Three patients with advanced cancer have become the first in the US to be treated with their own CRISPR-Cas9 modified T cells. The work, recently published in Science,1 details the CRISPR-Cas9–mediated knockout of 2 native T-cell receptors (TCRs) to increase efficacy and reduce toxicity of the T cells in 2 patients with multiple myeloma and 1 patient with liposarcoma. In addition, the team targeted the gene encoding PD-1 in the T cells to improve antitumor immunity, and introduced a synthetic T-cell receptor (TCR) targeting NY-ESO-1, a protein present on the tumors of the 3 patients.

All of the patients had metastatic disease and were heavily pretreated, both by chemotherapeutic regimens and transplants. Since the trial, 1 patient has died and the other 2 have seen their disease progress — but testing a potentially curative treatment was not the main goal of the trial, the researchers emphasized.

“These patients had no other treatment options left,” said Carl June, MD, director of the Center for Cellular Immunotherapies at the Perelman School of Medicine at the University of Pennsylvania and senior author of the paper. “The study wasn’t designed to test for efficacy — this was just a safety and feasibility study. For example, we wanted to know if the therapy would work at all in such heavily pretreated patients,” he added.

The main goals of the initial, small-scale trial were to test the feasibility of the multiplexed gene editing and also the safety profile of the treatment, with a main worry being that the patients’ immune systems would react to the Cas9 proteins expressed on the injected T cells after gene editing.


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“We showed that there was no problem with Cas9 immunogenicity,” said Dr June, and explained that there were concerns that individuals would experience an immune reaction to the Cas9 protein, due to previous exposure to Staphylococcus or Streptococcus bacteria. “The treatment was completely nontoxic,” he added, noting that the treatment could feasibly be done as an outpatient treatment with no fever or side effects related to the modified cells.

“This is a groundbreaking paper showing you can get away with this type of gene editing and can improve persistence and engraftment of these T cells,” said David Maloney, MD, PhD, medical director of cellular immunotherapy at the Immunotherapy Integrated Research Center at Fred Hutchinson Cancer Research Center in Seattle, Washington. “However, there was only minimal evidence of antitumor activity — but it appears to be safe,” he added.

The modified T cells also showed persistence and trafficking to the tumor sites, giving hope that these modifications introduced by the gene editing may help tackle the T-cell exhaustion2 that is often seen when treating solid tumors with chimeric antigen receptor T cells (CAR-T).

“In 1 of the patients, they took some blood after 4 months and showed the infused T cells ultimately formed memory T-cell phenotypes, which is what happens naturally when we respond to infections, instead of an exhausted phenotype seen in solid tumors,” said Astero Klampatsa, PhD, team leader in thoracic oncology immunotherapy at The Institute of Cancer Research in London, U.K.

Despite the therapies showing an excellent safety profile in the 3 patients treated, a major concern was whether there were mutations introduced in the T cells by the CRISPR-Cas9 gene editing. Theoretically, if mutations are introduced in an oncogene, for example, they could result in transformation of the T cells themselves, forming hematological malignancies.

“We used a very sensitive assay to look for off-target effects of the CRISPR-Cas9 editing. Almost all of the edits were on target and for the PD-1 guide, it was 100% on target. The least specific was the T-cell beta guide RNA and we’ve since made a better one,” said Dr June.

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Dr June is keen to stress that the CRISPR-Cas9 technology has evolved significantly since the group began using it for this particular study in 2016, believing that the frequency of mutations encountered in the edited T cells would be further reduced using more modern CRISPR-Cas9 techniques.

“Obviously, everything that involves DNA manipulation is a concern. With CRISPR in humans, we have to give it time and do studies with bigger pools of patients. We really don’t know what a lot of these mutations mean and if they are significant. If they are, we should see this after a few months in the [gene-edited] product,” said Dr Klampatsa.

Dr June also noted that more than 1000 patients have been treated with virus-modified T cells with no cases of new hematological malignancies so far. But even with improved technologies, are these gene-edited cells a concern if they are to have a future as a cancer therapy?