Directed evolution of human scFvs in DT40 cells

Lim, Alfred; Williams, Gareth T.; Rada, Cristina; Sale, Julian E.

doi:10.1093/protein/gzv058

Cited by 8 publications

(5 citation statements)

References 35 publications

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“…Both hybridoma and in vitro techniques sometimes lead to mAbs with lower than expected affinity and specificity (Foote and Eisen, 1995;González-Fernández et al, 2020). To alleviate this drawback, supplementary in vitro affinity maturation is used to increase the potency of the designed antibodies resulting in lower injected doses and side effects (Lim et al, 2016;Persson et al, 2018). The two approaches in use today in this respect include random mutagenesis (Tachioka et al, 2016) and chain shuffling/site-directed mutagenesis (Lou and Marks, 2010).…”

Section: Introductionmentioning

confidence: 99%

Towards an optimal monoclonal antibody with higher binding affinity to the receptor-binding domain of SARS-CoV-2 spike proteins from different variants

Neamţu

Mocci

Laaksonen

et al. 2022

Preprint

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A highly efficient and robust multiple scales in silico protocol, consisting of atomistic constant charge Molecular Dynamics (MD), constant-charge coarse-grain (CG) MD and constant-pH CG Monte Carlo (MC), has been used to study the binding affinities, the free energy of complexation of selected antigen-binding fragments of the monoclonal antibody (mAbs) CR3022 (originally derived from SARS-CoV-1 patients almost two decades ago) and 11 SARS-CoV-2 variants including the wild type. CR3022 binds strongly to the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, but chooses a different site rather than the receptor-binding motif (RBM) of RBD, allowing its combined use with other mAbs against new emerging virus variants. Totally 235,000 mAbs structures were generated using the RosettaAntibodyDesign software, resulting in top 10 scored CR3022-RBD complexes with critical mutations and compared to the native one, all having the potential to block virus-host cell interaction. Of these 10 finalists, two candidates were further identified in the CG simulations to be clearly best against all virus variants, and surprisingly, all 10 candidates and the native CR3022 did exhibit a higher affinity for the Omicron variant with its highest number of mutations (15) of them all considered in this study. The multiscale protocol gives us a powerful rational tool to design efficient mAbs. The electrostatic interactions play a crucial role and appear to be controlling the affinity and complex building. Clearly, mAbs carrying a lower net charge show a higher affinity. Structural determinants could be identified in atomistic simulations and their roles are discussed in detail to further hint at a strategy towards designing the best RBD binder. Although the SARS-CoV-2 was specifically targeted in this work, our approach is generally suitable for many diseases and viral and bacterial pathogens, leukemia, cancer, multiple sclerosis, rheumatoid, arthritis, lupus, and more.

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Section: Introductionmentioning

confidence: 99%

Towards an optimal monoclonal antibody with higher binding affinity to the receptor-binding domain of SARS-CoV-2 spike proteins from different variants

Neamţu

Mocci

Laaksonen

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…B cell lymphoma lines with misregulated AID-dependent somatic hypermutation have been used to evolve antibodies or fluorescent proteins (Arakawa et al, 2008; Lim et al, 2016; Wang et al, 2004), but the promiscuous mutagenesis in this system is relatively inefficient. In contrast, by directly recruiting AID, diversifying base editing systems (CRISPR-X and TAM) can facilitate efficient, targeted mutagenesis of key structural or functional domains of endogenous targets through carefully designed sgRNA pools.…”

Section: Section 2: Applications Of Base Editingmentioning

confidence: 99%

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

et al. 2017

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The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double stranded breaks and donor templates), and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

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“…After washing three times with 0.05% PBST and PBS, the bound antibody was detected by staining with DAB. 25,26 …”

Section: Analysis Of Selected Scfv Clones By Western Blottingmentioning

confidence: 99%

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology

et al. 2017

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The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

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Directed evolution of human scFvs in DT40 cells

Cited by 8 publications

References 35 publications

Towards an optimal monoclonal antibody with higher binding affinity to the receptor-binding domain of SARS-CoV-2 spike proteins from different variants

Towards an optimal monoclonal antibody with higher binding affinity to the receptor-binding domain of SARS-CoV-2 spike proteins from different variants

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology

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