3O - Generation of immune checkpoint knock-out human antigen-specific T cells via CRISPR/Cas9-mediated genetic engineering

Date 04 November 2016
Event ESMO Symposium on Immuno-Oncology 2016
Session Proffered paper session
Presenter Chi Zhang
Citation Annals of Oncology (2016) 27 (suppl_8): viii1-viii2. 10.1093/annonc/mdw525
Authors C. Zhang1, Y. Peng2, P. Hublitz2, T. Dong2
  • 1Nuffield Department Of Medicine, Weatherall Institute of Molecular Medicine (WIMM)University of Oxford, OX3 9DS - Oxford/GB
  • 2Nuffield Department Of Medicine, Weatherall Institute of Molecular Medicine (WIMM)University of Oxford, Oxford/GB



In response to the constant antigen stimulation given by tumours or chronic viral infections, T cells usually malfunction. It is now clear that tumours co-opt certain immune checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumour antigens. This study aims to establish a platform to efficiently disrupt immune checkpoints, such as pdcd1 (PD1), on the antigen-specific T cell clones or lines via CRISPR/Cas9 to improve T cell functions.


The antigen-specific T cells (CD4 and CD8) were isolated from patients by tetramer sorting and cultured in vitro using feeder cells plus PHA, and could be further enriched or cloned for functional study. Lentivirus (pL-CRISPR.SFFV.GFP, Addgene 57827) co-expressing Cas9, EGFP and sgRNA was used to transduce antigen-specific T cells and knock out immune checkpoint genes, such as pdcd1. Functional studies include cytokine intracellular staining and Ki67 intracellular staining were carried out to evaluate T cell functions.


Several PD1 knock-out antigen-specific T cell lines were generated and knocking out PD1 could restore T cell functions in PDL1-hi microenvironment.


This study is another aspect of immunotherapy and could be applied to genetically improve antigen-specific T cells functions in different disease types and therefore shed light on the therapeutic treatments of cancers or chronic viral infections. The merits of this technology include that it only targets antigen-specific T cells of interest, other immunity will be minimally disturbed and the engineered T cells naturally express co-stimulatory components and T-cell receptors, which direct the host to kill the target cells.

Clinical trial identification

Legal entity responsible for the study

University of Oxford


Medical Research Council (MRC)


All authors have declared no conflicts of interest.