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129 A novel CAR conducting antigen-specific JAK-STAT signals demonstrates superior antitumor effects with minimal undesired non-specific activation
  1. Sachiko Okamoto1,
  2. Yasunori Amaishi1,
  3. Mitsuki Shigeta1,
  4. Yu Okubo1,
  5. Yota Ohashi2,
  6. Naoto Hirano2 and
  7. Junichi Mineno1
  1. 1Takara Bio Inc., Kusatsu, Shiga, Japan
  2. 2Princess Margaret Cancer Centre, Toronto, Canada


Background Despite recent impressive successes in chimeric antigen receptor (CAR)-T cell therapy, there are still considerable clinical challenges. To improve T cell persistence and antitumor effect, which are critical for clinical responses, various efforts have been made to optimize the CAR design such as the inclusion of a costimulatory domain(s). It is known that non-specific activation of CAR-T cells is greatly influenced by the CAR design, and excessive T cell activation leads exhaustion of T cells and depletion of naïve/memory subsets important for durable clinical responses. Thus, the CAR construct needs to be optimized so that transduced T cells persist and induce potent antigen-specific response with reduced non-specific activation. For optimal T cell activation and proliferation, three signals including TCR (signal 1), co-stimulatory (signal 2), and cytokine (signal 3) signals, are essential. The conventional second and third generation CARs containing CD3ζ and a co-stimulatory domain such as a signal domain of CD28 and 4-1BB can conduct signal 1 and 2, but not signal 3. Recently, we have developed a new generation JAK-STAT CAR composed of a truncated cytoplasmic domain of the IL-2 receptor β chain and STAT3/5 binding motifs, CD28 co-stimulatory domain, and CD3ζ domain. The novel anti-CD19 JAK-STAT CAR-T cells showed antigen-specific activation of the JAK-STAT signaling pathway, enhanced proliferation, and limited terminal differentiation in vitro compared to second generation 28ζ CAR or 4-1BBζ CAR-transduced T cells. Furthermore, the anti-CD19 JAK-STAT CAR-T cells demonstrated superior in vivo persistence and antitumor effect in mouse models.1 In addition, we previously showed that a hinge region and the composition of a single chain variable fragment (scFv) such as the order of VH and VL regions critically influence not only antigen-dependent activation but also undesired antigen-independent activation known as tonic signaling.2

Methods In this study, we have optimized the scFv design in 28ζ CAR and JAK-STAT CAR constructs to show superior antigen-specific activation and reduced tonic signaling for several targets (CD19, CD20, Mesothelin, and GD2). And we have evaluated the feature of JAK-STAT CAR-T cells compared to 28ζ CAR-T cells.

Results JAK-STAT CAR-T cells showed superior antigen-specific proliferation with less differentiated status, whereas 28ζ CAR-T cells showed antigen-independent proliferation and displayed higher exhaustion marker expression after repetitive stimulations.

Conclusions These results suggest that our JAK-STAT-CARs with enhanced antigen-specific response with minimized tonic signaling targeting various antigens has the potential to demonstrate improved clinical efficacy.


  1. Kagoya Y, et al. A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects. Nat Med 2018;24:p352–359.

  2. Okamoto S, et al. Detail analysis of non-specific activation of CD19 CAR-T cells caused by CAR design. ASGCT ( 2015)

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