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Molecular Farming Antibodies in Plants: From Antibody Engineering to Antibody Production
Published in Maurizio Zanetti, J. Donald Capra, The Antibodies, 2002
Rainer Fischer, Ricarda Finnern, Olga Artsaenko, Stefan Schillberg
Ribosome display is a new technique for protein and antibody engineering that makes it possible to generate very large protein or peptide libraries on the surface of ribosomes, screen the library for binding affinities to a specific target molecule (ligand) and directly obtain the genetic information encoding the protein with the most desired biological function. As a complete in vitro system, ribosome display is the first selection system which requires no living cells. This gives ribosome display several potential advantages over phage display, which is today the preferred method for the construction and screening of protein libraries.
Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs
Published in mAbs, 2022
Andre A. R. Teixeira, Sara D’Angelo, M. Frank Erasmus, Camila Leal-Lopes, Fortunato Ferrara, Laura P. Spector, Leslie Naranjo, Esteban Molina, Tamara Max, Ashley DeAguero, Katherine Perea, Shaun Stewart, Rebecca A. Buonpane, Horacio G. Nastri, Andrew R. M. Bradbury
Library size is often a concern when performing in vitro evolution of any sort since one is limited by the number of transformants that can be conveniently obtained during library generation: 109–1010 for phage and E. coli display and 108–109 for yeast display in S. cerevisiae. We opted for a step-wise approach64 since it would allow us to explore the sequence space more effectively: in phase 1 we combined LCDR1 with LCDR2, HCDR1 with HCDR2, and used LCDR3 by itself, generating libraries that were larger than the corresponding theoretical combinatorial diversity. We have no reason to believe this is the optimal approach and did so because it was the most convenient set of combinations for library construction. Alternatives such as splitting HCDR1 and HCDR2 or combining them with LCDR1 or LCDR2 may provide advantages, although, given the affinity improvements observed here, this is not immediately obvious. Alternatively, one could use a purely molecular technique such as ribosome display, which is not limited by transformation efficiencies but presents its own challenges.
Strategies for targeting undruggable targets
Published in Expert Opinion on Drug Discovery, 2022
Gong Zhang, Juan Zhang, Yuting Gao, Yangfeng Li, Yizhou Li
However, two inevitable issues exist: one is incompatibility to toxic, insoluble, or misfolded proteins and the other is the limitation of library size (less than ~109 due to transformation or transfection efficiency). Accordingly, cell-free display systems including ribosome display and mRNA display have been developed (Table 3). These display systems harness the biological translation machinery but fully in vitro. Ribosome display requires a reconstituted transcription/translation system, allowing for nascent protein libraries to connect with their encoding mRNA in the protein-ribosome-mRNA complex upon translation (Figure 2a). Cycling from several rounds of transcription–translation–panning–washing–elution, ribosome display can accomplish enrichment of high-affinity binding members[52]. mRNA display library is constructed in vitro with puromycin attached at 3ʹ-end. Puromycin, structurally similar to the aminoacyl-tRNA, stalls the translational process to form a covalent linkage between the translation product and the encoding mRNA (Figure 2a). The selection round subsequently undergoes alternative peptide stabilization, reverse transcription, solid-phase selection, and PCR to amplify the enriched candidate. mRNA display is a monovalent library where each peptide is tagged by the corresponding coding mRNA and could accommodate modified unnatural amino acids to a certain extent[53]. In addition, owing to the cell-free property, ribosome display, and mRNA display technologies enable constructions of huge libraries with up to 1014 members (Table 3).
Abicipar pegol for neovascular age-related macular degeneration
Published in Expert Opinion on Biological Therapy, 2020
Rehan M. Hussain, Christina Y. Weng, Charles C. Wykoff, Raya A. Gandhi, Seenu M. Hariprasad
DARPins are derived from natural ankyrin repeat motifs, protein domains that mediate many well-characterized protein–protein interactions. Starting with large, diverse ribosome display DARPin libraries, and applying tools such as polymerase chain reaction, off-rate and specificity selection schemes allow for the identification of molecules with high specificity and affinity without some of the limitations of immunization-based technologies [46]. Additionally, DARPins exhibit high thermodynamic stability and solubility, with melting temperatures in the range of 80 − 100°C [47]. These properties allow for high-concentration formulation development and the evaluation and use of new application routes. Abicipar is a 14-kDa recombinant protein coupled to a 20-kDa polyethylene glycol (PEG) moiety to yield a 34-kDa molecule (Figure 1) [18]; this permits higher dosing on a molar basis compared to some of the commercially available anti-VEGF agents (excluding brolucizumab, which is a 26 kDA antibody fragment). Compared to ranibizumab, abicipar has a smaller molecular weight (34 kDa vs. 48 kDa) [48], reported higher target binding affinity (2 pM vs. 46 pM) [49] and longer ocular half-life [49,50]. These properties may contribute to favorable durability of action.