Published in The Journal for the ImmunoTherapy of Cancer: Persistence and CAR-T Product Optimization with Single-Cell Functional Proteomics

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 h1906BBzthe 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.

Want to learn more? Speak to an expert to see for yourself how IsoPlexis can accelerate your research.


  1. 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



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