6944635 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
Detection of Specific Glycan Moieties on the Cell Surface
细胞表面特定聚糖部分的检测
- 批准号:
8349512 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
Oligosaccharide substrate interactions with beta-1,4-Gal
寡糖底物与 β-1,4-Gal 的相互作用
- 批准号:
7291793 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
Oligosaccharide Substrate and Inhibitor Interactions with beta-1,4-Gal-T1
寡糖底物和抑制剂与 β-1,4-Gal-T1 的相互作用
- 批准号:
7965207 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
Oligosaccharide Substrate and Inhibitor Interactions with beta-1,4-Gal-T1
寡糖底物和抑制剂与 β-1,4-Gal-T1 的相互作用
- 批准号:
7732974 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
PRINCIPALS OF CONFORMATIONAL ANALYSIS OF CARBOHYDRATES - A TEXT BOOK
碳水化合物构象分析原理 - 教科书
- 批准号:
6289310 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
Structure-Function Studies and Design of Novel Glycosyltransferases
新型糖基转移酶的结构功能研究和设计
- 批准号:
7965164 - 财政年份:
- 资助金额:
$ 25.49万 - 项目类别:
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