| 1. |
- Blokzijl, Andries, et al.
(författare)
-
Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
- 2016
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 15:6, s. 1848-1856
-
Tidskriftsartikel (refereegranskat)abstract
- The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.
|
|
| 2. |
- Mersmann, Michael, et al.
(författare)
-
Towards proteome scale antibody selections using phage display
- 2010
-
Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 27:2, s. 118-128
-
Tidskriftsartikel (refereegranskat)abstract
- In vitro antibody generation by panning a large universal gene library with phage display was employed to generate antibodies to more than 60 different antigens. Of particular interest was a comparison of pannings on 20 different SH2 domains provided by the Structural Genomics Consortium (SGC). Streamlined methods for high throughput antibody generation developed within the 'Antibody Factory' of the German National Genome Research Network (NGFN) were demonstrated to minimise effort and provide a reliable and robust source for antibodies. For the SH2 domains, in two successive series of selections, 2668 clones were analysed, resulting in 347 primary hits in ELISA. Half of these hits were further analysed, and more than 90 different scFv antibodies to all antigens were identified. The validation of selected antibodies by cross-reactivity ELISA, western blot and on protein microarrays demonstrated the versatility of the in vitro antibody selection pipeline to generate a renewable resource of highly specific monoclonal binders in proteome scale numbers with substantially reduced effort and time.
|
|
