Preclinical findings from Stanford University may help explain the lack of efficacy with CAR T-cell therapies in some patients with large B-cell lymphomas. The researchers pinpointed the CD58–CD2 axis as a novel resistance mechanism, then figured out how next-generation CAR T cells could be engineered to overcome this issue.

Preclinical findings from Stanford University in California may help pinpoint why, in some patients with large B-cell lymphomas (LBCL), little efficacy has been seen with CD19-targeting chimeric antigen receptor (CAR) T-cell therapies. The data were presented by Robbie Majzner, MD, during the 2020 American Society of Hematology Annual Meeting, held virtually December 5–8.

Both axicabtagene ciloleucel (axi-cel; Yescarta; Gilead) and tisagenlecleucel (tisa-cel; Kymriah; Novartis) have “revolutionized the treatment” of LBCL, Majzner said, with 40% to 50% of patients responding completely and durably. That said, for the rest, “the outcome is really poor, with a median overall survival of less than 200 days. If we could just find the mechanisms of resistance and engineer around them, we'd see more long-term remissions.”

Collaborating with Stanford colleagues who developed the next-generation technology CAPP-Seq (CAncer Personalized Profiling by deep Sequencing), Majzner's group examined circulating tumor DNA (ctDNA) in samples from patients treated with axi-cel. They observed a common theme among those who responded only partially: aberrations in CD58, such as a K60E point mutation, or loss of CD58 protein expression, detected by immunohistochemistry.

That CD58 loss was observed upon disease recurrence, not before treatment, “suggests that it could be a marker of relapse,” said Marcela Maus, MD, PhD, of Massachusetts General Hospital Cancer Center in Boston. Pinpointing CD58 through ctDNA “validates this tool to discover and home in on candidate biomarkers,” she added.

Further probing their discovery, Majzner's team knocked out CD58 in a mouse model of B-cell leukemia. Three therapies were then assessed—two with the same CD19-targeting CAR constructs as axi-cel and tisa-cel, and a third outfitted with a CAR aimed at CD22. Across these studies, “we saw that the CAR T cells had reduced or ablated cytokine production and lost their ability to kill tumor cells,” Majzner said. “There were partial responses initially, but the disease came roaring back and the mice quickly died.” By contrast, mice with intact CD58 responded durably to treatment.

Next, the researchers turned their attention to CD58's ligand on T cells: CD2. A series of experiments indicated that “CD2 engagement by CD58 greatly enhances CAR T-cell function,” Majzner said. Mass spectrometry revealed that this interaction stimulates downstream proteins such as PAK2, which mediate cell adhesion and cytoskeletal rearrangements and are key in enabling tumor eradication by T cells.

The team began devising another way to turn on CD2 signaling—despite aberrant CD58—for full CAR T-cell activation. “First, we integrated a CD2 costimulatory domain in cis, within our CAR construct,” Majzner said. “We were heartened by in vitro data showing cytolytic activity in CD58 knockout lines. But in vivo, once again, these CAR T cells were unable to finish the job; we ended up seeing no prolongation of survival.”

Back to the drawing board: Rather than the CD2 domain being added directly in cis, it was incorporated in trans on a second CAR molecule. “Only when we adopted this setup did we achieve significant efficacy and improved survival,” Majzner said.

“This is a fascinating approach,” Maus remarked. “It would be very interesting to see if it holds true in other hematologic malignancies where CAR T cells have proven efficacy, such as multiple myeloma.”

To Marco Ruella, MD, of the University of Pennsylvania in Philadelphia, “the CD58–CD2 axis could be an important target for new CAR-T strategies.” Whether adding CD2 costimulation to CAR T cells is the optimal way to improve therapeutic response remains to be addressed in future trials, he added. –Alissa Poh

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