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ABSTRACT: High-throughput generation of antibodies for proteome research has become feasible by using antibody gene libraries and in vitro selection methods like phage display. Typically monovalent antibody fragments like scFv, Fab or scFab are obtained by this technology. To mimic the IgG molecule and gain avidity, resulting in stronger binding, multimerization domains can be fused to antibody fragments. Here we systematically analyzed different multimerization domains in respect to three key parameters, crucial for the high-throughput generation of binders. (i) The compatibility to be displayed on phage (assessed for at least three different antibody formats, scFv, Fab and scFab) in combination with five different multimerization domains; (ii) production yields and (iii) oligomerization properties were analyzed for three different scFv fragments. We found that the use of a biotin acceptor domain in combination with an in vivo biotinylation system performed best concerning the key parameters and thus would be a useful tool to generate multimeric antibody complexes on demand from phage display selected antibody fragments with the least effort.
New Biotechnology 08/2009; 26(6):314-21. · 2.76 Impact Factor
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ABSTRACT: Ribonucleases degrade RNA, now considered an important drug target. The parent member of this protein superfamily is bovine pancreatic RNase A that functions as a digestive enzyme. Other physiological roles and activities have been ascribed to more recently discovered members of this superfamily. Angiogenin was isolated by following angiogenic activity from cell culture media conditioned by colon cancer cells. ONCONASE kills tumor cells in vitro and in vivo and has advanced to a phase IIIb confirmatory clinical trial for the treatment of unresectable malignant mesothelioma. All three of these RNA degrading enzymes have been used to generate immunoRNases; chemical conjugates and ligand-RNase fusion proteins, for cancer therapy. The properties of each of these RNases are described along with the increasingly sophisticated construction of recombinant immunoRNases. The advantages of using RNase as an antibody payload is compared to using plant or bacterial toxins in the construction of immunotoxins, a related strategy for specifically killing malignant cells.
Current pharmaceutical design 02/2009; 15(23):2665-75. · 4.41 Impact Factor
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ABSTRACT: Vascular cell adhesion molecule 1 (VCAM-1) is involved in the recruitment of leukocytes to inflammatory sites. In this study we present the first functional knockdown of VCAM-1 using an ER retained antibody construct. We generated a knockdown construct encoding the VCAM-1 specific single chain variable fragment scFv6C7.1 fused to the C-terminal ER retention sequence KDEL. HEK-293:VCAM-YFP cells stably expressing a VCAM-YFP fusion protein were transiently transfected with the knockdown construct and showed down-regulation of surface VCAM-1. Knockdown efficiency of the system is time-dependent due to used transient transfection of the intrabody construct. Furthermore, intrabody mediated knockdown of HEK-293:VCAM-YFP cells also impaired cell-cell interaction with Jurkat cells that are endogenously expressing VLA-4, the physiological partner of VCAM-1. Posttranslational knockdown with ER retained antibodies seems to be a promising technique, as shown here for VCAM-1.
Journal of Immunological Methods 12/2008; 341(1-2):30-40. · 2.20 Impact Factor
