Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

Bahara, Nur Hidayah Hairul; Chin, Siang Tean; Choong, Yee Siew; Lim, Theam Soon

doi:10.1177/1087057115609144

Cited by 19 publications

(12 citation statements)

References 28 publications

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“…The availability of synthetic universal, single‐pot camelid, shark and human single domain antibody libraries displayed on phages or yeast cells enables selection of a huge number of different single domain intrabodies theoretically directed against any antigen and desired epitope . −18, −31, By using antibody affinity maturation strategies, binders with high affinity can be isolated from these nonaffinity‐maturated universal libraries by introducing CDR mutations, randomization of mutational hotspots or error prone PCR .…”

Section: Resultsmentioning

confidence: 99%

“…Synthetic human VH/VL libraries are constructed from a stable human VH/VL germline framework or from a VH/VL scaffold of a human monoclonal antibody. Randomization of the CDRs, especially of CDR3, leads to synthetic human VH libraries with high diversity . −16, VL single domain antibody libraries have been constructed similarly −18 (Fig.…”

Section: Non Camelid Sdab Librariesmentioning

confidence: 99%

“…Camelid and shark single domain antibodies possess good solubility, high thermal stability and refolding capability . In addition, stable human VH and VL domains can be constructed from human antibodies after framework mutations and randomization of CDRs . Camelid nanobodies can be stably expressed inside the cytoplasm even if the disulfide bridge is not formed .…”

Section: Introductionmentioning

confidence: 99%

“…In general nanobodies are selected from naïve, immune, synthetic camelid VHH single domain antibody repertoires whereas shark sdAbs and human VH and VL sdAbs are mainly selected from synthetic antibody libraries …”

Section: Introductionmentioning

confidence: 99%

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Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Böldicke

2017

Protein Science

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Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.

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Section: Resultsmentioning

confidence: 99%

Section: Non Camelid Sdab Librariesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Böldicke

2017

Protein Science

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“…Human single-domain antibody (sdAb) of about 15 kDa consists of only the variable domain of human antibody heavy chain and has no immunogenicity [17,18]. It has good tissue and tumor penetration [19]. It can access an epitope inaccessible to the large-size conventional mAb [20,21].…”

Section: Introductionmentioning

confidence: 99%

Novel single-domain antibodies against the EGFR domain III epitope exhibit the anti-tumor effect

Chen

Hong

et al. 2020

J Transl Med

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Background Monoclonal antibodies (mAbs) have been used for cancer therapy. They are large and have some disadvantages limiting their use. Smaller antibody fragments are needed as their alternatives. A fully human single-domain antibody (sdAb) has a small size of only 15 kDa and consists of only the variable domain of the human antibody heavy chain (VH). It has no immunogenicity. It can easily penetrate into tumor tissues, target an epitope inaccessible to mAb and be manufactured in bacteria for a low cost. Epidermal growth factor receptor (EGFR) is over-expressed in many cancer cells and is a good target for cancer therapy. Methods The EGFR protein fragment located on the EGFR extracellular domain III was chosen to screen a human sdAb library. Five human anti-EGFR sdAbs were identified. Their specific binding to EGFR was confirmed by ELISA, Western blotting and flow cytometry. Their anti-tumor effects were tested. Results Five novel fully human anti-EGFR sdAbs were isolated. They specifically bound to EGFR, not to the seven unrelated proteins as negative controls. They also bound to the three different human cancer cell lines, but not to the two cell lines as negative controls. They inhibited cell proliferation, migration and invasion and increased apoptosis of these three cancer cell lines. Two of them were tested for their anti-tumor effect in vivo and showed the anti-tumor activity in a mouse xenograft model for human lung cancer. Immunohistochemical staining of xenograft tumors also showed that their anti-tumor effects were associated with the inhibition of cancer cell proliferation and the promotion of cancer cell apoptosis. Conclusions This study clearly demonstrated that the anti-EGFR sdAbs could inhibit cancer cell growth in vitro and tumor growth in vivo. They could be potential therapeutics for the treatment of different human cancers.

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