CAR-T cells generated using Sleeping Beauty vectors and expanded with a lymphoblastoid cell line (LCL) display in vivo antitumor activity

Chicaybam, Leonardo; Carneiro, Mayra; Abdo, Luiza; Pereira, Bárbara Costa; Viegas, Mariana; Sousa, Priscila de; Carvalho, Leonardo César; Bonamino, Martín

doi:10.35259/isi.sact.2019_32823

Cited by 4 publications

(7 citation statements)

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“…Finally, we deployed the Tc Buster system for the production of CAR-T cells. CAR-T cells have been generated for preclinical and clinical use with a number of transposon systems including SB and PB 25–28,30–32,35,36,43 . Here, we demonstrated the feasibility and high efficiency of their production using Tc Buster and validated their subsequent effector function (Figure 4).…”

Section: Discussionmentioning

confidence: 99%

“…Clinically, the SB system has been used to introduce CD19-specific CARs to patient-and donor-derived T cells 27, 28 . Many preclinical studies and clinical trials thus far have introduced SB transposase and CAR by electroporation of bulk peripheral blood mononuclear cells (PBMCs) [29][30][31][32][33] . CAR-expressing T cells were subsequently expanded over several weeks in culture using feeder cells engineered to express the target antigen and co-stimulatory molecules 27 .…”

Section: Introductionmentioning

confidence: 99%

“…However, lessons can be learned from T cells on the use of transposons for CAR delivery to primary NK cells. The initial approach of electroporating PBMCs with transposon-based CARs suggests that NK cells could be selectively outgrown instead of T cells 29,30 . Indeed, some reports have shown outgrowth of NK cells reaching 50% of the PBMC population after co-culture with feeder cells 30 .…”

Section: Introductionmentioning

confidence: 99%

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Non-Viral Engineering of CAR-NK and CAR-T cells using theTc BusterTransposon System™

Pomeroy

Lahr

Chang

et al. 2021

Preprint

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Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome. Here, we used the newly developed hyperactive Tc Buster (Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function. Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Non-Viral Engineering of CAR-NK and CAR-T cells using theTc BusterTransposon System™

Pomeroy

Lahr

Chang

et al. 2021

Preprint

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“…A variety of transposon-based systems have been reported for CAR T-cell production, and these systems provide safe and reliable DNA transfer into T-cells. The sleeping beauty (SB) transposon system is currently being used as a substitute for viral-based vectors in the preparation of, for example, CD19+ CAR T-cells [23], with reported antitumor activity both in vitro and in vivo [24]. The main strength of this approach lies in the overall better integration profile of the transduced genetic material.…”

Section: Viral Vs Nonviral Methods Of Car T-cell Preparationmentioning

confidence: 99%

CAR T-Cell Production Using Nonviral Approaches

Lukjanov

Koutná

Šimara

2021

Journal of Immunology Research

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Chimeric antigen receptor T-cells (CAR T-cells) represent a novel and promising approach in cancer immunotherapy. According to the World Health Organization (WHO), the number of oncological patients is steadily growing in developed countries despite immense progress in oncological treatments, and the prognosis of individual patients is still relatively poor. Exceptional results have been recorded for CAR T-cell therapy in patients suffering from B-cell malignancies. This success opens up the possibility of using the same approach for other types of cancers. To date, the most common method for CAR T-cell generation is the use of viral vectors. However, dealing with virus-derived vectors brings possible obstacles in the CAR T-cell manufacturing process owing to strict regulations and high cost demands. Alternative approaches may facilitate further development and the transfer of the method to clinical practice. The most promising substitutes for virus-derived vectors are transposon-derived vectors, most commonly sleeping beauty, which offer great coding capability and a safe integration profile while maintaining a relatively low production cost. This review is aimed at summarizing the state of the art of nonviral approaches in CAR T-cell generation, with a unique perspective on the conditions in clinical applications and current Good Manufacturing Practice. If CAR T-cell therapy is to be routinely used in medical practice, the manufacturing cost and complexity need to be as low as possible, and transposon-based vectors seem to meet these criteria better than viral-based vectors.

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“…The Sleeping Beauty (SB) transposon vector provides a safe, effective and economic method to integrate genetic information through a gene transfer vector, which may overcome the shortcomings of viral vectors. However, the applicability of the SB transposon system in transferring CAR into primary NK cells remains to be verified (48)(49)(50). It is considered a safe and economic method to transfect CAR-NK cells with mRNA, which demonstrated significant cytotoxicity in xenotransplantation cancer models (33,51).…”

Section: Persistence Of Car-nk Cells In Vivomentioning

confidence: 99%

Chimeric antigen receptor‑natural killer cells: novel insight into immunotherapy for solid tumors (Review)

Peng

Chen

et al. 2021

Exp Ther Med

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The chimeric antigen receptor (CAR) is an artificially modified fusion protein consisting of an extracellular antigen-binding domain, transmembrane domain and intracellular signalling domain. CAR-T therapy has demonstrated remarkable clinical efficacy in hematological malignancies. However, cytokine release syndrome and other side effects have hindered its application in solid tumors. CAR-natural killer (NK) cells have attracted broad attention due to their safety in clinical applications, their mechanism in recognising cancer cells and the abundance of its clinical specimens. Preclinical and clinical trials of human primary NK cells and NK-92 cell lines demonstrated that CAR-NK cells are able to fight haematological malignancies and solid tumors. However, the implication of CAR-NK cell therapy also has certain challenges, including the expansion and activation of primary NK cells in vitro , selection of CAR targets, survival time of CAR-NK cells in vivo , storage and transportation of NK cells, and efficiency of NK cell transduction. This review focuses on the latest progress of CAR-NK cells in the treatment of solid tumors.

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CAR-T cells generated using Sleeping Beauty vectors and expanded with a lymphoblastoid cell line (LCL) display in vivo antitumor activity

Cited by 4 publications

References 0 publications

Non-Viral Engineering of CAR-NK and CAR-T cells using theTc BusterTransposon System™

Non-Viral Engineering of CAR-NK and CAR-T cells using theTc BusterTransposon System™

CAR T-Cell Production Using Nonviral Approaches

Chimeric antigen receptor‑natural killer cells: novel insight into immunotherapy for solid tumors (Review)

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