A team at Scripps Research reported the development of a method for rapidly discovering potential cancer-treating compounds that work by resurrecting anti-tumor activity in T cells. Cancerous tumors often thrive because they render T cells dysfunctional or “exhausted.” The new technique uncovers medicinal compounds that can restore the function of these T cells, making cancers vulnerable to them again, according to the research.

The approach (“Discovery of Small Molecules for the Reversal of T Cell Exhaustion”), described in Cell Reports, may also help restore T-cell responses to persistent infections from viruses or other pathogens. It, therefore, should speed the development of new cancer and infectious-disease immunotherapies, including those that can be combined with existing immunotherapy drugs to enhance their effects, according to Michael Oldstone, MD, professor emeritus in the department of immunology and microbiology at Scripps Research. The scientists demonstrated the potential utility of the approach by using it to rapidly screen a collection of more than 12,000 drug compounds and uncovering 19 that can reawaken exhausted T cells.

“Inhibitory receptors (IRs) function as critical regulators of immune responses by tempering T-cell activity. In humans, several persisting viruses, as well as cancers, exploit IR signaling by upregulating IR ligands, resulting in suppression of T-cell function (i.e., exhaustion). This allows escape from immune surveillance and continuation of disease. Here, we report the design, implementation, and results of a phenotypic high-throughput screen for molecules that modulate CD8+ T-cell activity,” the investigators wrote.

“We identify 19 compounds from the ReFRAME drug-repurposing collection that restore cytokine production and enhance the proliferation of exhausted T cells. Analysis of our top hit, ingenol mebutate, a protein kinase C (PKC) inducing diterpene ester, reveals a role for this molecule in overriding the suppressive signaling cascade mediated by IR signaling on T cells. Collectively, these results demonstrate a disease-relevant methodology for identifying modulators of T-cell function and reveal new targets for immunotherapy.”

“This new screening method should be particularly useful because we can use it not only to identify compounds that restore needed function to exhausted T cells, but also to quickly analyze these T cells to determine how these compounds work on them,” said Oldstone.

The novel screening system and, to some extent, the wider field of cancer immunotherapy, is based in part on research over the past several decades by Oldstone’s laboratory and several former lab members, along with other scientists that have conducted animal-based research on how the immune system responds to lymphocytic choriomeningitis virus (LCMV).

A unique variant of LCMV known as “clone 13” establishes a persistent infection by exhausting the virus-specific T cells that are required to clear the infection. It does this by boosting signals through T-cell receptors such as PD-1 and IL-10. The discovery that LCMV clone 13 can survive by switching off anti-LCMV T cells was quickly followed by the recognition that cancers often persist using the same trick.

Immunotherapies that block signaling from PD-1 or similarly acting receptors to restore T cells’ anticancer responses are among the most powerful cancer medicines available today. These therapies save many patients who in the past had seemingly untreatable tumors. But because treatment with these drugs typically works well for only a few cancers, including melanoma, and less often on other cancers, scientists suspect that cancers usually hijack multiple inhibitory T-cell pathways. This suggests that a combination of immunotherapies directed to different molecular pathways could be more effective than the current therapy.

“The idea now is to develop more immunotherapy drugs and find the best combinations of them,” Oldstone said.

The new system is designed to enable scientists to swiftly find such drugs—in this case, pharmacologically active small-molecule compounds that might work better than, or augment, the current injectable antibody immunotherapies now available. The system uses T cells that have been exhausted by LCMV clone 13 and detects signs of renewed activity in these cells when a tested compound works to reawaken them. An advantage of the new method is that it is specific and highly automated; thus, thousands of compounds can be tested within days, with the hits verified in experiments involving mice.

Oldstone and colleagues applied the new screening system to a drug repurposing library of more than 12,000 compounds that either are FDA-approved or have been tested as potential drugs. They quickly identified 19 hits, compounds that, at modest doses, can effectively resurrect the activity of exhausted T cells.

One of these compounds, ingenol mebutate, is a plant-derived molecule that is already used in gel form (Picato) to treat actinic keratosis, a precancerous skin condition. The researchers employed elements of their screening system to study the reactivated T cells and determined that ingenol mebutate restores function for these cells largely by activating signaling enzymes called protein kinase C enzymes, a known pathway of activity for this compound.

Co-first authors of the study, postdoctoral fellows Brett Marro, PhD, and Jaroslav Zak, PhD, in the department of immunology and microbiology, are currently collecting and exploring the therapeutic potential of other reported hits that may work in combination with treatments that block PD-1 and another T-cell-inhibitory receptor, CTLA-4. Indeed, one such hit in combination with antibody to PD-L1 is already undergoing evaluation in patients.

Oldstone noted that the new screening approach is flexible enough to adapt for finding compounds that have other effects on T cells, such as reducing T-cell activity to treat autoimmune conditions.

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