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Cocktail & Poster Display session

12P - CRISPR/Cas9-induced knock-out of DGKαζ in TAG-72 CAR-T cells improves function and persistence in ovarian cancer


06 Mar 2023


Cocktail & Poster Display session


Vera Evtimov


Annals of Oncology (2023) 8 (1suppl_2): 100900-100900. 10.1016/esmoop/esmoop100900


V. Evtimov, M. Hammett, N. Nhu-Y, J. Zhuang, I. Nisbet, A. Trounson, R. Boyd, R. Shu

Author affiliations

  • Research, Cartherics Pty Ltd, 3168 - Notting Hill/AU


This content is available to ESMO members and event participants.

Abstract 12P


The efficacy of chimeric antigen receptor (CAR-) T cells against solid tumours to date has been limited. This could be attributed, in part, to the endogenous tumour microenvironment (TME) - a complex milieu of immunosuppressive factors and ligands. To mitigate the impact of the immunosuppressive TME on CAR-T function, we have generated second-generation, tumour associated glycoprotein (TAG)-72 targeting CAR-T cells devoid of diacylglycerol (DAG) kinase α and ζ (DGKαζ). This deletion increases the intracellular level of DAG allowing the cells to be more metabolically active.


TAG-72 CAR lentivirus transduction was performed on healthy donor T cells followed by CRISPR/Cas9-mediated deletion of DGKαζ to give rise to CTH-004 CAR-T cells; T cells (no CAR) and TAG-72 CAR-T cells were generated in parallel. The phenotype for all groups was characterised by flow cytometry. The cytotoxic effect of CTH-004 CAR-T cells was evaluated in vitro using the real time impedance-based assay, xCELLigence®. In vivo CAR-T cell function was assessed in a NSG mouse model, xenografted with human ovarian cancer cell lines where flank tumours reached >100mm3 before intravenous administration of CAR-T cells. Persistence of CAR-T cells at the tumour site was assessed by immunohistochemistry.


Deletion of DGKαζ did not impact the viability or proliferative potential of CTH-004 CAR-T cells. Further, there were no significant differences in CTH-004 phenotype observed relative to T cell (no CAR) or TAG-72 CAR-T cell controls. CTH-004 CAR-T cells were able to selectively kill TAG-72hi (OV-90) and TAG-72mid (OVCAR-3) but not TAG-72low (MES-OV) expressing ovarian cancer cell lines in vitro. In the OVCAR-3 xenograft model we observed a significant reduction in the size of pre-established tumours with TAG-72 CAR-T cells for 30-40 days after treatment. In contrast, administration of CTH-004 CAR-T cells completely ablated the tumours, with CAR-T cells still observable at the remnants of the tumour site 100 days following treatment.


The combination of TAG-72 CAR-T cells with deletion of DGKαζ provides improved tumour control, probably via either increased T cell killing and / or enhanced longevity.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.


Cartherics Pty Ltd.


V. Evtimov, M. Hammett, N. Nhu-Y, J. Zhuang, I. Nisbet, A. Trounson, R. Boyd, R. Shu: Financial Interests, Personal, Full or part-time Employment: Cartherics Pty Ltd.

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