A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Xi, Biao; Berahovich, Robert; Zhou, Hui; Xu, Shuning; Wei, Yuehua; Guan, Jasper; Harto, Hizkia; Guan, Jian; Wu, Lijun; Ana, David Santa; Cerignoil, Fabio; Lamarche, Brandon J.; Abassi, Yama; Golubovskaya, Vita M.

doi:10.3791/59033

Cited by 7 publications

(3 citation statements)

References 37 publications

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“…In Treg, which have high levels of GITR expression, GITR signaling participates in regulation of proliferation and modulation of suppressive function ( 5 , 20 , 21 ). Targeting GITR via anti-GITR agonistic antibody ( 22 , 23 , 24 , 25 ), recombinant Fc-GITRL proteins ( 26 , 27 , 28 ), or engineered Chimeric Antigen Receptor T cells (CAR-T) expressing recombinant receptors with the GITR cytosolic domain ( 29 , 30 , 31 ) can exert potent therapeutic activities. However, GITR-mediated intracellular signaling remains only partially characterized.…”

mentioning

confidence: 99%

Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

Hostager

Arkee

et al. 2021

Journal of Biological Chemistry

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Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) plays context-specific roles in multiple receptor-mediated signaling pathways in different cell types. Mice lacking TRAF3 in T cells display defective T-cell-mediated immune responses to immunization and infection and demonstrate defective early signaling via the TCR complex. However, the role of TRAF3 in the function of GITR/TNFRSF18, an important costimulatory member of the TNFR superfamily, is unclear. Here we investigated the impact of T cell TRAF3 status on both GITR expression and activation of specific kinases in the GITR signaling pathway in T cells. Our results indicate that TRAF3 negatively regulates GITR functions in several ways. First, expression of GITR protein was elevated in TRAF3-deficient T cells, resulting from both transcriptional and posttranslational regulation that led to greater GITR transcript levels, as well as enhanced GITR protein stability. TRAF3 associated with T cell GITR in a manner dependent upon GITR ligation. TRAF3 also inhibited several events of the GITR mediated early signaling cascade, in a manner independent of recruitment of phosphatases, a mechanism by which TRAF3 inhibits signaling through several other cytokine receptors. These results add new information to our understanding of GITR signaling and function in T cells, which is relevant to the potential use of GITR to enhance immune therapies.

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confidence: 99%

Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

Hostager

Arkee

et al. 2021

Journal of Biological Chemistry

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“…We observed differential cytolysis of GD2+ GSCs and CHLA20 neuroblastoma cells by two anti-GD2 CAR T cell products across a range of E:T ratios tested. Impedance sensing has several advantages over conventional 51 Cr release assays, including the ability to perform real-time label-free analysis without radioactive isotopes while facilitating additional downstream analyses for potency assessment [ 50 , 51 ]. This is important for detecting cytotoxicity when the onset of killing might be delayed due to low E:T ratios or potential low-affinity T-cell interactions.…”

Section: Discussionmentioning

confidence: 99%

Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products

Logun¹,

Colonna²,

Mueller³

et al. 2023

Cytotherapy

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“…In general, cytotoxicity assays involve co-culture of target and effector cells to mimic in vivo cell function. There are various outputs to measure the cytotoxic effect, such as bioluminescence and impedance [ 9 , 10 ]. Alternatively, using vital dyes, flow cytometry is also a sensitive method for detecting target cell death.…”

Section: Introductionmentioning

confidence: 99%

In vitro CAR-T cell killing: validation of the potency assay

Piccinini,

Carloni,

Arienti

et al. 2024

Cancer Immunol Immunother

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For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 −) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

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A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Cited by 7 publications

References 37 publications

Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products

In vitro CAR-T cell killing: validation of the potency assay

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