January 22, 2021 -- A new chimeric antigen receptor (CAR) T-cell system that targets two tumor antigens may be effective at treating difficult solid tumors in children in cases where other CAR T therapies have not been effective. Findings from the preclinical evaluation of the novel CAR system were published in Nature Communications on January 21.
CAR T-cell therapy has revolutionized the treatment of B-cell malignancies, but the therapy has not been effective for treating solid tumors such as childhood neuroblastoma. One of the barriers to developing effective CAR T therapies for solid tumors is toxic off-tumor side effects where tumor-associated antigen expression in normal tissues can have devastating effects.
To counteract this in CAR T therapies for adults, researchers have designed terminating switches that when activated efficiently eliminate CAR T cells but also result in reduced efficacy of the therapy.
"CAR T therapy is incredibly powerful, but for solid tumors it has significant barriers," said first author Dr. Babak Moghimi, physician at Children's Hospital Los Angeles and associate professor at the Keck School of Medicine of the University of Southern California, in a statement. "We needed a way to boost the CAR T-cells to make them fight harder and smarter against the cancer. But we also want to save brain cells and other healthy tissue."
A novel approach to CAR T therapy design is the use of synthetic notch (SynNotch) as a gating strategy, where expression of a chimeric antigen receptor for one tumor-associated antigen is dependent on a signal from another tumor-associated antigen. The gate is created by fusing a single-chain variable fragment (scFv) directed against a tumor-associated antigen to a SynNotch receptor. When the nongated CAR binds to the first tumor-associated antigen, the SynNotch site is cleaved and induces the gated expression of a CAR against the second tumor-associated antigen.
In this system, the expression of the second CAR is dependent on the gate and CAR-T cytotoxicity is dependent on the presence of the second tumor-associated antigen. The SynNotch system spatially and temporally separates activation signals, to eliminate the risk of partial activation, according to the authors.
To determine if the SynNotch design could generate specific and efficacious CAR T-cells against neuroblastoma, researchers from Children's Hospital Los Angeles built a gated CAR T-cell targeting neuroblastoma and evaluated its safety, specificity, and efficacy against neuroblastoma cell lines and mouse models.
First, studies in preclinical in vitro mouse models conducted by the team found that costimulatory domains, which are secondary signals that generate immune responses in the presence of an antigen-presenting cell, can create unpredictable toxicity and provided the impetus to evaluate gated disialoganglioside (GD2) CAR systems in neuroblastoma mouse models, specifically those directed against GD2 as the gate and B7H3 as the target.
GD2 is a highly expressed tumor-associated antigen found on neuroblastomas, so the researchers chose it as the gating tumor-associated antigen in the construction of the SynNotch receptor. Furthermore, B7H3 (CD276) is an immune checkpoint molecule expressed at high levels on several adult and pediatric solid tumors. Human B7H3 CAR T cells exhibit efficacy in preclinical models of neuroblastoma, so it was chosen as the target tumor-associated antigen for the CAR construct.
The researchers tested the newly constructed GD2-B7H3 CAR T cells (GD2 as SynNotch gate for B7H3 CAR) with a number of in vitro and in vivo preclinical tests to demonstrate that the cells exhibited greater metabolic fitness, a lower exhaustion profile, and superior in vivo anti-tumor efficacy after repeated in vitro stimulation compared to conventional B7H3 CAR T cells.
"With normal CAR T therapy, the CAR T-cells burn out and are no longer active after some time," said Dr. Shahab Asgharzadeh, a physician scientist at the Cancer and Blood Disease Institute of Children's Hospital Los Angeles. "But we discovered the synNotch CAR T-cells are more metabolically stable because they are not activated constantly."
This means the CAR T cells use less energy, which allows them to continue to fight the cancer for a longer period of time.
Importantly, the researchers found that GD2- B7H3 T cells (B7H3 SynNotch controls the expression of GD2 CAR) prevented disease progression in xenografted metastatic neuroblastoma mice and none of the mice exhibited evidence of neurotoxicity. They attributed this to decay of CAR expression post infiltration into the central nervous system.
Overall, the researchers favored the alternative design with GD2 as the gate because B7H3-GD2 T cells can still infiltrate the central nervous system, whereas there was limited evidence of this in the GD2-B7H3 construction.
"The way it works is really unique," explained Moghimi.
The researchers hope to continue to explore the possibilities of the SynNotch system, with particular interest in multiple infusions of gated CAR T cells and explaining possible tumor escape risks associated with the system.
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