Year
Month
Title
Journal
Information
2023
Production and applications of fluorobody from redox-engineered Escherichia coli
Srila W., Min T.T., Sumphanapai T., Rangnoi K., Berkmen M., Yamabhai M.
Applied Microbiology and Biotechnology
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Abstract:
Abstract: Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3) Propionibacterium acnes, 4) Pseudomonas aeruginosa, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different Escherichia coli (E. coli) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three E. coli hosts tested, an engineered E. coli B strain called SHuffle B that promotes disulfide bond formation in the cytoplasm appeared to be the most appropriate host. The recombinant fluorobodies were well expressed (2–8 mg/L), possessed the fluorescence property of EmGFP, and retained the ability to bind to their cognate targets. Their specific bindings were demonstrated by ELISA, fluorescence-linked immunosorbent assay (FLISA), flow cytometry, and fluorescent microscope imaging. The fluorobody expression platform in this study could be further adopted as a one-step immunostaining technique based on scFv, isolated from phage display library to numerous desired targets. Key points: • E. coli SHuffle express T7 is a suitable expression host for scFv-EmGFP (fluorobody) • Only the clones harboring scFv-EmGFP plasmid will show bright fluorescent signal • This platform can be used to produce fluorobodies for numerous purposes Graphical abstract: [Figure not available: see fulltext.] © 2023, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Keyword: E. coli SHuffle; Emerald green fluorescent protein (EmGFP); Fluorobody; Fusion; Immunofluorescence; Single-chain variable fragment (scFv)
Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85147315895&doi=10.1007%2fs00253-023-12395-6&partnerID=40&md5=328dc371afb7f41d6fff0568e7ef8510
DOI: 10.1007/s00253-023-12395-6
2023
Correction to: Production and applications of fluorobody from redox-engineered Escherichia coli (Applied Microbiology and Biotechnology, (2023), 107, 5-6, (1959-1970), 10.1007/s00253-023-12395-6)
Srila W., Min T.T., Sumphanapai T., Rangnoi K., Berkmen M., Yamabhai M.
Applied Microbiology and Biotechnology
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Abstract:
The article “Production and applications of fluorobody from redox‑engineered Escherichia coli” was originally published Online First without open access. After publication in volume 107, issue 5-6, page 1959–1970, the author decided to opt for Open Choice and to make the article an open access publication. © 2023, The Author(s).
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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85151139944&doi=10.1007%2fs00253-023-12494-4&partnerID=40&md5=e6a6959b65ac6962fbc4b8a130937fb3
DOI: 10.1007/s00253-023-12494-4
2022
Targeting acute myeloid cell surface using a recombinant antibody isolated from whole-cell biopanning of a phage display human scFv antibody library
Sumphanapai T., Chester K., Sawatnatee S., Yeung J., Yamabhai M.
Medical Oncology
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To discover new therapeutic antibodies for treatment of acute myeloid leukemia (AML) without the requirement of a known antigen, a human single-chain variable fragment (scFv) library was used to isolate novel antibody fragments recognizing HL-60 AML cells. After three rounds of biopanning, scFv-expressing phages were selected to test for binding to the target cell by flow cytometry. The clone with highest binding specificity to HL-60 cells (designated y1HL63D6) was further investigated. Fluorescent staining indicated that y1HL63D6 scFv bound to a target located on the cell surface. Whole immunoglobulin, IgG-y1HL63D6 was then generated and tested for the binding against bone marrow mononuclear cells (BMMCs) from AML patients. Significantly higher fluorescent signals were observed for some patient samples when compared to normal BMMCs or non-AML patients’ BMMCs. Next, the IgG-y1HL63D6 format was tested for antibody-dependent cell cytotoxicity (ADCC). The results demonstrated that IgG-y1HL63D6 but not the control antibody, trastuzumab, could mediate specific killing of HL-60 target cells. In conclusion, our results indicate that specific antibodies can be isolated by biopanning whole cells with a non-immunized human scFv antibody phage display library and that the isolated antibody against HL-60 cells showed therapeutic potential. © 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
Keyword: Acute myeloid leukemia; Antibody; Antibody-dependent cell-mediated cytotoxicity; Cell surface; Immunotherapy; Phage display technology
Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85138855666&doi=10.1007%2fs12032-022-01806-9&partnerID=40&md5=2ca3e1849a6172c68b611a930657b324
DOI: 10.1007/s12032-022-01806-9
2022
Determination of a distinguished interferon gamma epitope recognized by monoclonal antibody relating to autoantibody associated immunodeficiency
Yasamut U., Wisitponchai T., Lee V.S., Yamabhai M., Rangnoi K., Thongkum W., Chupradit K., Tayapiwatana C.
