Development of a simple new flow cytometric antibody-dependent cellular cytotoxicity (ADCC) assay with excellent sensitivity

Tanaka, Miho; Ishige, Akiko; Yaguchi, Masami; Matsumoto, Takehisa; Shirouzu, Mikako; Yokoyama, Shigeyuki; Ishikawa, Fumihiko; Kitabayashi, Issay; Takemori, Toshitada; Harada, Michishige

doi:10.1016/j.jim.2018.10.014

Cited by 5 publications

(5 citation statements)

References 36 publications

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“…To prepare immunizing antigens and reagents for antibody screening by flow cytometry, we transduced Daudi lymphoma and HepG2 cell lines with a cDNA encoding human NTCP in a retroviral vector ( 31 ) and established Daudi-NTCP and HepG2-NTCP cell lines that both expressed NTCP on the cell surface and bound preS1 at significant levels (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…To generate human NTCP-expressing cells, Daudi, HepG2, or Huh7 cells were transduced with a retroviral vector encoding NTCP or Flag tag-NTCP with 10 μg/mL of Polybrene (Sigma-Aldrich, St. Louis, MO). The retroviral vectors were generated by modification of a previously described method ( 31 ). Briefly, the retroviral expression plasmid and the envelope expression plasmid p10A1 were cotransfected into GP2-293 cells using the Retro-X universal packaging system (TaKaRa Bio Inc., Shiga, Japan).…”

Section: Methodsmentioning

confidence: 99%

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Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection

Takemori

Sugimoto-Ishige

Nishitsuji

et al. 2022

J Virol

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Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1-domain of the viral L protein to the Na + /Taurocholate Cotransporting Polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, Myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/HDV infection and has been approved as Hepcludex® in Europe for the treatment of patients with chronic hepatitis D virus (HDV) infection. We established a monoclonal antibody (mAb) N6HB426-20 that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo , administration of the N6HB426-20 mAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84-87 in ECL1 and aa 157-165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope-mapping and the 3D model of the NTCP structure suggested that the N6HB426-20 mAb may recognize aa 276/277 at the tip of ECL4 and interfere with an binding of HBV to the aa 84-87 region. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleos(t)ide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic mAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these mAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations and the presence of subviral particles. Although N6HB426-20 requires a higher dose than Myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time post administration in proportion to the half-life of an IgG mAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection

Takemori

Sugimoto-Ishige

Nishitsuji

et al. 2022

J Virol

Self Cite

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“…Each of these methods utilizes fluorescent molecules to distinguish target cells from effector cells and to measure cell viability as indicator of efficiency of tumor killing. Several other groups have recently published variations on these experimental protocols (Alrubayyi et al, 2018;Carter et al, 2019;González-González et al, 2019;Kamen et al, 2019;Tanaka et al, 2019;van der Haar Àvila, Marmol, Cany, Kiessling, & Pico de Coaña, 2019;Yamashita et al, 2016). These techniques have several advantages over other methods, including the ability to simultaneously measure the amount of target antibody bound to the cells and cell viability.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions

Miller

Finn

2020

Methods in Enzymology

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Monoclonal antibody-based therapies are increasingly being used to treat cancer. Some mediate their therapeutic effects through modifying the function of immune cells globally, while others bind directly to tumor cells and can recruit immune effector cells through their Fc regions. As new direct-binding agents are developed, having the ability to test their Fc-mediated functions in a high-throughput manner is important for selecting antibodies with immune effector properties. Here, using monoclonal anti-CD20 antibody (rituximab) as an example and the CD20 + Raji cell line as tumor target, we describe flow cytometry-based assays for determining an antibody's capacity for mediating antibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). These assays are sensitive, reliable, affordable and avoid the use of radioactivity.

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“…PLG was the first biopolymer to be tested in the attempts to develop single-dose vaccines. As discussed above, although several technical limitations so far have prevented widespread clinical application of PLG-based vaccines, PLG microspheres have been widely published and clinically validated for the controlled release of proteins, peptides, and small molecules 36 , 82 , 126 , 128 , with the potential of co-delivering antigens and adjuvants 129 – 131 . A single-dose vaccine of stabilized PLG microparticles may allow for a pulsatile release of the antigen and the timing of the “bursts” can be potentially modulated by changing the biochemical and biophysical properties of the PLG microparticles 36 , 132 ; however, a continuous release kinetic is also possible 133 – 135 .…”

Section: Modulating the Release Kinetics Of The Vaccine Componentsmentioning

confidence: 99%

Technological approaches to streamline vaccination schedules, progressing towards single-dose vaccines

Lofano¹,

Mallett²,

Bertholet³

et al. 2020

npj Vaccines

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Vaccines represent the most successful medical intervention in history, with billions of lives saved. Although multiple doses of the same vaccine are typically required to reach an adequate level of protection, it would be advantageous to develop vaccines that induce protective immunity with fewer doses, ideally just one. Single-dose vaccines would be ideal to maximize vaccination coverage, help stakeholders to greatly reduce the costs associated with vaccination, and improve patient convenience. Here we describe past attempts to develop potent single dose vaccines and explore the reasons they failed. Then, we review key immunological mechanisms of the vaccine-specific immune responses, and how innovative technologies and approaches are guiding the preclinical and clinical development of potent single-dose vaccines. By modulating the spatio-temporal delivery of the vaccine components, by providing the appropriate stimuli to the innate immunity, and by designing better antigens, the new technologies and approaches leverage our current knowledge of the immune system and may synergize to enable the rational design of next-generation vaccination strategies. This review provides a rational perspective on the possible development of future single-dose vaccines.

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Development of a simple new flow cytometric antibody-dependent cellular cytotoxicity (ADCC) assay with excellent sensitivity

Cited by 5 publications

References 36 publications

Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection

Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection

Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions

Technological approaches to streamline vaccination schedules, progressing towards single-dose vaccines

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