Double knockout strategy could boost CAR-T in solid tumours

CAR-T therapy

A new approach that delivers a ‘one-two punch’ to help T cells attack solid tumours is the focus of a preclinical study by researchers from the Perelman School of Medicine at the University of Pennsylvania.

The findings showed that targeting two regulators that control gene functions related to inflammation led to at least 10 times greater T cell expansion in models, resulting in increased antitumour immune activity and durability.

CAR-T cell therapy was pioneered by Carl H June, the Richard W Vague Professor in Immunotherapy at Penn Medicine and Director of the Center for Cellular Immunotherapies (CCI) at Abramson Cancer Center. His work led to the first approved CAR-T cell therapy for B-cell acute lymphoblastic leukaemia in 2017.

Since then, personalised cellular therapies have revolutionised blood cancer treatment, but remained stubbornly ineffective against solid tumours, such as lung cancer and breast cancer.

“We want to unlock CAR-T cell therapy for patients with solid tumours, which include the most commonly diagnosed cancer types,” said June, the new study’s senior author. “Our study shows that immune inflammatory regulator targeting is worth additional investigation to enhance T cell potency.”

T cell exhaustion

One of the challenges for CAR-T cell therapy in solid tumours is a phenomenon known as T cell exhaustion, where the persistent antigen exposure from the solid mass of tumour cells wears out the T cells to the point that they aren’t able to mount an antitumour response.

The research team, including lead author David Mai, a Bioengineering graduate student in the School of Engineering and Applied Science, hypothesised that targeting the related, but independent Roquin-1 regulator at the same time could boost responses further.

“Each of these two regulatory genes has been implicated in restricting T cell inflammatory responses, but we found that disrupting them together produced much greater anticancer effects than disrupting them individually,” Mai said. “By building on previous research, we are starting to get closer to strategies that seem to be promising in the solid tumour context.”

CRISPR gene editing

The team used CRISPR-Cas9 gene editing to knock out Regnase-1 and Roquin-1 individually and together in healthy donor T cells with two different immune receptors that are currently being investigated in Phase I clinical trials: the mesothelin-targeting M5 CAR (mesoCAR) and the NY-ESO-1-targeting 8F TCR (NYESO TCR).

Neither engineered T cell product targets CD19, the antigen targeted by most approved CAR-T cell therapies, as this antigen is not present in solid tumours.

After CRISPR editing, the T cells were expanded and infused in solid tumour mice models. Researchers observed the double knockout led to at least 10 times as many engineered T cells compared to disabling Regnase-1 alone.

They also observed increased antitumour immune activity and longevity of the engineered T cells. In some mice, it also led to overproduction of lymphocytes, causing toxicity.

“CRISPR is a useful tool for completely ablating the expression of target genes like Regnase and Roquin, resulting in a clear phenotype, however, there are other strategies to consider for translating this work to the clinical setting, such as forms of conditional gene regulation,” said co-corresponding author Neil Sheppard, Head of the CCI T Cell Engineering Lab.

“We’re certainly impressed by the antitumour potency that was unleased by knocking out these two non-redundant proteins in combination. In solid tumour studies, we often see limited expansion of CAR-T cells, but if we’re able to make each T cell more potent, and replicate them to greater quantities, we expect T cell therapies to have a better shot at attacking solid tumours.”

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