Scientific Reports
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Anti-interferon gamma autoantibodies (anti-IFN-γ autoAbs) neutralize the IFN-γ-mediated functions, contributing to immunodeficiency. A particular autoAb in patient serum had been previously demonstrated to recognize the same determinant on IFN-γ as the neutralizing anti-IFN-γ monoclonal antibody clone B27 (B27 mAb). This study explored the epitope recognized by B27 mAb. The specific peptide sequence recognized by B27 mAb, TDFLRMMLQEER, was retrieved from a phage display random peptide library. Sequence alignment and homology modeling demonstrated that the queried phage peptide sequence and structure were similar to amino acids at position 27–40 (TLFLGILKNWKEES) of the human IFN-γ. This determinant resides in the contact surface of IFN-γ and interferon gamma receptor 1. To elucidate the crucial amino acids, mutations were introduced by substituting T27 and T27F29L30 with alanine or deleting the amino acid residues T27–L33. The binding of B27 mAb to IFN-γ T27A using western blotting was lesser than that to wild-type. The interaction with triple mutant and T27–L33 deletion mutant using western blotting and sandwich ELISA was abolished. The finding demonstrated that T27, F29, and L30 are critical residues in the B27 antigenic determinant. Identification of the functional domain of IFN-γ decrypted the relevance of neutralizing autoAb in adult-onset immunodeficiency. © 2022, The Author(s).
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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85129557677&doi=10.1038%2fs41598-022-11774-9&partnerID=40&md5=5982693b352a0972409804eeecdd7ee2
DOI: 10.1038/s41598-022-11774-9
2022
Immunoreactivity of humanized single-chain variable fragment against its functional epitope on domain 1 of CD147
Intasai N., Rangnoi K., Yamabhai M., Pamonsupornwichit T., Thongkum W., Yasamut U., Chupradit K., Takheaw N., Nimmanpipug P., Tayapiwatana C.
Scientific Reports
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Abstract:
Domain 1 of CD147 participates in matrix metalloproteinase (MMP) production and is a candidate for targeted therapy to prevent cancer invasion and metastasis. A functional mouse anti-CD147 monoclonal antibody, M6-1B9, was found to recognize domain 1 of CD147, and its respective mouse single-chain variable fragment (ScFvM61B9) was subsequently generated. The EDLGS epitope candidate for M6-1B9 was identified using the phage display peptide technique in this study. For future clinical applications, humanized ScFv specific to domain 1 of CD147 (HuScFvM61B9) was partially adopted from the hypervariable sequences of parental mouse ScFvM61B9 and grafted onto suitable human immunoglobulin frameworks. Molecular modelling and simulation were performed in silico to generate the conformational structure of HuScFvM61B9. These results elucidated the amino acid residues that contributed to the interactions between CDRs and the epitope motif. The expressed HuScFvM61B9 specifically interacted with CD147 at the same epitope as the original mAb, M6-1B9, and retained immunoreactivity against CD147 in SupT1 cells. The reactivity of HuScFvM61B9 was confirmed using CD147 knockout Jurkat cells. In addition, the inhibitory effect of HuScFvM61B9 on OKT3-induced T-cell proliferation as M6-1B9 mAb was preserved. As domain 1 is responsible for cancer invasion and metastasis, HuScFvM61B9 would be a candidate for cancer targeted therapy in the future. © 2022, The Author(s).
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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85128855102&doi=10.1038%2fs41598-022-10657-3&partnerID=40&md5=74903df9a4bd8ece283b230fe112d010
DOI: 10.1038/s41598-022-10657-3
2022
Codon and signal peptide optimization for therapeutic antibody production from Chinese hamster ovary (CHO) cell
Srila W., Baumann M., Borth N., Yamabhai M.
Biochemical and Biophysical Research Communications
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2022
Anti-inflammatory activity of well-defined chitooligosaccharides (chos) derived from enzymatic hydrolysis of chitosan
Min T.T., Yamabhai M.
