Combinatorial CAR design improves target restriction

Köksal, Hakan; Dillard, Pierre; Juzeniene, Asta; Kvalheim, Gunnar; Smeland, Erlend B.; Myklebust, June Helen; Inderberg, Else Marit; Wälchli, Sébastien

doi:10.1074/jbc.ra120.016234

Cited by 8 publications

(7 citation statements)

References 40 publications

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“…In addition, ECAR measures the rate of extracellular acidification, which mainly comes from the accumulation of lactic acid in the medium (glycolytic pathway) [64]. Among the studies reported, some used the Seahorse technology to explore the influence of CAR signaling tails (4-1BB vs. CD28) [49,65], while others focused on comparing CARs with either different designs or targets [52,[66][67][68][69], the additional secretory functions [53,70], the combination with PD-1/PD-L1 blockage [71], the polarization of the T cells [53,72], or the influence of co-expressing enzymes alongside the CARs in T cells [73,74]. The second injection of Oligomycin, an ATP synthase inhibitor, permits, through the measurement of ECAR, assessment of the maximum glycolytic capacity.…”

Section: Study Of Metabolism In Car T Cellsmentioning

confidence: 99%

“…In particular, they performed RNA sequencing and showed that their CD19-sPD1 CAR T cells were less differentiated and less exhausted and activated less glycolytic pathway-related genes. We have recently presented an approach to improving the efficacy of CD19 CAR T cells by combining CD19 CAR with an anti-IgKappa (IGK) CAR, called Kz19BB [66]. With this combinatorial CAR design, we showed that we could keep the specificity of IGK CAR while attenuating its serum sensitivity.…”

Section: Effect Of the Combination Of Car T Cells And Combinatorial D...mentioning

confidence: 99%

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How CAR T Cells Breathe

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Casey

et al. 2022

Cells

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The manufacture of efficacious CAR T cells represents a major challenge in cellular therapy. An important aspect of their quality concerns energy production and consumption, known as metabolism. T cells tend to adopt diverse metabolic profiles depending on their differentiation state and their stimulation level. It is therefore expected that the introduction of a synthetic molecule such as CAR, activating endogenous signaling pathways, will affect metabolism. In addition, upon patient treatment, the tumor microenvironment might influence the CAR T cell metabolism by compromising the energy resources. The access to novel technology with higher throughput and reduced cost has led to an increased interest in studying metabolism. Indeed, methods to quantify glycolysis and mitochondrial respiration have been available for decades but were rarely applied in the context of CAR T cell therapy before the release of the Seahorse XF apparatus. The present review will focus on the use of this instrument in the context of studies describing the impact of CAR on T cell metabolism and the strategies to render of CAR T cells more metabolically fit.

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Section: Study Of Metabolism In Car T Cellsmentioning

confidence: 99%

Section: Effect Of the Combination Of Car T Cells And Combinatorial D...mentioning

confidence: 99%

How CAR T Cells Breathe

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Joaquina

Casey

et al. 2022

Cells

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“…It is noteworthy that this platform eliminates the need for cumbersome transport and washing procedures, which are usually inevitable in downstream cell spheroid analysis [ 31 ], as washing and staining after co-culture of immune cells and tumor spheroids are usually required. Unlike other devices, 3DHSP does not require both expensive fluorescent staining and washing steps for spheroid size measurement [ 2 , 24 , 32 ] (Table 1 ). In addition, this platform enables real-time visualization of HER2-CAR T cells infiltrating into the tumor spheroid.…”

Section: Resultsmentioning

confidence: 99%

3D hanging spheroid plate for high-throughput CAR T cell cytotoxicity assay

et al. 2022

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Background Most high-throughput screening (HTS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor microenvironment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CAR T cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CAR T and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays. Results The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CAR T cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20°. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300–350 μm in diameter after 2 days in the 3DHSP. The cytotoxic effects of T cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR. Conclusions The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids. Graphical Abstract

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“…Frontiers in Bioengineering and Biotechnology frontiersin.org (Köksal et al, 2019;Köksal et al, 2020;Pierre et al, 2021). The T cells were kept at a concentration of 1 × 10 6 cells/mL throughout the culture period, with fresh medium added every second day.…”

Section: Figurementioning

confidence: 99%

Determination of CAR T cell metabolism in an optimized protocol

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Caulier

et al. 2023

Front. Bioeng. Biotechnol.

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Adoptive transfer of T cells modified to express chimeric antigenic receptors (CAR) has emerged as a solution to cure refractory malignancies. However, although CAR T cell treatment of haematological cancers has now shown impressive improvement in outcome, solid tumours have been more challenging to control. The latter type is protected by a strong tumour microenvironment (TME) which might impact cellular therapeutic treatments. Indeed, the milieu around the tumour can become particularly inhibitory to T cells by directly affecting their metabolism. Consequently, the therapeutic cells become physically impeded before being able to attack the tumour. It is therefore extremely important to understand the mechanism behind this metabolic break in order to develop TME-resistant CAR T cells. Historically, the measurement of cellular metabolism has been performed at a low throughput which only permitted a limited number of measurements. However, this has been changed by the introduction of real-time technologies which have lately become more popular to study CAR T cell quality. Unfortunately, the published protocols lack uniformity and their interpretation become confusing. We herein tested the essential parameters to perform a metabolic study on CAR T cells and propose a check list of factors that should be set in order to draw sound conclusion.

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Combinatorial CAR design improves target restriction

Cited by 8 publications

References 40 publications

How CAR T Cells Breathe

How CAR T Cells Breathe

3D hanging spheroid plate for high-throughput CAR T cell cytotoxicity assay

Determination of CAR T cell metabolism in an optimized protocol

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