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)与各种分子的糖缀合用于癌症诊断和治疗:为了将我们的生物缀合工作扩展到免疫脂质体制剂,我们采用了针对CD 22和HER-2蛋白的单链抗体(scFv)。抗CD 22和HER-2蛋白的scFv的cDNA从Dimitrovs博士组获得。对这些cDNA进行操作,使得单链抗体在E.大肠杆菌具有含有1、3或17个苏氨酸(Thr)残基的C-末端融合多肽。在E.大肠杆菌主要以包涵体的形式存在,而作为可溶性蛋白的含量很低,从可溶性组分中仅获得微克量。因此,已经开发了一种体外折叠方法,该方法从1升细菌培养物中产生近20 mg的每种。竞争性ELISA检测表明体外折叠的抗HER-2单链抗体是正确折叠的活性蛋白。此外,这些重组scFv融合蛋白的C-末端延伸融合多肽用作人多肽-R-nu-乙酰半乳糖胺转移酶II(h-ppGalNAc- T2)的受体底物,所述人多肽-R-nu-乙酰半乳糖胺转移酶II将GalNAc或2-酮基-Gal(具有化学手柄的修饰半乳糖)从它们各自的UDP-糖转移至Thr残基的侧链羟基。蛋白酶切割后,糖基化C-末端融合多肽的MALDI-TOF光谱显示,具有单个Thr残基的糖基化scFv融合蛋白被单个2-酮基-Gal完全糖基化,而具有3个和17个Thr残基的糖基化scFv融合蛋白分别被发现为2-3个和5-8个2-酮基-Gal糖基化融合蛋白的等量混合物。然后将这些具有修饰的半乳糖的融合scFv蛋白与携带正交反应基团的荧光探针Alexa 488缀合。荧光标记的scFv蛋白特异性结合过表达HER 2受体的人乳腺癌细胞系(SK-BR-3),表明体外折叠的scFv融合蛋白是生物活性的,并且在其C末端存在缀合的多个Alexa 488探针不干扰其与抗原的结合。在E.大肠杆菌,并已在体外糖基化与超过50糖与GalNAc,使用ppGalNAc-T1。因此,使用本发明的位点特异性和多位点缀合方法,具有C-末端muc 6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缀合。预期此类复合物携带不只是几个而是几十个与scFv分子缀合的生物活性分子。本文描述的方法可以产生位点特异性和多位点缀合的抗体-生物活性分子,其对于靶向MRI图像造影剂和靶向药物递送系统的开发是非常需要的。将脂质分子1,2-二棕榈酰-sn-甘油基-3-磷酸乙醇胺(DPPE)转化成DPPE-氨基氧基或DPPE-炔衍生物,用于与携带具有正交反应基团的糖部分的scFV缀合。携带脂质分子的scFV分子接下来将用于(与Blumenthals博士和Dimitrovs博士小组的合作项目中),用于靶向药物递送的脂质体的配制。
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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