Recently, a paper published in Science Signaling reported that those previously neglected parts of CAR-T cells have a striking effect on the behavior of CAR-T when killing cancer cells, which even influences treatment safety and efficacy.

This neglected part is the T cell signaling unit in the CAR structure called the costimulatory domain. Two CAR-T cell products, Yescarta and Kymriah, approved last year for the treatment of leukemia and lymphoma represent two different form — CD28 and 4–1BB. It is worth noting that this latest study from Fred Hutch is the first in the industry to compare in detail two common CAR designs used in clinical applications.

Although CAR-T cell therapy has shown strong therapeutic potential in the field of malignant hematologic malignancies, so far, researchers have not fully understood the potential biological pathways that this breakthrough therapy can play an anti-cancer response. Understanding these pathways is important for designing future generations of CAR-T cells, which may help to reduce their side effects, prevent recurrence after treatment, and make them effective against more common cancers, such as solid tumors.

CARs are synthetic receptors that are part of engineered T cells aiming to help T cells accurately recognize cancer cells. It should be noted that the CAR structure in different CAR-T cells is different, and this may lead to differences in the safety and efficacy of cancer treatment. Fred Hutch’s team studied the differences between the two most commonly used co-stimulatory domains.

Or more specifically, they studied how these two CAR designs (called CD28 and 4–1BB) signal their T cell mobilization against cancer, and how they affect the behavior and effectiveness of T cells on human cancer cells in vitro and in mice.

As a result, the researchers found that both types of CAR triggered the same signaling pathway, but the timing and intensity of the signal varied, with the CD28 CAR design showing faster and stronger activity, and the 4–1BB CAR showing slower and gentler activation. Moreover, further testing in a mouse model of lymphoma showed that 4–1BB CAR is more effective at clearing cancer cells.


The researchers also found that:
1. A signaling protein called Lck in T cells is capable of modulating the intensity of T cell responses in the CD28 CAR design, and researchers can manipulate it to slightly tune the response of CD28 CAR.

2. 4–1BB CAR-T cells show higher expression of genes associated with T cell memory, suggesting that 4–1BB CAR signaling can produce T cells that last longer and are able to maintain their anticancer effects.

In addition, the study used mass spectrometry to perform a comprehensive analysis of proteins involved in T cell signaling, which is unprecedented, because the co-author, Dr. Amanda Paulovich of Fred Hutch, is an expert in the field. Paulovich said that he hopes to develop a more effective CAR-T cell therapy for patients by developing a targeted detection of phosphorylated proteins for T cell signaling to help advance the development of immunotherapy.

Develop a better CAR structure

Currently, Novartis and Gilead/Kite are actively seeking a wider range of hematologic indications for their CAR-T cell products, with Gilead preferring to challenge solid tumors. Although Gilead’s Yescarta uses the CD28 costimulatory domain, the company has reached a clinical trial collaboration with Pfizer to assess the safety and efficacy of the combination therapy between utomilumab (a fully humanized 4–1BB agonist monoclonal antibody) and Yescarta in patients with refractory large B-cell lymphoma. In preclinical models, this antibody has been shown to enhance T cell mediated immune responses.

In addition, at this year’s ASCO annual meeting, scientists from Beijing’s biotechnology startup, ImmunoChina Pharmaceutical, reported the results of clinical trials of 47 patients with B-cell acute lymphoblastic leukemia who underwent CAR-T therapy. This is the first time in the world to conduct clinical research on the clinical safety and efficacy of two different CAR molecules under the same process. All 28 patients underwent objective remission (ORR 100%) after receiving 4–1BB CAR-T treatment. Fifteen of the 17 patients who received CD28 CAR-T experienced objective remission (ORR 88%).

Moreover, the peak of CAR-T in 4–1BB peripheral blood is significantly higher than that in CD28 group, which may indicate a sustained effect. In terms of safety, CRS of grade III-IV occurred in 5 patients in the CD28 group.

While Gilead and Novartis maintain ongoing research on optimizing co-stimulatory molecules in CAR-T cells, academic laboratories like the Michel Sadelain Lab are also involved in this common pursuit. With all these concerted efforts, we believe that safer and more efficient CAR-T cell products will be designed in the near future.

Types of Cancer Immunotherapy
Non-antigen-specific and antigen-specific therapies are the two main types of immunotherapy, while the former includes nonspecific immune stimulation and immune checkpoint inhibitors, the latter includes adoptive cell transfer of autologous cancer-specific T cells and various therapeutic vaccination approaches. Both are intensively researched in the past decades as there is an urgent need to find a cure for cancer. Among all immune checkpoint inhibitors, TIGIT Inhibitor and Galectins Inhibitor recently interest an increasing group of researchers.

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