A Spheroid Killing Assay by CAR T Cells

Dillard, Pierre; Köksal, Hakan; Inderberg, Else Marit; Wälchli, Sébastien

doi:10.3791/58785

Cited by 17 publications

(10 citation statements)

References 12 publications

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“…All cell lines were maintained in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS (HyClone, Logan, UT, USA) and gentamycin 0.05 mg/mL (Thermo Fisher Scientific). Target cells used in the BLI killing assay, in vivo experiments or in microspheroid assays were transduced with a retrovirus encoding a luciferase-GFP fusion transgene [ 19 ]. They were sorted for GFP positivity and maintained as a pure line.…”

Section: Methodsmentioning

confidence: 99%

NK cells specifically TCR-dressed to kill cancer cells

et al. 2019

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BackgroundAdoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome.MethodsWe herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells.FindingsThis is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells.InterpretationThese results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution.FundThis work was supported by of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the .

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Section: Methodsmentioning

confidence: 99%

NK cells specifically TCR-dressed to kill cancer cells

et al. 2019

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Section: Discussionmentioning

confidence: 99%

“…Tumor cells clustered into spheroids from a single cell line or heterogeneous tumor cell types have been used to assess antibody ( 29 ), natural killer cell ( 29 – 31 ), cytotoxic T cell ( 32 ) and CAR T cell ( 33 – 35 ) immunotherapy in vitro . Highly variable spheroid size increases experimental variability if endpoints are quantified, and endpoint analysis often requires sophisticated visual monitoring ( 33 ) for quantification. The stereolithographic bioprinting of human SK-N-BE(2) neuroblastoma cells within a photoactivatable methacrylated gelatin matrix that we use here creates a group of identically viable copies with which to conduct sophisticated CAR T cell testing in vitro .…”

Section: Discussionmentioning

confidence: 99%

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization

et al. 2021

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Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

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“…In order to proceed with the evaluation of Kz-19BB, we performed 3D tumor spheroid killing assays because they mimic some of the in vivo tumor properties ( 30 , 31 ). Despite the expansion downsides, both IGK CAR and Kz-19BB still demonstrated significant cytotoxicity against BL-41 spheroids and maintained their selectivity against Granta-519 spheroids.…”

Section: Discussionmentioning

confidence: 99%

Combinatorial CAR design improves target restriction

Köksal

Dillard

Juzeniene

et al. 2021

Journal of Biological Chemistry

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CAR T cells targeting the B-lymphocyte antigen CD19 have led to remarkable clinical results in B-cell leukemia and lymphoma, but eliminate all B-lineage cells, leading to increased susceptibility to severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain κ or λ, we designed a second-generation CAR targeting Igκ, IGK CAR. This construct demonstrated high target specificity, but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB co-stimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of sIgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.

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A Spheroid Killing Assay by CAR T Cells

Cited by 17 publications

References 12 publications

NK cells specifically TCR-dressed to kill cancer cells

NK cells specifically TCR-dressed to kill cancer cells

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization

Combinatorial CAR design improves target restriction

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