Construction of a Large Synthetic Human scFv Library with Six Diversified CDRs and High Functional Diversity

Yang, Hye Young; Kang, Kyung Jae; Chung, Julia Eunyoung; Shim, Hyunbo

doi:10.1007/s10059-009-0028-9

Cited by 64 publications

(79 citation statements)

References 27 publications

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“…51 Using yeast surface display technology, 52 the C12 Fab was further engineered by randomization of VH-CDR3 to isolate a high affinity anti-KRS scFv, the binding of which was primarily mediated via the VH domain. The resulting C20 Fab with the engineered VH was formatted into IgG1 by subcloning the VH and VL genes into the plasmids of pcDNA 3.4-HC and pcDNA 3.4-LC, respectively, generating C20 IgG mAb.…”

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confidence: 99%

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Choi¹,

Bae

Shin

et al. 2014

mAbs

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These authors contributed equally to this work.Keywords: cell-penetrating antibody, cytosolic protein targeting, cellular internalization, endosomal release, IgG antibody, intracellular trafficking, next-generation antibodyAbbreviations: IgG, immunoglobulin G; VL, light chain variable domain; VH, heavy chain variable domain; LC, light chain; HC, heavy chain; CDR, complementarity-determining region.Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were coexpressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira Ò ) and bevacizumab (Avastin Ò ), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.

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confidence: 99%

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Choi¹,

Bae

Shin

et al. 2014

mAbs

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“…A half volume of 50% glycerol was subsequently added and thoroughly mixed, and 1 ml aliquots were frozen in liquid nitrogen and kept at -80°C. Phage libraries were rescued from the frozen E. coli stocks as previously described (Yang et al, 2009) and then combined into a single large rabbit scFv library.…”

Section: Library Preparationmentioning

confidence: 99%

“…Panning and ELISA screening against passively adsorbed antigens (proteins and protein-conjugated peptides/small molecules) were performed as previously described (Yang et al, 2009). Biotinylated peptide antigens were first captured on M-480 paramagnetic streptavidin-conjugated beads (Invitrogen) by mixing 50 µl of the beads with 1 µg of the peptide in 1 ml of PBS, followed by incubation for 15 min with gentle rotation.…”

Section: Library Panning and Screeningmentioning

confidence: 99%

“…The beads were then washed (once for the first round, three times for the subsequent rounds) with TBST, and the bound phages were eluted with 1 ml of 100 mM triethylamine. Subsequent steps were performed as previously described (Yang et al, 2009). After four rounds of panning, ELISA screening was performed on the biotinylated peptide antigen captured by surface-coated avidin (10 µg/ml in PBS).…”

Section: Library Panning and Screeningmentioning

confidence: 99%

“…For immunoblotting and ELISA experiments, purified scFv (Yang et al, 2009) or unpurified E. coli periplasmic extract containing scFv was used as a primary antibody. For immunoprecipitation of a target antigen in cell lysate, the scFv gene was cloned into a pcDNA3.1-based scFv-Fc expression vector.…”

Section: Analysis Of Selected Clonesmentioning

confidence: 99%

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Antibodies against Non-Immunizing Antigens Derived from a Large Immune scFv Library

Moon

Lee³

et al. 2011

Molecules and Cells

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A robust antibody discovery platform for difficult‐to‐express G protein‐coupled receptors

et al. 2022

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G protein-coupled receptors (GPCRs) are in the spotlight as drug targets due to the fact that multiple research results have verified the correlation between the activation of GPCRs and disease indications. This is because the GPCRs are present across the cell membranes, which interact with either extracellular ligands or other types of compartments and simultaneously mediate intracellular signaling. Despite the importance of the GPCRs as drug targets, they are too difficult to express in soluble forms. Currently, the difficulty of preparing functional GPCRs and the lack of efficient antibody screening methods are the most challenging steps in the discovery of antibodies targeting GPCRs. In this study, we developed a powerful platform that facilitates isolating GPCRspecific antibodies by obviating difficulties in GPCR preparation. The strategies include (i) conjugation of the P9 peptide, an envelope protein of Pseudomonas phi6, to the N-terminus of GPCRs to improve the expression level of the GPCRs in Escherichia coli, (ii) stabilization of the GPCRs in their active forms with amphiphilic poly-γ-glutamate (APG) to shield the seven hydrophobic transmembrane domains, and (iii) further limiting the size of the APG complex to improve the chance to isolate antibodies targeting the proteins-of-interest. Capitalizing on the above strategies, we could prepare GPCR proteins in their active forms as facile as other general-soluble antigen proteins. Furthermore, this protocol was validated to be successful in discovering three individual GPCR-specific antibodies targeting glucagon-like peptide-1 receptor, C-X-C chemokine receptor type 4, and prostaglandin E2 receptor 4 in this study. K E Y W O R D S amphiphilic poly-γ-glutamate, antibody discovery, G protein-coupled receptors, phage display, purification 1 | INTRODUCTION G protein-coupled receptors (GPCRs) are one of the membrane receptor proteins that generate intracellular signal transduction in response to various extracellular signals. The cell signaling by GPCRs is generally mediated through ligand-induced activation, which makes changes in the structure of the GPCRs and activates proteins that interact with their intracellular domains inside the cell, triggering multiple intracellular events such as Nam Hyuk Kim and Sumin Kang contributed equally to this work.

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Construction of a Large Synthetic Human scFv Library with Six Diversified CDRs and High Functional Diversity

Cited by 64 publications

References 27 publications

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Antibodies against Non-Immunizing Antigens Derived from a Large Immune scFv Library

A robust antibody discovery platform for difficult‐to‐express G protein‐coupled receptors

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