CAR-T cell therapies have been heralded as a game changer for blood cancers, but those therapies approved so far have been focused on a few subtypes of leukemia, lymphoma and myeloma. Now, a new strategy that leverages CRISPR to create CAR-Ts that target a ubiquitous antigen has shown potential to treat all forms of the three hematological diseases.
In an article published Aug. 31 in Science Translational Medicine, a research team from the University of Pennsylvania’s Perelman School of Medicine—co-led by CAR-T pioneer Carl June, M.D.—described how it developed CAR-T cells that target CD45, a protein found on the surface of nearly all blood cancer cells. A proof-of-concept study in cell cultures and mouse models showed that the anti-CD45 CAR-Ts could eliminate acute myeloid leukemia (AML) cells within three weeks.
“One drawback of the current approach to CAR-T cell therapy is that each therapy must be developed individually based on the targets for that cancer type,” June said in a press release. “This study lays the groundwork for a more universal approach that could potentially expand CAR-T cell therapy to all blood cancers.”
The six CAR-T cell therapies so far approved by the FDA have highly specific target antigens, like CD19 for B-cell leukemia and lymphoma, BCMA for myeloma and CD33 for AML. Targeting CD45, a pan-hematologic antigen—that is, one that is present across all blood cancer cells—has proven challenging, as it’s found on healthy blood stem cells and T cells, too.
Without a way to target anti-CD45 CAR-Ts at cancerous cells only, such a therapy risks causing the CAR-T cells to kill each other before they can be infused into the patient. And even if it did get past the first step, it could wipe out the patient’s blood cells completely.
To get around this, the researchers took an approach that’s “essentially a blood stem cell transplant paired with CAR-T cell therapy,” as lead author Nils Wellhausen put it in the release. Dubbed “epitope editing” by the researchers, the technique works by using CRISPR to edit a single nucleotide on the CD45 epitope, the portion where a CAR-T cell would bind, on both the CAR-T cells themselves and engineered blood stem cells that would be infused into the patient. That way, the patient’s new blood cells won’t be targeted by the anti-CD45 CAR-Ts.
The mutation is small enough that it doesn’t affect the function of CD45 but large enough that it makes non-cancerous cells unrecognizable to anti-CD45 CAR-T cells. Thus, when the engineered cells are engrafted, the anti-CD45 CAR-Ts kill the malignant cells, but not each other or the new blood stem cells, which begin making new blood cells, Wellhausen explained.
After observing in cell cultures that the anti-CD45 CAR-Ts could proliferate and were effective against AML cells, the researchers tested them in mice. They engrafted the AML cells from patients into mice, then treated them with either the edited CAR-Ts or control cells. Within three weeks, the leukemia cells were eradicated, and 75% of the treated mice lived to the end of the 65-day experiment. In contrast, all of the mice in the control group died before Day 20.
The anti-CD45 CAR-Ts remained primed to fight cancer for the long term, too. When the surviving mice were again engrafted with leukemia cells three months after the initial dose, they remained tumor-free.
“This suggested that the [epitope-edited anti-CD45 CAR-T cells] could persist and maintain anti-leukemia activity with long-term immunosurveillance,” the researchers wrote in the paper.
To show that the anti-CD45 CAR-Ts could also work against other types of blood cancers, the researchers cultured them with B-cell lymphoma, AML and T-cell acute lymphoblastic leukemia cells. For comparison, they cultured the cancer cells with anti-CD33 or anti-CD19 CAR-Ts as well. The scientists found that while these CAR-Ts could only target cancer cells with their specific lineage, the anti-CD45 CAR-Ts worked against all of them.
Next, the team looked at whether the epitope-edited blood stem cells would be both functional and protected from anti-CD45 CAR-Ts. After finding that they were capable of proliferating both in the lab and in mice, they engrafted another set of mice with either the edited stem cells or unedited stem cells, then followed up with a dose of the anti-CD45 CAR-Ts. The edited blood stem cells survived, while the unedited ones were eliminated by the CAR-Ts.
Finally, the team put it all together. They engrafted either edited or unedited blood stem cells in mice, injected them with AML cells, and treated them with the anti-CD45 CAR-Ts. Mice with the unedited blood stem cells quickly developed leukopenia, or a condition where the body doesn’t have enough immune cells; the mice with the edited stem cells did not. Meanwhile, the treatment killed off the cancerous cells and improved survival in both groups.
The results come with a few caveats, namely due to the nature of the models. For instance, because the amino acid sequences in the edited epitope differ between mice and humans, testing in nonhuman primates will be necessary to know whether epitope editing affects the function of the infused blood stem cells, the scientists noted in the paper.
“Ultimately, the safety and efficacy of this approach needs to be tested in human clinical trials,” they wrote. To this end, the team is currently conducting additional studies in preparation to request FDA permission to get their program into the clinic.