The Application of HL60-IL-6 Assay for in vitro Pyrogen Detection of CAR-T Cells Products

Author(s): Can Wang, Mingren Wang, Gaomin Li, Ziqiang Wang, Hong Shao, Gang Chen*

Journal Name: Current Pharmaceutical Biotechnology

Volume 22 , Issue 1 , 2021


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Graphical Abstract:


Abstract:

Objective: To detect the pyrogen in CAR-T cells product employing the HL60-IL-6 assay.

Methods: The HL60 cells were incubated with CAR-T cells injection or endotoxin standard for 48 hours. After then, the secreted cytokine interleukin-6 (IL-6) from HL60 cells was determined by ELISA. According to the four-parameter logistic curve fitted by Optical Density (OD) value corresponding to IL-6 and endotoxin standard concentration, the endotoxin equivalents of pyrogen content in the CAR-T cells products can be measured. Then, the method was validated, including the limit of detection (LOD), limit of quantitation, the recovery rate and the comparison of the determined results by HL60-IL-6 assay with that by the conventional pyrogen test, the Rabbit Pyrogen Test (RPT).

Results: The HL60-IL-6 assay applied to pyrogen test in CAR-T cells products has been established and validated, The LOD was 0.03 EU/mL while the LOQ was 0.07 EU/mL, the recovery rates were 121.4% and 94.5% respectively. The results determined by HL60-IL-6 assay were consistent with that by the RPT.

Conclusion: The HL60-IL-6 assay can be employed in CAR-T cell products in vitro pyrogen test.

Keywords: HL60-IL-6 assay, CAR-T, pyrogen, LOD, limit of quantitation, the recovery rate, the RPT.

[1]
Boyiadzis, M.M.; Dhodapkar, M.V.; Brentjens, R.J.; Kochenderfer, J.N.; Neelapu, S.S.; Maus, M.V.; Porter, D.L.; Maloney, D.G.; Grupp, S.A.; Mackall, C.L.; June, C.H.; Bishop, M.R. Chimeric Antigen Receptor (CAR) T therapies for the treatment of hematologic malignancies: Clinical perspective and significance. J. Immunother. Cancer, 2018, 6(1), 137.
[http://dx.doi.org/10.1186/s40425-018-0460-5] [PMID: 30514386]
[2]
Human Vaccines & Immunotherapeutics: News. Hum. Vaccin. Immunother., 2017, 13(10), 2202-2203.
[http://dx.doi.org/10.1080/21645515.2017.1389553] [PMID: 29045220]
[3]
Mullard, A. Second anticancer CAR T therapy receives FDA approval. Nat. Rev. Drug Discov., 2017, 16(12), 818.
[http://dx.doi.org/10.1038/nrd.2017.249] [PMID: 29180733]
[4]
Piscopo, N.J.; Mueller, K.P.; Das, A.; Hematti, P.; Murphy, W.L.; Palecek, S.P.; Capitini, C.M.; Saha, K. Bioengineering solutions for manufacturing challenges in CAR T cells. Biotechnol. J., 2018, 13(2)
[http://dx.doi.org/10.1002/biot.201700095] [PMID: 28840981]
[5]
Prajitha, N.; Athira, S.S.; Mohanan, P.V. Pyrogens, a polypeptide produces fever by metabolic changes in hypothalamus: Mechanisms and detections. Immunol. Lett., 2018, 204, 38-46.
[http://dx.doi.org/10.1016/j.imlet.2018.10.006] [PMID: 30336182]
[6]
Yao, X.J.; Yin, J.A.; Xia, Y.F.; Wei, Z.F.; Luo, Y.B.; Liu, M.; Feleder, C.; Dai, Y. Puerarin exerts antipyretic effect on lipopolysaccharide-induced fever in rats involving inhibition of pyrogen production from macrophages. J. Ethnopharmacol., 2012, 141(1), 322-330.
[http://dx.doi.org/10.1016/j.jep.2012.02.038] [PMID: 22401764]
[7]
da Silva, C.C.; Presgrave, O.A.; Hartung, T.; de Moraes, A.M.; Delgado, I.F. Applicability of the Monocyte Activation Test (MAT) for hyperimmune sera in the routine of the quality control laboratory: Comparison with the Rabbit Pyrogen Test (RPT). Toxicol. In Vitro, 2016, 32, 70-75.
[http://dx.doi.org/10.1016/j.tiv.2015.12.004] [PMID: 26688320]
[8]
Wang, C.; Dong, S.S.; Zhao, H.B.; Wang, Z.Q.; Zhou, Q.; Wu, L.H.; Shao, H.; Chen, G. A novel monocyte-based pyrogen test based on the mechanism of human fever reaction. Curr. Pharm. Anal., 2016, 12(3), 227-233.
[http://dx.doi.org/10.2174/1573412912666151211200203]
[9]
Wang, M.R.; Dong, S.S.; Shao, H.; Wang, C.; Chen, G. The Optimization of HL60-IL6 assay and its application in the pyrogen detection of monoclonal antibody. Curr. Pharm. Anal., 2018, 14, 1.
[http://dx.doi.org/10.2174/1573412914666180627142302]
[10]
Dong, S.S.; Wang, C.; Zhou, Q.; Wu, L.H.; Shao, H.; Chen, G. The application of HL60-IL6 assay in the pyrogen detection of vaccine. China Pharmacist, 2016, 5, 870-873.
[11]
Romero, D. Haematological cancer: Favourable outcomes with CAR T cells. Nat. Rev. Clin. Oncol., 2018, 15(2), 65.
[http://dx.doi.org/10.1038/nrclinonc.2017.208] [PMID: 29297506]
[12]
Gomes-Silva, D.; Atilla, E.; Atilla, P.A.; Mo, F.; Tashiro, H.; Srinivasan, M.; Lulla, P.; Rouce, R.H.; Cabral, J.M.S.; Ramos, C.A.; Brenner, M.K.; Mamonkin, M. CD7 CAR T cells for the therapy of acute myeloid leukemia. Mol. Ther., 2019, 27(1), 272-280.
[http://dx.doi.org/10.1016/j.ymthe.2018.10.001] [PMID: 30391141]
[13]
Utescher, C.L.A.; Buosi, K.L.; Botosso, V.F.; Quintilio, W. Monocyte Activation Test (MAT) as a possibility of replacement for the rabbit pyrogen test in hyperimmune sera. Braz. J. Pharm. Sci., 2018, 54.
[http://dx.doi.org/10.1590/s2175-97902018000217530]


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Article Details

VOLUME: 22
ISSUE: 1
Year: 2021
Published on: 31 December, 2019
Page: [176 - 181]
Pages: 6
DOI: 10.2174/1389201021666200101110731
Price: $65

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