Chitooligosaccharides: Prevention and Control of Diseases
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Abstract:
Chitooligosaccharides (COS or CHOS) are one of the most promising bioproducts with tremendous appealing functions for human well-being. Despite their great potential in various health sectors, there are discrepancies in their specification and effectiveness. This ambiguity happened because COS with different sizes and structures seem to possess dissimilar biological activities and the structure of COS varied greatly, depending on the type of substrate and the methodologies used to degrade the chitin/chitosan polymer. To assure high quality of the COS products and elucidate their precise mechanism of actions in the prevention and control of diseases, a method to create well-defined COS must be established. Biological approach using chitinolytic enzymes is an effective strategy to create homogeneous COS because the enzyme is highly specific, and the reactions can be tightly controlled. This approach is also attractive because it is environmentally friendly. In this chapter, bioconversion of chitosan into COS using various enzymes and a process for manufacturing COS will be described. In addition, biological activities of various COS products, focusing on their anti-inflammatory activity, which is related to the prevention and control of various noncommunicable diseases (NCDs) will be elucidated. This information is crucial for determining an effective dosages of COS products. © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022. All rights reserved.
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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85156206811&doi=10.1007%2f978-3-030-92806-3_14&partnerID=40&md5=fe9a76527f75184b535d3ee8d0bdf6b2
DOI: 10.1007/978-3-030-92806-3_14
2022
Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains
Wutti-in Y., Sujjitjoon J., Sawasdee N., Panya A., Kongkla K., Yuti P., Yongpitakwattana P., Thepmalee C., Junking M., Chieochansin T., Poungvarin N., Yamabhai M., Yenchitsomanus P.-T.
Frontiers in Oncology
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Second-generation anti-CD19-chimeric antigen receptor T cells (anti-CD19-CAR2 T cells) are effective for treating B-cell malignancies; however, anti-CD19-CAR2 T cells can induce human anti-mouse immune responses because anti-CD19 single-chain variable fragment (scFv) in the CAR molecules is derived from a murine FMC63 (mFMC63) monoclonal antibody. Consequently, the persistence of mFMC63-CAR2 T cells and their therapeutic efficiency in patients are decreased, which results in tumor relapse. In an attempt to remedy this shortcoming, we generated a new anti-CD19-CAR T cells containing fully human anti-CD19 scFv (Hu1E7-CAR4 T cells) to pre-clinically evaluate and compare with mFMC63-CAR4 T cells. The human anti-CD19 scFv (Hu1E7) was isolated from a human scFv phage display library and fused to the hinge region of CD8α, the transmembrane domain of CD28, three intracellular costimulatory domains (CD28, 4-1BB, and CD27), and a CD3ζ signaling domain (28BB27ζ). Compared to mFMC63-CAR2 T cells (BBζ) and mFMC63-CAR3 (BB27ζ), the mFMC63-CAR4 T cells (28BB27ζ) exerted superior anti-tumor activity against Raji (CD19+) target cell. The Hu1E7-CAR4 and mFMC63-CAR4 T cells demonstrated comparable cytotoxicity and proliferation. Interestingly, compared to mFMC63-CAR4 T cells, the Hu1E7-CAR4 T cells secreted lower levels of cytokines (IFN-γ and TNF-α), which may be due to the lower binding affinity of Hu1E7-CAR4 T cells. These findings demonstrated the successfulness in creation of a new CAR T cells containing a novel fully human-derived scFv specific to CD19+ cancer cells. In vivo studies are needed to further compare the anti-tumor efficacy and safety of Hu1E7-CAR4 T cells and mFMC63-CAR4 T cells. Copyright © 2022 Wutti-in, Sujjitjoon, Sawasdee, Panya, Kongkla, Yuti, Yongpitakwattana, Thepmalee, Junking, Chieochansin, Poungvarin, Yamabhai and Yenchitsomanus.
Keyword: adoptive T cell therapy; anti-CD19 CAR T cells; B-cell malignancies; human single-chain variable fragment; scFv
Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85123872156&doi=10.3389%2ffonc.2021.802876&partnerID=40&md5=f8e10176e12a3c9321388941f903c62a
DOI: 10.3389/fonc.2021.802876
2021
Repeated exposure to dengue virus elicits robust cross neutralizing antibodies against Zika virus in residents of Northeastern Thailand
Hattakam S., Elong Ngono A., McCauley M., Shresta S., Yamabhai M.
