Author: Dr Mustafa M. Munye, 6th May 2020
A recent paper from Carl June’s lab published in the journal Science¹ described results of a University of Pennsylvania clinical trial that administered in three patients T-cells engineered to target cancer cells using a virus whilst simultaneously engineered using CRISPR to stop expression of 3 genes. The aim of the clinical trial was to assess the safety and feasibility of this approach more so than efficacy. In this regard, the therapy was well tolerated but that is just the beginning.
Carl June was one of the pioneers who led on the development of Kymriah- one of two approved CAR-T cell products that have produced remarkable results in treating specific blood cancers. His lab now has sights set on treating solid tumours. In 2015 the group published the results of a clinical trial where 24 patients had their T-cells modified to target and kill cells with the NY-ESO-1 antigen- a tumour associated antigen found to be highly expressed in many tumour types. The engineered T-cells were shown to be effective in homing to and killing cancer cells, but the effects were short lived. The T-cells did not persist for long and patients relapsed².
In this recent study¹ the team again used a virus to instruct the patients T-cells to express a T-cell receptor (TCR) that recognises the NY-ESO-1 antigen. T-cells have an endogenous TCR composed of an alpha and beta chain. To make sure the new TCR assembles correctly the team used CRISPR to knockout the genes that encode the endogenous TCR alpha and beta chains (TRAC and TRBC respectively). Going further, the group also knocked-out the PDCD1 gene which encodes the PD-1 protein. Cancer cells can engage PD-1 and trigger the T-cells to stop killing.
The team treated fewer patients in this trial than in the first study, but the engineered T-cells persisted for far longer; up to 9 months in the current study. The average half-life of the cells was 83.9 days compared to ~1 week in the first study. Notably, cells where TRAC and/or PDCD1 genes were disrupted displayed sustained persistence. The impact of the engineered T-cells on the tumours however were less pronounced in this study.
Of the three patients treated, two had stable disease for 4 months with no progression of their tumour but also no regression. A third patient had an ~50% decrease in a large abdominal mass indicating tumour killing. This reduction was sustained for four months. However, other sites of tumour in this patient continued to progress. In all three patients the cancer did eventually progress with one patient unfortunately succumbing to the disease.
Biopsies of the tumours revealed that the tumour cells had lowered the levels of the NY-ESO-1 so making them less visible to the engineered T-cells. The team confirmed the engineered T-cells were still able to kill tumour cells at 3 and 9 months so long as the NY-ESO-1 antigen was present.
Whilst the clinical efficacy here was modest there are learnings for those contemplating a similar approach.
In total six patients were enrolled to this trial and had their cells taken to be engineered. However, a satisfactory product could only be made for four of the patients highlighting how complex it is to successfully manufacture a therapy with multiple manipulations. Unfortunately, the cancer for one of the four patients progressed rapidly whilst waiting for their cells to be engineered and they were no longer eligible for the trial. In all then only three patients were given the therapy.
Where manufacturing was successful the group ran a battery of tests to characterise the cells produced. Around 2-5% of the total cells produced were successfully engineered to express the NY-ESO-1 targeting TCR. This is surprisingly low considering the lentiviral vector used are normally very good at getting into T-cells. The low efficiency may be due to the manufacturing protocol used where two days prior to the virus being added the cells had undergone electroporation to introduce the Cas9 protein for the CRISPR knockout.
Electroporation is a technique whereby an electrical field is applied to cells to increase their permeability by creating temporary pores on the cell membrane. This may have altered the cells such that they were less permissive to the virus; or made the addition of the virus the straw that broke the camel’s back leading to the cell’s death.
Having a low proportion of cells engineered to target cancer cells could impact the efficacy of the therapy but T-cells, once they engage their target, trigger a cascade where they multiply to make more of themselves. All is not lost. In all three patients this is exactly what happened with NY-ESO-1 TCR engineered T-cell numbers increasing over time and found enriched at sites of tumour.
With regards to the CRISPR gene knockout it was easiest to edit the TRAC gene with ~45% of the cells lacking the gene post engineering. TRBC and PDCD1 were both edited with an efficiency of ~15 and ~20% respectively. Given these numbers it should be clear that the cells given back to the patient were a heterogenous mix. This includes engineered and “wild-type” cells; those that went through the process but were not modified.
Of the engineered cells some had only one of the three genes knocked out, fewer had two and fewer still had all three knocked out. When factoring in that only 2-5% of the cells had the NY-ESO-1 TCR, <1% of the cells given to the patients had the desired NY-ESO-1 TCR in addition to knock-out of TRAC, TRBC and PDCD1. From one patient whose cells had undergone in-depth genetic analysis this rare population of cells, with all 4 modifications, could no longer be detected at 113 days post infusion suggesting these cells did not have a survival advantage.
Doing multiple genetic modifications is not without risk. CRISPR gene editing using Cas9 works by introducing a double-stranded break in the DNA. Doing this increases the risk that large chunks of DNA will move from on chromosome to another. These events are known as translocations and can themselves lead to cancer.
Trying to simultaneously edit three different genes all on different chromosomes only increases the chances of translocations happening and the team at the University of Pennsylvania did find translocation events in some of the engineered cells. This was not specific to a patient as all three had translocations in a proportion of engineered T-cells. Encouragingly both prior to infusion and several months post infusion the T-cells remained a heterogenous population suggesting that none of the translocation events led to uncontrolled cell division that would have led to that clone dominating.
Of course, this study included only 3 patients so we must be careful to not draw too many conclusions nor extrapolate the findings. The risk remains that translocations can lead to cancer so a risk/benefit analysis must always be conducted before proceeding with similar trials. An acceptable risk in one patient population may not be acceptable in another.
Whilst this study did show the approach to be safe in the three patients treated as part of the clinical trial, the authors noted “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.”
It is important to note that the technology used in this trial is based on what was available in 2016. Not only has the gene editing technology moved on, so has the electroporation technology used to get the Cas9 into the cells as well as the culture technology to keep the engineered cells in prime condition. It will not be long before we see clinical trials taking advantage of these advances generating highly engineered T-cells that with any luck provide sustained regression of solid tumours that has so far been elusive.
References 1. Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science. 367, (2020). 2. Rapoport, A. P. et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat. Med. 21, 914–921 (2015).