Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Bangphoomi, Kunan; Poungpair, Ornnuthchar; Maneewatch, Santi; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

doi:10.1371/journal.pone.0049254

Cited by 55 publications

(83 citation statements)

References 32 publications

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“…Phage clones displaying NS5BD55-bound human scFvs and the scFv characteristics C-terminally 55 amino acid deleted recombinant NS5B protein, i.e., NS5BD55, with inherent RdRp activity of the native HCV polymerase 13 was used as antigen in the phage bio-panning for selecting the NS5B-bound phage clones from a previously established human na€ ıve scFv phage display library.…”

Section: Resultsmentioning

confidence: 99%

“…[20][21][22][23][24][25] The engineered antibodies can also be made into intact four-chain molecules when the biological activities of the Fc fragment are needed for the therapeutic effect. Alternatively, smaller antibody fragments that are devoid of the Fc fragment or constant domains, e.g., scFv 21,22 or single domain antibodies (nanobodies/VH/ V H H) 13,[23][24][25] can be used to increase the tissue penetrating ability and reduce tissue inflammation. The small antibodies have high solubility, reproducible refolding capacity and thermal stability, which ease the production process.…”

Section: Discussionmentioning

confidence: 99%

“…13 Phage clones that displayed the NS5BD55 bound-scFvs were selected from a human scFv phage display library constructed previously.…”

Section: Methodsmentioning

confidence: 99%

“…Binding of the scFvs by individual E. coli clones to the NS5BD55 protein was determined by indirect ELISA and western blot analysis using BSA as control antigen. 13 The restriction fragment length polymorphism (RFLP) of the scfvs coding for the NS5BD55 specific-scFvs was determined by digesting the scfv PCR amplicons with MvaI restriction endonuclease. 13 The digested DNA fragments were subjected to a 14% PAGE and ethidium bromide staining.…”

Section: Methodsmentioning

confidence: 99%

“…8 Recently, humanized, cell penetrable single domain antibodies (VH/V H H) that bound specifically to epitopes located around the HCV polymerase catalytic groove and inhibited the RdRp activity leading to the suppression of the virus replication were reported. 13 In this study, cell penetrable human single chain variable antibody fragment (scFv) molecules that bound to epitopes different from the previously reported NS5B specific-VH/V H H were produced. A cocktail of human/humanized small antibodies to HCV NS5B polymerase that simultaneously interferes with different regions of the protein could be another future STAT-C candidate.…”

Section: Introductionmentioning

confidence: 99%

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Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Thueng-in

Thanongsaksrikul

Jittavisutthikul

et al. 2014

mAbs

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dodecyl sulfate; SOC, standard-of-care; STAT-C, specifically targeted anti-viral therapy for hepatitis C; SVR, sustained virologic response; VH, variable heavy chain domain of conventional four-chain IgG; V H H, variable heavy chain domain of heavy chain antibody; VL, variable light chain domainA new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…13 Phage clones that displayed the NS5BD55 bound-scFvs were selected from a human scFv phage display library constructed previously.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Thueng-in

Thanongsaksrikul

Jittavisutthikul

et al. 2014

mAbs

Self Cite

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Cell‐penetrable nanobodies (transbodies) that inhibit the tyrosine kinase activity of EGFR leading to the impediment of human lung adenocarcinoma cell motility and survival

Tabtimmai

Suphakun

Srisook

et al. 2019

J of Cellular Biochemistry

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Most patients suffering from non–small cell lung cancer (NSCLC) have epidermal growth factor receptor (EGFR) overexpression. Currently, EGFR tyrosine kinase inhibitors (TKIs) that act as the ATP‐analogs and monoclonal antibodies (MAbs) to EGFR‐ectodomain that block intracellular signaling are used for the treatment of advanced NSCLC. Unfortunately, adverse effects due to the TKI off‐target and drug resistance occur in a significant number of the treated patients while some NSCLC genotypes do not respond to the therapeutic MAbs. Thus, a more effective remedy for the treatment of EGFR‐overexpressed cancers is deemed necessary. In this study, VH/VHH displayed‐phage clones that are bound to recombinant EGFR‐TK were fished‐out from a humanized‐camel VH/VHH phage display library. VH/VHH of three phage‐infected Escherichia coli clones (VH18, VHH35, and VH36) were linked molecularly to nonaarginine (R9) for making them cell penetrable. R9‐VH18, R9‐VHH35, and R9‐VH36 were cytotoxic to human adenocarcinomic alveolar basal epithelial cells (A549) at the fifty percent inhibitory concentration (IC50) 0.181 ± 0.132, 0.00961 ± 0.00516, and 0.00996 ± 0.00752 μM, respectively, which were approximately 1000‐fold more effective than small molecular TKIs. R9‐VH18 and R9‐VH36 also delayed cancer cell migration in a scratch‐wound assay. Computerized homology modeling and intermolecular docking revealed that VH18 and VHH35 used CDR3 to interact with EGFR‐TK residues close to the catalytic site, which might sterically hinder the ATP‐binding of the TK; VH36 used CDR2 to bind at the asymmetric dimerization surface, which might disrupt EGFR dimerization leading to inhibition of intracellular signaling. The humanized‐cell penetrable nanobodies have a high potential for developing further towards a clinical application.

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COVID‐19 cooling: Nanostrategies targeting cytokine storm for controlling severe and critical symptoms

Zheng,

Li,

et al. 2023

Medicinal Research Reviews

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As severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants continue to wreak havoc worldwide, the “Cytokine Storm” (CS, also known as the inflammatory storm) or Cytokine Release Syndrome has reemerged in the public consciousness. CS is a significant contributor to the deterioration of infected individuals. Therefore, CS control is of great significance for the treatment of critically ill patients and the reduction of mortality rates. With the occurrence of variants, concerns regarding the efficacy of vaccines and antiviral drugs with a broad spectrum have grown. We should make an effort to modernize treatment strategies to address the challenges posed by mutations. Thus, in addition to the requirement for additional clinical data to monitor the long‐term effects of vaccines and broad‐spectrum antiviral drugs, we can use CS as an entry point and therapeutic target to alleviate the severity of the disease in patients. To effectively combat the mutation, new technologies for neutralizing or controlling CS must be developed. In recent years, nanotechnology has been widely applied in the biomedical field, opening up a plethora of opportunities for CS. Here, we put forward the view of cytokine storm as a therapeutic target can be used to treat critically ill patients by expounding the relationship between coronavirus disease 2019 (COVID‐19) and CS and the mechanisms associated with CS. We pay special attention to the representative strategies of nanomaterials in current neutral and CS research, as well as their potential chemical design and principles. We hope that the nanostrategies described in this review provide attractive treatment options for severe and critical COVID‐19 caused by CS.

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Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

Cited by 55 publications

References 32 publications

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Cell‐penetrable nanobodies (transbodies) that inhibit the tyrosine kinase activity of EGFR leading to the impediment of human lung adenocarcinoma cell motility and survival

COVID‐19 cooling: Nanostrategies targeting cytokine storm for controlling severe and critical symptoms

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