Scientific Reports
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Zika virus (ZIKV) and dengue virus (DENV) are antigenically related mosquito-borne flaviviruses. ZIKV is becoming increasingly prevalent in DENV-endemic regions, raising the possibility that pre-existing immunity to one virus could modulate the response to a heterologous virus, although whether this would be beneficial or detrimental is unclear. Here, we analyzed sera from residents of a DENV-endemic region of Thailand to determine the prevalence of DENV-elicited antibodies capable of cross-neutralizing ZIKV. Sixty-one participants who were asymptomatic and unselected for viral serostatus were enrolled. Among them, 52 and 51 were seropositive for IgG antibody against DENV or ZIKV E proteins (ELISA assay), respectively. Notably, 44.23% (23/52) of DENV seropositive participants had serological evidence of multiple exposures to DENV, and these subjects had strikingly higher titers and broader reactivities of neutralizing antibodies (NAbs) against ZIKV and DENV heterotypes compared with participants with serological evidence of a single DENV infection (25/52, 48.1%). In total, 17 of the 61 participants (27.9%) had NAbs against ZIKV and all four DENV serotypes, and an additional 9 (14.8%) had NAbs against ZIKV and DENV1, 2, and 3. NAbs against DENV2 were the most prevalent (44/61, 72.1%) followed by DENV3 (38/61, 62.3%) and DENV1 (36/61, 59.0%). Of note, anti-ZIKV NAbs were more prevalent than anti-DENV4 NAbs (27/61, 44.3% and 21/61, 34.4%, respectively). Primary ZIKV infection was detected in two participants, confirming that ZIKV co-circulates in this region. Thus, residents of DENV-endemic regions with repeated exposure to DENV have higher titers of NAbs against ZIKV than individuals with only a single DENV exposure. © 2021, The Author(s).
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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85105397472&doi=10.1038%2fs41598-021-88933-x&partnerID=40&md5=be2b398b1e246bdf3584640bb4ecf30e
DOI: 10.1038/s41598-021-88933-x
2021
Binding Characteristic of Various Antibody Formats against Aflatoxins
Rangnoi K., Rüker F., Wozniak-Knopp G., Cvak B., O'Kennedy R., Yamabhai M.
ACS Omega
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The application of recombinant antibodies for the analysis of foods and food contaminants is now a major focus, given their capacity to be engineered to tailor their specificity, enhance their stability, and modify their structural formats to fit the desired analytical platform. In this study, human scFv antibody fragments generated against aflatoxin B1 (AFB1) were selected as the model antibody to explore the effect of antibody formats on their binding activity and to evaluate their potential use as immunoreagents for food contaminant analysis. Four human scFv antibody fragments against aflatoxin B1 (AFB1), previously isolated and engineered by chain shuffling, were converted into various formats, that is, scFv-AP fusions, scFv-Fc, and whole IgG molecules. The result indicated that the effects of the antibody format on the binding property varied, depending on the sequence of scFv. For all of the scFv clones, the scFv-AP fusion format showed the highest sensitivity by competitive ELISA, while the effects on the binding activity after conversion to scFv-Fc or IgG format varied, depending on the amino acid sequence of the antibodies. The sAFH-3e3 antibodies that showed the best performance by competitive ELISA were selected for further investigation. The sAFH-3e3 was converted to the scFv-GFP format and tested by fluorescence-linked immunosorbent assay (FLISA), which showed that its binding property was equivalent to those of scFv-Fc and IgG formats. The potential applications of the sAFH-3e3 in a rapid test kit format based on ELISA (scFv-AP) and in a lateral flow immunochromatography assay (LFIA) (IgG) were demonstrated. A comparison of methods for the extraction of AFB1 from matrices for use with these assay formats indicated that PBS and TBST are better than 70% methanol. © 2021 The Authors. Published by American Chemical Society.
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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85116699487&doi=10.1021%2facsomega.1c03044&partnerID=40&md5=c5b81d99df3bbd743509fd9c89487446
DOI: 10.1021/acsomega.1c03044
