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:
  • 项目状态:
    未结题

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)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

Pradman K Qasba其他文献

Pradman K Qasba的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ 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 Interactions with Proteins
低聚糖与蛋白质的相互作用
  • 批准号:
    6559116
  • 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:
Utilizing Glycosyltransferases for Bioconjugation
利用糖基转移酶进行生物共轭
  • 批准号:
    8552799
  • 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:

相似海外基金

EukaryoticHopanoids: Deciphering the regulatory network behind unusual lipids in eukaryotes
真核Hopanoids:破译真核生物异常脂质背后的调控网络
  • 批准号:
    EP/Y024702/1
  • 财政年份:
    2024
  • 资助金额:
    $ 25.49万
  • 项目类别:
    Fellowship
Conference: 26th International Symposium on Plant Lipids
会议:第26届国际植物脂质研讨会
  • 批准号:
    2416127
  • 财政年份:
    2024
  • 资助金额:
    $ 25.49万
  • 项目类别:
    Standard Grant
Glucagon-like peptide 1 and 2 double receptor knockout (GLPDRKO) mice have higher post-prandial lipids and glucose in a sex- and meal-dependent manner
胰高血糖素样肽 1 和 2 双受体敲除 (GLPDRKO) 小鼠具有较高的餐后血脂和血糖,且具有性别和膳食依赖性
  • 批准号:
    495444
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
Breath biopsy technology targeting for volatile oxidized lipids
针对挥发性氧化脂质的呼吸活检技术
  • 批准号:
    23K06080
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Development and evaluation of a method to assess nutritional quality of dietary lipids in food
食品中膳食脂质营养质量评估方法的开发和评价
  • 批准号:
    2890339
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
    Studentship
Integral Membrane Proteins and Lipids Ejected from the Membranes of Native Tissues
从天然组织膜中排出的完整膜蛋白和脂质
  • 批准号:
    EP/Y029259/1
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
    Research Grant
Development and evaluation of a method to assess the nutritional quality of dietary lipids in food
食品中膳食脂质营养质量评估方法的开发和评价
  • 批准号:
    BB/Y512382/1
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
    Training Grant
The Role of Lipids in Alzheimer's Disease and Related Dementias among Black Americans: Examining Lifecouse Mechanisms
脂质在美国黑人阿尔茨海默病和相关痴呆中的作用:检查生命机制
  • 批准号:
    10643344
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
Whole genome sequence interpretation for lipids to discover new genes and mechanisms for coronary artery disease
脂质的全基因组序列解释,以发现冠状动脉疾病的新基因和机制
  • 批准号:
    10722515
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
Impact of inflammatory lipids on Yersinia pestis infection
炎性脂质对鼠疫耶尔森菌感染的影响
  • 批准号:
    10722648
  • 财政年份:
    2023
  • 资助金额:
    $ 25.49万
  • 项目类别:
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了