The first U.S. Phase I clinical trial to evaluate CRISPR-Cas9-edited T cells in humans with advanced cancer has reported that the patients experienced no negative side effects, and that the engineered T cells persisted in their bodies for months. The trial included three advanced cancer patients who received T cells that had been engineered using CRISPR technology to carry three different genetic changes. It represents the first sanctioned study to evaluate the clinical application of multiple CRISPR edits to the human genome.

Lab tests have confirmed that the gene-edited T cells recovered from the trial participants months after they received the treatment were still able to kill cancer cells. The results have been reported by the trial’s investigators at the Abramson Cancer Center of the University of Pennsylvania, led by veteran immunotherapy physician Carl June, MD.

The new findings, published today in Science, suggest that the gene editing approach is both feasible and safe, and represent a key important milestone towards the ultimate goal of using gene editing to help the body’s own immune system recognize and attack cancer.

June tells GEN: “CRISPR technology has proven safe in patients with advanced refractory and metastatic cancer. Our results demonstrate the ability to precisely edit the DNA code at three different genes.”

“These findings provide a guide for the safe production and non-immunogenic administration of gene-edited somatic cells,” write Jennifer Hamilton, PhD, and CRISPR pioneer Jennifer Doudna, PhD, in an accompanying perspective. “The clinically validated long-term safety of CRISPR-Cas9 gene-edited cells reported [here] paves the way for next-generation cell-based therapies.”

June, who is the Richard W. Vague Professor in Immunotherapy and director of the Center for Cellular Immunotherapies in the Abramson Cancer Center and director of the Parker Institute for Cancer Immunotherapy at the Perelman School of Medicine at the University of Pennsylvania, says his team’s data from these three patients “demonstrate two important things that, to our knowledge, no one has ever shown before.”

“First, we can successfully perform multiple edits with precision during manufacturing, with the resulting cells surviving longer in the human body than any previously published data have shown. Second, thus far, these cells have shown a sustained ability to attack and kill tumors.”

June is senior author of the report, entitled “CRISPR-engineered T cells in patients with refractory cancer.” Publication of the data follows an initial report last year, confirming that researchers were able to use CRISPR-Cas9 technology to successfully edit three cancer patients’ immune cells. Penn is conducting the ongoing study in cooperation with the Parker Institute for Cancer Immunotherapy, and Tmunity Therapeutics (a biotech company June co-founded).

Down to a T

Gene-editing technologies offer the potential to correct DNA mutations and could feasibly be used to treat or eliminate countless human genetic diseases, the authors wrote. “Recent advances in CRISPR-Cas9 technology have also permitted efficient DNA modifications in human T cells, which holds great promise for enhancing the efficacy of cancer therapy.”

The strategy taken for the reported Phase I trial is closely related to CAR (chimeric antigen receptor) T cell therapy, in which a patient’s own T cells are engineered to express a receptor that can specifically detect and kill tumor cells. However, there are differences. Rather than directly arming patient-derived T cells with a receptor to a protein such as CD19, the researchers first used CRISPR to remove three genes from T cells taken from each of the three trial patients. The first two edits removed a T cell’s natural receptors so that they could then be reprogrammed to express a synthetic T cell receptor (NY-ESO-1) and so allow these cells to look for and destroy tumors. The third edit removed PD-1, a natural checkpoint that sometimes blocks T cells from doing their job.

The authors explained, “Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ  (TRBC) were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding PD-1 (PDCD1), was performed to improve anti-tumor immunity.”

In principle, this combination of editing would allow increased expression of the TCR and limit the development of something known as T cell exhaustion, which can be triggered when using checkpoint ligands. Once the three genes were knocked out, a fourth genetic modification was then carried out using a lentivirus to insert the cancer-specific synthetic T cell receptor, which instructs the edited T cells to target an antigen called NY-ESO-1. The modified T cells were then reinfused back into the patients.

The patients were treated by Edward A. Stadtmauer, MD, section chief of Hematologic Malignancies at Penn, who is the co-lead author on the study along with Joseph A. Fraietta, PhD, an assistant professor of Microbiology at Penn. The co-senior author is Simon F. Lacey, PhD, director of the Translational and Correlative Studies Laboratory in the Center for Cellular Immunotherapies.

In contrast with CAR-T cells, the CRISPR-edited T cells used for the trial were not active on their own. Rather, they required the cooperation of a molecule known as HLA-A*02:01, which is only expressed in a subset of patients. This meant that patients had to be screened to ensure that they were a match for the treatment approach. Participants who met the requirements received clinically indicated therapy as needed while their engineered T cells were produced. Once that process was completed, all three patients received their gene-edited cells in a single infusion after a short course of chemotherapy.

Several months after the infusion, researchers isolated the CRISPR-edited cells from the patients’ blood for study, and demonstrated through lab tests that the cells were still able to kill tumors. Whereas previously published data had found that these cells typically would only survive for less than a week, the new analysis confirmed that the edited cells used for the trial persisted, with the longest follow up at nine months.

In one patient, “a frequency of 30% of di-genic and tri-genic editing was achieved in the infused cell population, and 20% of the TCR transgenic T cells in circulation 4 months later had persisting di-genic and tri-genic edits,” the investigators added.

Normally these cells lose function within days, “so the fact that the CRISPR-edited cells in this study retained anti-tumor function for a significantly longer period of time after a single infusion is very encouraging,” June said.

The new analysis “confirmed that the manufactured cells contained all three edits, providing proof of concept for this approach. This is the first confirmation of the ability of CRISPR-Cas9 technology to target multiple genes at the same time in humans and illustrates the potential of this technology to treat many diseases that were previously not able to be treated or cured.”

Nothing adverse

While none of the patients actually responded to the therapy, there were no treatment-related serious adverse events. Bone marrow and tumor biopsies also showed that the NYCE engineered cells were transported to the tumor sites in all patients.

“The best clinical responses were stable disease in two patients,” the investigators noted. One of the patients demonstrated a mixed response, with a ~50% decrease in a large abdominal mass that was sustained for four months, but this occurred alongside the progression of other lesions. The team confirmed that by last December, all of the patients exhibited progressive disease, and one had died.

The investigators also acknowledge that while this initial trial has indicated that the technique has demonstrated acceptable safety, “experience with more patients given infusions of CRISPR-engineered T cells with higher editing efficiencies, and longer observation after infusion, will be required to fully assess the safety of this approach.” Nevertheless, based on the Phase I study, they confirmed that “multiplex CRISPR-Cas9 editing of the human genome is possible at clinical scale.”

Engineered T cells were manufactured using methods available back in 2016, but that technology has progressed significantly and it is possible that gene-edited T cells manufactured using today’s methods may be more effective, increasing efficiencies and lowering off-target editing.

Getting regulatory approval to carry out the trial in patients took two years, involving a comprehensive series of institutional and federal regulatory approval steps, including approval by the National Institutes of Health’s Recombinant DNA Research Advisory Committee and review by the FDA, as well as Penn’s institutional review board and institutional biosafety committee.

The Penn team says their data will open the door for future studies—of which it has several planned—to extend the approach beyond cancer. Penn notes that it has already pioneered a number of “first uses” of engineered T cells over the past decades.

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