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A Cell Assay for Detecting Anti-PEG Immune Response against PEG-Modified Therapeutics

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Abstract

Purpose

Immunogenicity of PEGylated proteins and nanomedicines represents a potential impediment against their development and use in clinical settings. The purpose of this study is to develop a method for detecting anti-PEG immunity of PEGylated proteins and/or nanomedicines using flow cytometry.

Methods

The binding of fluorescence-labeled mPEG-modified liposomes to HIK-G11 cells, PEG-specific hybridoma cells, or spleen cells was evaluated by flow cytometry for detecting immunogenicity of PEGylated therapeutics.

Results

The fluorescence-labeled methoxy PEG (mPEG)-modified liposomes were efficiently bound to HIK-G11 cells. Such staining with fluorescence-labeled mPEG-modified liposomes was significantly inhibited in the presence of either non-labeled mPEG-modified liposomes or mPEG-modified ovalbumin (OVA) but not polyglycerol-modified liposomes. In addition, we found that mPEG-modified liposomes, highly immunogenic, caused proliferation of PEG-specific cells, while hydroxyl PEG-modified liposomes, less immunogenic, scarcely caused. Furthermore, after intravenous injection of mPEG-modified liposomes, the percentage of PEG-specific cells in the splenocytes, as determined by flow cytometry, corresponded well with the production level of anti-PEG antibodies, as determined by ELISA.

Conclusions

PEG-specific B cell assay we introduced may become a useful method to detect an anti-PEG immune response against PEGylated therapeutics and clarify the mechanism for anti-PEG immune responses.

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Abbreviations

ABC:

Accelerated blood clearance

Chol:

Cholesterol

DiI:

1,1’-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

ELISA:

Enzyme-linked immunosorbent assay

HEPC:

Hydrogenated egg phosphatidylcholine

HSPC:

Hydrogenate soy phosphatidylcholine

IFNα:

Interferon alpha

l-OHP:

Oxaliplatin

mPEG:

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (CH3O-PEG2000-DSPE)

MPS:

Mononuclear phagocyte system

OVA:

Ovalbumin

PEG:

Polyethylene glycol

PG:

Polyglycerol

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Mr. J. L. McDonald for his helpful advice in preparing this manuscript. This work was partially supported by the Japan Society for the Promotion of Science through a Grant-in-Aid for Young Scientists (B) (15 K18921), by the Japan Society for the Promotion of Science through a Grant-in-Aid for Scientific Research (B) (15H04639), by the Takeda Science Foundation, by the Uehara Memorial Foundation, by the Takahashi Industrial and Economic Research Foundation, and through a research program for the development of an intelligent Tokushima artificial exosome (iTEX) from Tokushima University.

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Authors and Affiliations

  1. Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, 1-78-1, Sho-machi, Tokushima, 770-8505, Japan

    Taro Shimizu, Amr S. Abu Lila, Mizuki Awata, Yukiyo Kubo, Yu Mima, Yosuke Hashimoto, Hidenori Ando, Keiichiro Okuhira, Yu Ishima & Tatsuhiro Ishida

  2. Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt

    Amr S. Abu Lila

  3. Department of Pharmaceutics, College of Pharmacy, Hail University, Hail, 81442, Saudi Arabia

    Amr S. Abu Lila

Authors
  1. Taro Shimizu
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  2. Amr S. Abu Lila
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  3. Mizuki Awata
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  4. Yukiyo Kubo
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  5. Yu Mima
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  6. Yosuke Hashimoto
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  9. Yu Ishima
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  10. Tatsuhiro Ishida
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Corresponding author

Correspondence to Tatsuhiro Ishida.

Electronic supplementary material

Suppl. Fig. 1

Linearity of PEG-specific B cell assay. HIK-G11 was spiked with naïve spleen cells at various concentrations. Following staining with DiI-labeled mPEG-modified liposomes, the percentage of DiI-positive cells was measured by flow cytometry. (PPTX 59 kb)

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Shimizu, T., Abu Lila, A.S., Awata, M. et al. A Cell Assay for Detecting Anti-PEG Immune Response against PEG-Modified Therapeutics. Pharm Res 35, 223 (2018). https://doi.org/10.1007/s11095-018-2505-3

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