Chimeric Antigen Receptors (CARs) are hybrid antigen receptors composed of an extracellular antigen-binding domain, hinge, and transmembrane, as well as intracellular activation motifs. When this activation domain contains more than one co-stimulatory domain, it is known as a third-generation CAR.
The most common co-stimulatory domains are CD28 or 4-1BB derived. CD28-based CARs have faster cytotoxicity and improved antitumor efficacy, while 4-1BB-based CARs show increased persistence and decreased exhaustion, favoring longer-term CAR T cell persistence in patients.
In a recent study published in Journal for ImmunoTherapy of Cancer1, researchers wanted to determine if a third-generation CAR containing 4-1BB and a mutated CD28 [with only PYAP signaling motif] (mut06) would provide beneficial aspects of both cytotoxicity and persistence. The team designed five CD19 specific CAR-T cells with either 4-1BB or mut06 together with the combination of both and evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells.
CAR-T cells were stimulated with target cells at a 5:1 E:T ratio. For several experiments, total CAR-T cells were plated and analyzed with IsoPlexis’ IsoLight instrument; a hub for comprehensive functional profiling of each cell type across a large assay menu of single-cell chip and software. The team used the IsoPlexis highly multiplexed proteomic assay to analyze hundreds of CAR-T cells at the single-cell level for frequency and intensity of 28 secreted cytokines.
Combined third-generation CAR-T cells showed improved antitumor activity, persistence, and polyfunctionality
The combined third-generation CAR-T cells showed increased central memory phenotype, polyfunctionality, expansion and cytotoxicity. Both CAR-Ts had increased LCK recruitment, but differences in LCK phosphorylation suggest that LCK recruitment is dependent on the positioning of the two co-stimulatory domains, the authors wrote.
These differences also pointed to the following conclusions:
- h19BB06z (4-1BB proximal to cell membrane): greatest cytotoxicity, significantly higher secretion of IL-2, significant secretion of effector cytokines TNFa, IFNy
- h1906BBz (mut06 proximal to cell membrane): lower proliferation, cytotoxicity, and IL-2 production when compared to h19BB06z
The bi-specific CAR targeting CD19/CD20, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers. The bi-specific CAR-T cells also exhibited improved in vivo antitumor activity with increased persistence and decreased exhaustion in Raji and Nalm6 studies.
In addition, h19BB06z CAR-T cells (highest PSI subset) were better able to control tumor growth compared to untreated and h1906BBz—the latter only showing a slight increase in CAR+ cells and slight decrease in CD19+ cells.
The results obtained from the IsoPlexis platform indicated that the combined third-generation CARs H1906BBz (mut06 domain proximal to the cell membrane), and H19BB06z (4-1BB proximal to the cell membrane) exhibited the highest PSI. By combining 4-1BB and mut06, CAR-T cell polyfunctionality was enhanced. In addition, BB06z CAR-T cells (highest polyfunctionality and number of cytokines secreted) demonstrated the greatest performance against Raji tumor cells.
Single-cell proteomics reveals high quality CAR-T cells associated with improved anti-tumor response
The results from this study revealed that co-stimulation, combining 4-1BB with an optimized form of CD28, is a promising approach to optimize CAR-T cell function. In addition, IsoPlexis’ single-cell technology demonstrated that highly polyfunctional CAR-T cells performed the greatest against tumor cell lines.
“The ability to produce a wider range of cytokines at a single-cell level is associated with an improved clinical response,” the authors stated.
This study, together with work that is currently being performed, demonstrates that the activity of CAR-T cells can be enhanced by fine-tuning the signaling domains, specifically, by combining 4-1BB and an optimized form of CD28 co-stimulation. Plans to evaluate the clinical relevance of these findings in patients with cancer are currently underway.
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- Roselli E, Boucher JC, Li G, et al. 4-1BB and optimized CD28 co-stimulation enhances function of human monospecific and bi-specific third generation CAR T cells. Journal for ImmunoTherapy of Cancer 2021;9:e003354. doi:10.1136/ jitc-2021-003354