Using Glycosyltransferases for Conjugation of Single-Chain Antibodies and Lipids

< < < < < >< & b&gt；单链抗体（scFv）与各种分子糖偶联用于癌症诊断和治疗：&lt；/i>< </b&gt；为了将我们的生物偶联工作扩展到免疫脂质体制剂，我们已经研究了针对CD22和HER-2蛋白的单链抗体（scFv）。scFv抗CD22和HER-2蛋白的cdna从Dr. dimitrov组获得。这些cdna经过处理，使得在大肠杆菌中表达的单链抗体具有含有1、3或17个苏氨酸残基的c端融合多肽。这些scFv抗体在大肠杆菌中主要在包涵体中表达，作为可溶性蛋白的表达能力很差，可溶性部分的表达量仅为微克。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近20毫克的每一种。竞争性酶联免疫吸附试验表明，体外折叠的抗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端扩展融合多肽被用作人多肽- r -&amp；#925的受体底物；-乙酰半乳糖氨基转移酶II (h-ppGalNAc- T2)，将GalNAc或2-酮缩半乳糖（一种化学修饰的半乳糖）从它们各自的udp糖转移到苏氨酸残基的侧链羟基上。在蛋白酶裂解后，糖基化的c端融合多肽的MALDI-TOF光谱显示，含有1个苏氨酸残基的糖基化scFv融合蛋白被1个2-酮氨酸完全糖基化，而含有3个和17个苏氨酸残基的糖基化scFv融合蛋白分别是2-3和5-8个2-酮氨酸糖基化融合蛋白的等量混合物。这些与修饰的半乳糖融合的scFv蛋白然后与携带正交反应基团的荧光探针Alexa488结合。荧光标记的scFv蛋白特异性结合到过表达HER2受体的人乳腺癌细胞系（SK-BR-3）上，表明体外折叠的scFv融合蛋白具有生物活性，并且在其c端存在共轭的多个Alexa488探针不会干扰其与抗原的结合。在大肠杆菌中表达为可溶性蛋白，并使用ppGalNAc-T1在体外用超过50个糖基化GalNAc。因此，使用我们目前的位点特异性和多位点偶联方法，具有c端muc6融合蛋白的scFv蛋白可以与修饰糖基化并与生物活性分子偶联。这样的配合物预计将携带不只是几个，而是几十个生物活性分子偶联到scFv分子。这里描述的方法可以生成位点特异性和多位点偶联的抗体生物活性分子，这对于开发靶向MRI图像造影剂和靶向药物传递系统非常需要。</p>< < < < < < < >< >< >< >< >& gt;& gt;& gt;& gt;& gt;& gt;& gt;& gt;& gt;& gt;& gt;& gt;& gt;& gt1,2-二棕榈酰基- n-甘油-3-磷酸乙醇胺（DPPE）转化为DPPE-氨基或DPPE-炔衍生物，与带糖片段的正交反应基团的scFV偶联。携带脂质分子的scFV分子接下来将用于（与Blumenthals博士和dimitrov博士团队的合作项目中），用于制定靶向药物递送的脂质体。

<P><i><b>Glycoconjugation of single chain antibodies (scFv) with various molecules for cancer diagnosis and treatment:</i></b> 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-Ν-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.</p><p>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.</p><P><i><b>Synthesis of lipids carrying aminooxy or alkyne group for linking with a glycoprotein that has a sugar moiety linked with an orthogonal reactive group:</i></b> 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.</p>