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ePoster Display

36P - In-vitro tonic signalling profiling of CAR-T cells generated to support pre-clinical studies for solid tumour targets


16 Sep 2021


ePoster Display


Basic Science;  Translational Research

Tumour Site


Suchete Hunjan


Annals of Oncology (2021) 32 (suppl_5): S361-S375. 10.1016/annonc/annonc684


S.S. Hunjan1, R. Sampson1, J. Evans1, H. Chenoweth1, I. Garrobo-Calleja1, S. Lekomtsev1, J. Zhang1, S. Zona1, J. Breuning1, R. Oren1, M. Davies1, A. Di-Tullio2, J. Euesden3, J. Kennedy3, C. Kay4, J. Colebrook4, B.-. Kloke5, T. Southgate5, J. Lee1

Author affiliations

  • 1 Oncology Cell Therapy Oncology Cell Therapy Research Unit, GlaxoSmithKline, SG1 2NY - Stevenage/GB
  • 2 Immuno-oncology Functional Genomics, GlaxoSmithKline, SG1 2NY - Stevenage/GB
  • 3 Research Statistics, GlaxoSmithKline, SG1 2NY - Stevenage/GB
  • 4 Cell & Gene Therapy, Cell Process Development, GlaxoSmithKline, SG1 2NY - Stevenage/GB
  • 5 Oncology R&d, GlaxoSmithKline, SG1 2NY - Stevenage/GB


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Abstract 36P


Chimeric antigen receptors (CARs) are synthetic receptors that redirect T-Cell specificity, function and persistence. CARs are composed of the antigen specific region of an antibody single chain fragment (scFv) fused to the T-cells activating domain (CD3ζ) and a co-stimulatory domain (CD28ζ or 4-1BBζ). CAR-T cells promote antigen specific activation and enhance proliferation and anti-apoptotic functions (June, 2018). Tonic signalling can be defined as constitutive activation of T-Cells in the absence of a ligand. CAR-T cells that exhibit tonic signalling lead to impaired in vitro cell function, exhaustion and inferior in vivo efficacy (Long et al., 2015). Tonic signalling can be influenced by a combination of features including the scFv, linker or hinge, signalling domains, surface expression locus and CAR expression levels (Ajina et al. 2018).


Tonic signalling was assessed using in vitro assays for two CAR-T cell assets targeting pan-epithelial solid tumour targets. Humanised CAR-T cells with the BBζ cytosolic domain and as a low-tonic signalling control CD19-BBζ CAR-T cells were generated by lentivector transduction of T-cells on a phospho-glycerate kinase (PGK) promoter with a low affinity nerve growth factor (LNGFR) detection domain. Similarly, a GD2-28ζ-LNGFR (14g2a scFv) CAR-T, that has been described to exhibit tonic signalling (Long et al., 2015), with an IgG1 (CH2CH3) hinge was generated using a lentivector on an elongation factor (EF1a) promoter. The propensity of the LNGFR enriched test CAR-T cells to present tonic signalling was determined by basal levels of interferon gamma (IFNγ) secreted in CAR-T cell culture supernatants, activation (CD69) and exhaustion (PD-1 and TIM-3) phenotyping and determination of CAR specific phosphorylated-CD3ζ (pCD3ζ) from cell lysates.


From the results obtained, the CAR-T cells confirmed responses similar to the non-tonic signalling CD19-BBζ CAR-T cell.


The tonic signalling results for the tested CAR-T cells were significantly lower than the observed responses for the high tonic signalling GD2-28CAR-T cells.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study





S.S. Hunjan, R. Sampson, J. Evans, H. Chenoweth, I. Garrobo-Calleja, S. Lekomtsev, J. Zhang, S. Zona, J. Breuning, R. Oren, M. Davies, A. Di-Tullio, J. Euesden, J. Kennedy, C. Kay, J. Colebrook, B.P. Kloke, T. Southgate, J. Lee: Financial Interests, Personal, Full or part-time Employment: GlaxoSmithKline; Financial Interests, Personal, Stocks/Shares: GlaxoSmithKline.

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