Gene-edited healthy donor CAR T cells show superior anti-tumour activity compared to CAR T cells derived from patients with lymphoma in an in vivo model of high-grade lymphoma

Graham, Charlotte; Jóźwik, Agnieszka; Quartey‐Papafio, Ruby; Ioannou, Nikolaos; Metelo, Ana M.; Scala, Carlo; Dickson, Glenda J.; Stewart, Orla; Almena-Carrasco, Maria; Peranzoni, Elisa; Ramsay, Alan G.; Patten, Piers; Pertel, Thomas; Farzaneh, Farzin; Dupouy, Sandra; Buggins, Andrea Pepper née; Benjamin, Reuben

doi:10.1038/s41375-021-01324-z

Cited by 14 publications

(6 citation statements)

References 15 publications

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“…Three consecutive cell production batches resulted in >80% CAR + , approximately 100-fold expansion, range of 4x10 9 total viable T cells, similar rations of CD8 + and CD4 + T cells and approximately two vector copies per genome. These results are comparable to CAR-T cells transduced with other lentiviral vectors (18,19).…”

Section: Discussionsupporting

confidence: 77%

GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

Zhang¹,

Wang²,

Zhu³

et al. 2023

Front. Immunol.

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IntroductionEpstein-Barr virus (EBV) is a widely spread pathogen associated with lymphoproliferative diseases, B/ T/ NK cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). EBV lytic reactivations contribute to the genomic instability, inflammation and tumorigenesis of NPC, promoting cancer progression. Patients with NPC refractory to standard therapies show dismal survival. EBV gp350 is an envelope protein detectable in NPC specimens intracellularly and on the cell membrane of malignant cells, and is a potential viral antigen for T cell-directed immunotherapies. The potency of T cells engineered with a chimeric antigen receptor (CAR) targeting gp350 against EBV+ lymphoproliferative disease was previously shown.MethodsHere, we advanced towards preclinical and non-clinical developments of this virus-specific CAR-T cell immunotherapy against NPC. Different gp350CAR designs were inserted into a lentiviral vector (LV) backbone.ResultsA construct expressing the scFv 7A1-anti−gp350 incorporating the CD8 transmembrane and CD28.CD3ζ signaling domain (ZT002) was selected. High titer ZT002 (~1x108 TU/ml) was manufactured in HEK 293T/17 suspension cells in serum free media as large-scale production under good manufacturing practices (GMP). A LV multiplicity of infection (MOI) of 1 resulted in high frequencies of functional gp350CAR+ T cells (>70%) at a low (<2) vector copy numbers in the genome. ZT002 was therefore used to establish gp350CAR-T batch run production methods. GMP upscaling and validation of T cell transduction and expansion in several runs resulted in average 3x109 gp350CAR-T cells per batch. >80% CD3+ gp350CAR-T cells bound to purified gp350 protein. In vitro cytotoxicity and cytokine secretion assays (IFN-γ and TNF-α) confirmed the specificity of gp350CAR-T cells against gp350+ NPC, GC and lymphoma cell targets. Immunocompromised B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) were challenged s.c. with a EBV+ NPC C666.1 cell line expressing gp350 and then treated with escalating doses of gp350CAR-T cells or with non-transduced T cells. gp350CAR-T cells promoted antitumor responses, bio-distributed in several tissues, infiltrated in tumors and rejected gp350+ tumor cells.DiscussionThese results support the use of gp350CAR-T cells generated with ZT002 as an Innovative New Drug to treat patients with solid and liquid EBV-associated malignancies.

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Section: Discussionsupporting

confidence: 77%

GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

Zhang¹,

Wang²,

Zhu³

et al. 2023

Front. Immunol.

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“…19,22 The high and sustained CAR expression levels achieved with the SB transposon-CRISPR-Cas9 platform also compares favorably with previous findings typically attained with use of g-retroviral or lentiviral vectors for CAR-T cell generation. 5,26,[35][36][37][38][39][40] Moreover, the use of the 2-week expansion protocol with early CAR+ enrichment still yielded up to 70-fold expansion of the transposon-engineered CAR/TCR KO T cells with predominant memory phenotypes, consistent with the CAR/TCR KO T cell profiles generated from viral vector-based systems. 60 This improved protocol is also highly relevant for current clinical-grade CAR T cell manufacturing methods, which typically require 2 weeks of cell expansion before harvest.…”

Section: Discussionsupporting

confidence: 64%

“…Particularly for pediatric and heavily treated patients who previously received chemotherapies and/or stem cell transplantation, the resulting lymphopenic conditions may lead to inadequate T cell numbers, suboptimal CAR T cell functions, and unsuccessful CAR T cell pro-duction. [5][6][7][8] Although use of allogeneic CAR T cells could overcome some of the limitations of autologous, patient-specific CAR T cells, 9,10 allogeneic T cell transplantation triggers graft-versushost disease (GvHD) because of HLA-mismatched a/b T cells. [11][12][13][14][15] Of note, depletion of a/b T cells alleviates GvHD effects, 16,17 and TCR ablation in T cells prevents GvHD in mice.…”

Section: Introductionmentioning

confidence: 99%

Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy

Tipanee¹,

Samara-Kuko²,

Gevaert³

et al. 2022

Molecular Therapy

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“…However, there may also be significant batch-to-batch product differences due to donor characteristics. Of note, one study could demonstrate that healthy donor-derived CD19CAR-T cells outperformed autologous leukemia patient-derived CAR-T cells in an in vivo xenograft model (96). Multiple reasons could explain the phenomenon: i) damage introduced by prior chemotherapy regimen, because patients were refractory to standard of care; ii) patient-intrinsic defects in effector immunity, which contributed to cancer development in the first place.…”

Section: Design Of Clinical Trials For Testing Allogeneic Gene-edited...mentioning

confidence: 99%

Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

et al. 2022

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T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

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Gene-edited healthy donor CAR T cells show superior anti-tumour activity compared to CAR T cells derived from patients with lymphoma in an in vivo model of high-grade lymphoma

Cited by 14 publications

References 15 publications

GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy

Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

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