A team at Cardiff University in the U.K. has used CRISPR/Cas9 genome editing to generate engineered killer T cells that are up to a thousand times more sensitive to cancer cell antigens than T cells engineered using more conventional approaches, and which allowed far better targeting of T cells to cancer cell lines and patient-derived leukemia cells.

The new approach exploits the genome editing technology to remove the T cells’ endogenous T cell receptors (TCRs), and simultaneously replace them with cancer antigen-specific TCRs. The researchers, led by Cardiff University School of Medicine’s Professor Andrew Sewell and Dr. Matesuz Legut, hope that the new method will enable the development of far more effective anticancer immunotherapies, and offer up a new experimental system for identifying novel cancer targets.

“The T-cells we made using genome editing do not have any of their own T-cell receptors left, and therefore the only receptor they can use is the one specific for cancer,” Dr. Legut comment. “As a result, these cells can be a thousand times better at seeing and killing cancer than the cells prepared using the current methodology.”

The researchers report on their studies in Blood, in a paper entitled, “CRISPR-mediated TCR replacement generates superior anticancer transgenic T-cells.”

TCRs in vertebrates exist as heterodimers composed of either αβ or γδ TCR chains. While the αβ TCRs generally recognize foreign antigenic peptides presented by major histocompatibility complex (MHC) molecules on antigen presenting cells, γδ TCRs tend to recognize cell surface targets, including cancer antigens, the researchers explain.

Traditional approaches to engineering T cells for cancer immunotherapy involve the transduction of cells with a chimeric antigen receptor (CAR) or a TCR for a specified antigen. The methods used effectively add the cancer antigen receptor to T cells that already express their own native receptors. But the presence of these pre-existing endogenous TCRs reduces the number of cancer-specific TCRs that can be inserted into the T cells, and also creates the potential for generating hybrid TCRs that can trigger potentially fatal autoimmunity.

“Up until now, T cells engineered to fight cancer had two kinds of receptors – the therapeutic one that was added in the lab, and their own naturally existing one,” notes Dr. Legut. “Since there is only limited 'space' on a cell for receptors, cancer-specific ones need to compete with the cell's own receptors to perform their function. More often than not, the cell's own receptors win that competition, and leave 'space' for only a very limited number of newly introduced, cancer-specific receptors, which means that T-cells engineered with the current technology never reach their full potential as cancer killers.”

Rather than just adding the new cancer-specific TCRs to T cells, the University of Cardiff researchers instead used CRISPR/Cas9 editing to simultaneously knock out the cells’ endogenous αβ TCR, and replace it with a cancer antigen-specific γδ TCRs. “This approach enhanced the expression of the transduced TCR at the T-cell surface and resulted in TCR transductants that displayed substantially improved antigen sensitivity,” they report.

When tested in the laboratory, the CRISPR-edited and transduced T cells demonstrated far better in vitro and ex vivo reactivity to primary blood cancers, compared with T cells expressing both endogenous and transgenic TCRs. “Transduction with a pan-cancer reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4+ and CD8+ T-cells against a panel of established blood cancers and primary, patient-derived B acute lymphoblastic leukemia blasts compared to standard TCR transfer,” the team states. “The improvement in the sensitivity of cancer recognition that can be achieved by editing out the existing natural receptor and then replacing it with one that sees cancer cells is remarkable,” professor Sewell adds.

The authors hope that the ability to generate cancer-specific γδ TCR T cells will allow the design of far more effective TCR-based immunotherapies, without concerns about side-effects. “γδ T-cells offer an attractive tool for cancer immunotherapy, due to their ability to recognize ubiquitously expressed targets and no evidence of MHC restriction,” they note. “This feature allows such γδ Tcells to respond to cancer from any individual and also eliminates the risk of graft versus host disease.”

They envisage that the technology could also help more fundamental research. “TCR replacement is preferable to TCR transfer for functional characterizations of TCRs of interest especially where these TCRs compete poorly with endogenous TCRs for surface expression or have a relatively low affinity for cognate antigen.” The system could in addition be used to generate high-throughput, whole genome screens to identify new TCR ligands and potential therapeutic targets.

“Immunotherapy – harnessing the body´s own immune cells – has become the most potent and promising new treatment for a range of cancers and represents one of the biggest breakthroughs in cancer treatment in memory,” concludes study co-author professor Oliver Ottmann, head of Haematology at Cardiff University and co-lead of the Cardiff Experimental Cancer Medicine Centre (ECMC). “I believe that our improved method of making cancer-specific T-cells will guide a new generation of clinical trials and be used by researchers in the laboratory to discover new cancer-specific T-cell receptors and new targets for cancer therapy.”


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