NEW YORK (GenomeWeb) – As genome-editing technologies continue to rapidly develop and spread throughout academic labs, they're beginning to make a serious impact in the realm of therapeutics. Though direct clinical applications appear to still be anything but close, gene editing is proving essential to developing one of the most promising areas of cancer treatment, T-cell therapy.
In the last year, major players in the pharmaceutical industry have formed alliances to access gene-editing experts to help them develop chimerical antigen receptor (CAR) T cells. These immune system-derived killers are cells that have been genetically reengineered to express receptors that allow them to recognize and eliminate cancerous cells.
"There have been some profound clinical responses over the last few years with CAR T cells, success where all the other drug classes didn't succeed" in treating hematological cancers, Robert Friesen, head of biologics research at Janssen, told GenomeWeb. "This has turned out to be a very effective way to deal with a lot of cancers."
With numerous receptors to target, CAR T cells could be engineered every which way to target many kinds of cancer — there's even evidence they could be used to treat solid tumors. But so far they only work as an autologous treatment, where the T cells are isolated from the patient, engineered ad hoc, grown to great numbers, and reintroduced to that same patient. Otherwise, if the T cells came directly from another patient, they would likely turn against the body, inducing graft versus host disease (GVHD).
"From an operational standpoint, it's almost a nightmare," Friesen said of autologous CAR T cell treatment. "It's very difficult and commercially that's not a very viable proposition."
Both Janssen and Cellectis, a Paris-based firm that recently opened a CAR T-cell research center in New York, are pursuing development of an off-the-shelf, or allogeneic, CAR T-cell product that would not cause GHVD. To do this, they'll need to engineer cells so that they don't attack the cells in the body and vice versa, and to make it happen they've both made bold decisions on genome-editing technologies. Janssen recently partnered with Transposagen to use CRISPR/Cas9, while Cellectis is going a different route with transcription activator-like effector nucleases (TALENs).
CAR T cells were developed in the academic medical environments of the National Cancer Institute and the University of Pennsylvania's Perelman School of Medicine. In 2013, researchers from Penn reported that CAR T cell therapy for pediatric acute lymphoblastic leukemia (ALL) resulted in 19 of 22 patients achieving complete remission. This particular therapy targeted CD19, a protein found on the surface of B cells. Novartis is currently running a clinical trial on the therapy and has published some promising results.
Phil Gotwals, of the Novartis Institutes for BioMedical Research, told GenomeWeb in an email that the firm is investigating the application of CRISPR and other gene-editing systems for future generations of T cell therapies, but declined to comment further.
Other companies that have announced CAR T cell projects include Kite, Bluebird, and Sangamo Biosciences. Sangamo is trying to engineer T cells to eliminate HIV, using its exclusive zinc finger nuclease gene-editing technology.
Not to be left out, Janssen, a division of Johnson & Johnson, recently signed a deal with Transposagen in November 2014 giving it access to CRISPR.
For now, Friesen said that Janssen is still in the first stage of its three-step plan: finding the right T cells to later engineer, ones with enough regenerative power to withstand growing in culture for a long time. After that, though, it will be time to put CRISPR to work.
To avoid the host killing the introduced cells, Friesen said Janssen needs to eliminate marks identifying the T cells as foreign, namely, human leukocyte antigen (HLA) proteins. "We somehow need to tinker with these HLA molecules on the cells we receive from the donor," he said. Janssen will use CRISPR to delete expression of the entire class of HLA type II molecules. "We know that is possible; that has been done. Your T cell function doesn't depend on these molecules."
However, there are also type I HLA proteins, which cannot be indiscriminately knocked out of the T cell. If there are no HLA molecules on a cell at all, the immune system also recognizes them as foreign, Friesen said. "That's the difficulty. We have to strike a balance between decreasing the number of HLA molecules on the surface of these T cells without having them be recognized as foreign."
Friesen added that when the time comes to actually add the receptor that allows the T cell to kill cancerous cells, Janssen plans to use CRISPR to introduce the CAR receptor as well. Janssen doesn't have any targets in mind yet, but Friesen said it's just getting started, having had a kickoff meeting with Transposagen in recent months.
But Janssen — and anybody else who is using CRISPR to engineer T cells — may run into problems, André Choulika, CEO of Cellectis, told GenomeWeb.
"It's a new technology for underfunded academic labs, but it's not a technology for industrial therapeutic labs," he said. "The technology is not ready yet."
Cellectis plans to use TALENs in its CAR T cell development, a technology for which it has acquired an exclusive license from the University of Minnesota. Dan Voytas, a professor there and inventor of TALENs, is the CSO of Cellectis' plant sciences division.
But Choulika said the company has done its due diligence and prefers TALENs for a variety of reasons. "We've tried them all — Meganucleases, ZFNs, CRISPR. We benchmarked them, comparing all the different technologies," he said, adding that TALENs come out on top in a number of important metrics for engineering CAR T cells.
"The most difficult thing for gene editing is vectorization in vivo," Choulika said. Cellectis acquired vectorization technology from Cyto Pulse when it decided to move into CAR T cells in 2010, providing it with an effective way to load TALENs into the cells without killing them.
More importantly, though, the benefits of CRISPR/Cas9, namely that it's cheap and flexible, don't mesh with Cellectis' plan to engineer CAR T cells. Unlike Janssen, which plans to knock out many HLA genes to avoid GVHD, Cellectis is only going after one gene, the TCR alpha chain.
Choulika said Cellectis can routinely get TCR alpha knockout efficiency of 80 to 95 percent with TALENs. Cellectis measured CRISPR/Cas9 knockout efficiency at around 25 percent, thus any cost savings would be partially lost due to lower effectiveness.
Another problem at this stage in CRISPR/Cas9 technology development is the off-target effects. "You can get your knockouts, but the T cell will lose one thing, which is t-cell expansion. Most of the cells won't be able to amplify," Choulika said.
"Most of the genes of the cells are composed of genes involved in amplification and cell division," he said. "Most of the time, a gene involved in cell division is hit," by off-target effects, which is undesirable for a technology that depends on T cells being able to multiply to great quantities after being introduced to the patient.
Cellectis already has some off-the-shelf T cells in development, with a CD19-targeting therapy that the firm hopes to push into clinical trials later this year. It also has products targeting amyloid leukemia and multiple myeloma that it hopes will enter trials in the next few years.
One area of interest for the company is introducing multiple genome edits to the T cells. Cellectis has 12 receptor targets it's working on for itself, and it's working on 13 others in collaboration with Pfizer. Some cancer cells are able to avoid elimination by expressing PDL1, a checkpoint receptor that pairs with the PD1 receptor in T cells. Knocking out the PD1 gene could render this cloaking tactic ineffective.
But Choulika said that as more engineering is introduced, the knock out efficiency goes down, and that's something that the entire field needs to resolve.
"We're still at the prehistory of this technology. We've faced hurdles, but the fact that sometimes you hit hurdles does not mean that these will not be overcome. I'm still convinced there is a path for this," Choulika said. "Gene editing is compulsory to take this technology into the 21st century."