Chimeric antigen receptor T cell (CAR-T) therapies have revolutionized treatment for various blood cancers. When traditional treatments fail, CAR-T therapies provide a second highly effective option. They are improving care and building hope for many.
The therapies involve taking T cells from a patient’s own blood and genetically modifying them to express a chimeric antigen receptor (CAR) that recognizes a specific protein on the surface of cancer cells. The modified cells are then transfused back into the patients, where they multiply, traffic throughout the body, and target and kill the cancerous cells.
There are several CAR-T therapies approved to treat B cell malignancies like large B cell lymphoma, B cell acute lymphoblastic leukemia, and multiple myeloma. These therapies are incredibly effective at reducing cancer in even hard-to-treat cases.
For a while, that is. But over 60% of patients eventually relapse after B cell CAR-T therapy. The challenge is that the CAR-T cells decline rapidly after their initial proliferation. Once the cells are gone, so too is their treatment efficacy.
Researchers at Fred Hutch are tackling this issue. A central focus in Dr. Christopher Peterson’s lab in the Translational Science and Therapeutics Division, “is to identify and overcome barriers to CAR T-cell function.” Dr. Hans-Peter Kiem’s lab is also dedicated to innovating CAR-T cell approaches. Together they are working to develop new CAR-T cell designs with improved durability.
The design of the CAR impacts everything from target recognition to downstream cell signaling and cell persistence. CARs are composed of several protein parts, called domains, that each contribute to the protein’s overall function. For example, the antigen recognition domain dictates what protein to target while the activation domain instructs the T cell what to do once it binds its target. Prior research has suggested that the costimulatory domain is important for CAR-T cell durability. Several variants exist within each domain type, but the functional differences between some of these variants are poorly defined.
In a recent study published in Blood, researchers in the Peterson and Kiem labs engineered and evaluated a collection of 20 unique CARs with different combinations of hinge, transmembrane, and costimulatory domain variants. The CARs all shared the same antigen recognition domain – targeting CD20, a protein selectively expressed on B cells – and the same activation domain so that CAR-T cell function could be directly compared.
The team manufactured CAR-T cells from pig-tailed macaques for each of the 20 CAR designs. They began by characterizing CAR-T cell responses to CD20 (the target protein) exposure ex vivo. Immediately, the CAR-T designs with the MyD88-CD40 costimulatory domain stood out. They proliferated both with and without CD20 stimulation while other CAR-T cells grew only in the presence of CD20. They also had higher expression of activation markers and lower expression of exhaustion markers compared to the other CAR-T cells. Not only were the MyD88-CD40 CAR-T cells more abundant, but they were also more primed to attack.
To determine how these newly engineered CAR-T cells function and persist in living animals, the team administered a pool of all 20 of the new CAR-T cell designs back into the same pig-tailed macaques from which the cells were originally derived. They observed an expansion of overall CAR-T cells with a concurrent decline of B cells, suggesting the CAR-T cells were detecting and killing their target cells as hoped. The MyD88-CD40 CAR-T designs were the most abundant, both out-proliferating and outlasting the other CAR-T designs in the pool. Excitingly, the CAR-T cells were detectable in blood until necropsy in all 3 of the animals studied – a period of up to 100 days – and the MyD88-CD40 CAR-T cells were present in tissues throughout the body.
“We clearly show that this costimulatory domain confers superior proliferation, durability, and tissue trafficking,” commented Dr. Peterson. He is excited about further characterizing the efficacy of this CAR. “We predict that the increased proliferation and durability of this CAR will result in more efficient killing of antigen-expressing target cells, even when those targets are hard to see (i.e. the number of antigen molecules at the cell surface is low).” The Kiem lab plans to use these findings to improve the design for their in vivo generation of CAR-T cells.
Proliferation, efficient killing, trafficking, persistence. These are the characteristics of the ideal CAR-T cell therapy – one that builds a large army to find and kill cancer cells wherever and whenever they pop up.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Keith Jerome and Hans-Peter Kiem contributed to this research.
The spotlighted research was funded by the National Institutes of Health.
Maynard LH, Cavanaugh EJ, Zhu H, Starke CE, Doherty SM, Einhaus T, Pérez-Osorio AC, Stensland L, Blair C, Roche AM, Everett JK, Murnane RD, Hoffman M, Nelson V, Herrin S, Littlewood C, Camou K, Wilson E, Wessel C, Bushman FD, Jerome KR, Kiem H-P, Peterson CW. 2025. Pooled CAR-T screening in pig-tailed macaques identifies designs with enhanced proliferation, trafficking, and persistence. Blood. doi: 10.1182/blood.2025028683
Science Spotlight writer Ashley Person is a PhD candidate in the Cohn lab in the Vaccine and Infectious Disease Division at Fred Hutch. She studies how HIV-infected cells persist over time in people living with HIV on long term treatment.
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