Automated production of CCR5-negative CD4+-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients

Schwarze, Lea Isabell; Sonntag, Tanja; Wild, Stefan; Schmitz, Sarah; Uhde, Almut

doi:10.1038/s41434-021-00259-5

Cited by 9 publications

(6 citation statements)

References 56 publications

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“… 6 In order to scale up our process, we initially focused on the identification of transfection conditions that permit high gene-transfer rates and low electroporation-associated toxicities. In accordance with prior studies reported by Alzubi et al 10 and Schwarze et al , 24 we modified pulse strength, length, mode and direction to arrive at a bi-pulse shock that permitted high frequencies of stable gene-transfer and high viability in activated CD8 and CD4 T cells. We then performed titration experiments to determine the optimal amount of MC DNA for electroporation and defined a sweet spot with maximum yield of CAR-expressing T cells.…”

Section: Discussionmentioning

confidence: 60%

Automated, scaled, transposon-based production of CAR T cells

Lock

Monjezi

Brandes

et al. 2022

J Immunother Cancer

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BackgroundThere is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition.MethodsWe used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR.ResultsWe defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1–3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns.ConclusionsWe report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.

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Section: Discussionmentioning

confidence: 60%

Automated, scaled, transposon-based production of CAR T cells

Lock

Monjezi

Brandes

et al. 2022

J Immunother Cancer

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“…Immunophenotyping of cells expanded in the CliniMACS Prodigy was performed to further characterize the T cell subpopulations in the final product. In principle, engineered T cells should be long-lived to ensure protection for an extended period of time; however, the survival and self-renewal rates are heterogeneous among different T cell subpopulations ( 35 ). Naïve T cells (Tn) can be distinguished from memory T cells, which can be further subdivided into central memory (Tcm) and effector memory T cells (Tem) ( 17 ).…”

Section: Resultsmentioning

confidence: 99%

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products

et al. 2022

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Cellular immunotherapies based on T cell receptor (TCR) transfer are promising approaches for the treatment of cancer and chronic viral infections. The discovery of novel receptors is expanding considerably; however, the clinical development of TCR-T cell therapies still lags. Here we provide a pipeline for process development and clinical-scale manufacturing of TCR-T cells in academia. We utilized two TCRs specific for hepatitis C virus (HCV) as models because of their marked differences in avidity and functional profile in TCR-redirected cells. With our clinical-scale pipeline, we reproduced the functional profile associated with each TCR. Moreover, the two TCR-T cell products demonstrated similar yield, purity, transduction efficiency as well as phenotype. The TCR-T cell products had a highly reproducible yield of over 1.4 × 109 cells, with an average viability of 93%; 97.8–99% of cells were CD3+, of which 47.66 ± 2.02% were CD8+ T cells; the phenotype was markedly associated with central memory (CD62L+CD45RO+) for CD4+ (93.70 ± 5.23%) and CD8+ (94.26 ± 4.04%). The functional assessments in 2D and 3D cell culture assays showed that TCR-T cells mounted a polyfunctional response to the cognate HCV peptide target in tumor cell lines, including killing. Collectively, we report a solid strategy for the efficient large-scale manufacturing of TCR-T cells.

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“…The main TALEN tool developed, CCR5-Uco-hetTALEN, includes a heterodimeric Fok1-cleavage domain and almost completely reduces off-target effects, with the notable exception of the highly homologous CCR2 ( 145 ). This technology has advanced so much that it is now automated and can reliably generate the CCR5 knockdown in frequencies above 60% within primary T cells, 40% of which can be biallelic CCR5 mutations ( 146 ).…”

Section: Mechanisms Of Targeting Ccr5 To Inhibit Hiv-1 Disease Progressionmentioning

confidence: 99%

Targeting CCR5 as a Component of an HIV-1 Therapeutic Strategy

et al. 2022

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Globally, human immunodeficiency virus type 1 (HIV-1) infection is a major health burden for which successful therapeutic options are still being investigated. Challenges facing current drugs that are part of the established life-long antiretroviral therapy (ART) include toxicity, development of drug resistant HIV-1 strains, the cost of treatment, and the inability to eradicate the provirus from infected cells. For these reasons, novel anti-HIV-1 therapeutics that can prevent or eliminate disease progression including the onset of the acquired immunodeficiency syndrome (AIDS) are needed. While development of HIV-1 vaccination has also been challenging, recent advancements demonstrate that infection of HIV-1-susceptible cells can be prevented in individuals living with HIV-1, by targeting C-C chemokine receptor type 5 (CCR5). CCR5 serves many functions in the human immune response and is a co-receptor utilized by HIV-1 for entry into immune cells. Therapeutics targeting CCR5 generally involve gene editing techniques including CRISPR, CCR5 blockade using antibodies or antagonists, or combinations of both. Here we review the efficacy of these approaches and discuss the potential of their use in the clinic as novel ART-independent therapies for HIV-1 infection.

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Automated production of CCR5-negative CD4+-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients

Cited by 9 publications

References 56 publications

Automated, scaled, transposon-based production of CAR T cells

Automated, scaled, transposon-based production of CAR T cells

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products

Targeting CCR5 as a Component of an HIV-1 Therapeutic Strategy

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