Rapid Generation of Epstein-Barr Virus–Specific T Cells for Cellular Therapy

Hao, Qingfei; Yang, Ying; Wang, Y.; Chen, L.-M.; Sheng, Guang-Yao; Luan, Zhiyong

doi:10.1016/j.transproceed.2013.04.015

Cited by 2 publications

(2 citation statements)

References 28 publications

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“…Multiple rounds of stimulation with irradiated autologous LCL are required to induce EBV-specific T cell expansion to a suitable therapeutic dose [12]. The introduction of rapid expansion protocols in development of new T cell therapies [13] indicates a need for both robust, rapid methods for EBV-specific T cell manufacture, and improved analytic methods to assess the quality of the material throughout.…”

Section: Introductionmentioning

confidence: 99%

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Cooper

Kowalczuk

Wilkie

et al. 2021

Preprint

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Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved GMP-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analyzing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL: T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analyzed using t-Stochastic Neighbour Embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using IL-2, TNF-α and IFN-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

show abstract

Section: Introductionmentioning

confidence: 99%

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Cooper

Kowalczuk

Wilkie

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Multiple rounds of stimulation with irradiated autologous LCL are required to induce EBV‐specific T cell expansion to a suitable therapeutic dose [ 12 ]. The introduction of rapid expansion protocols in development of new T cell therapies [ 13 ] indicates a need for both robust, rapid methods for EBV‐specific T cell manufacture and improved analytical methods to assess the quality of the material throughout.…”

Section: Introductionmentioning

confidence: 99%

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy

Cooper

Kowalczuk

Wilkie

et al. 2021

Clinical and Experimental Immunology

View full text Add to dashboard Cite

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoproteinfree GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multiparameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

show abstract

Rapid Generation of Epstein-Barr Virus–Specific T Cells for Cellular Therapy

Cited by 2 publications

References 28 publications

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy

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