Since their discovery, personalized cellular therapies have revolutionized blood cancer treatment, but remain ineffective against solid tumors—largely due to the loss of inflammatory effector functions.

The lab of CAR T cell pioneer Carl June, MD, professor in immunotherapy at the Perelman School of Medicine, University of Pennsylvania, explores strategies to improve T-cell therapies for solid tumors. Their latest study shows that targeting two inflammatory regulators delivered a “one-two punch” that led to at least 10 times greater T-cell expansion in preclinical models.

More specifically, the researchers targeted a regulatory axis of T-cell inflammatory responses, Regnase-1 and Roquin-1, to “enhance antitumor responses in human T cells engineered with two clinical-stage immune receptors.” The findings show that a knockout of either Regnase-1 or Roquin-1 alone enhances antitumor function in solid tumor models. But the double knockout increases function more than either one alone.

This work is published in PNAS in the paper, “Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells.

“We want to unlock CAR T cell therapy for patients with solid tumors, which include the most commonly diagnosed cancer types,” said June. “Our study shows that immune inflammatory regulator targeting is worth additional investigation to enhance T-cell potency.”

One of the challenges for CAR T cell therapy in solid tumors is T-cell exhaustion—where the persistent antigen exposure from the solid mass of tumor cells wears out the T cells to the point that they aren’t able to mount an antitumor response.

Previous observational studies hinted at the inflammatory regulator Regnase-1 as a potential target to indirectly overcome the effects of T-cell exhaustion because it can cause hyperinflammation when disrupted in T cells—reviving them to produce an antitumor response. The research team hypothesized 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,” said David Mai, a bioengineering graduate student in the School of Engineering and Applied Science. “By building on previous research, we are starting to get closer to strategies that seem to be promising in the solid tumor context.”

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 tumors.

After CRISPR editing, the T cells were expanded and infused in solid tumor mice models, where researchers observed the double knockout led to at least 10 times as many engineered T cells compared to disabling Regnase-1 alone, as well as increased antitumor 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 Neil Sheppard, DPhil, head of the T Cell Engineering Lab at the Center for Cellular Immunotherapies (CCI) at Abramson Cancer Center. “We’re certainly impressed by the antitumor potency that was unleashed by knocking out these two non-redundant proteins in combination. In solid tumor 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 tumors.”

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