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103 Quality improvement of anti-CD38-JAK/STAT CAR-T cells by suppressing CD38 expression and inhibition of tyrosine kinase
  1. Yasunori Amaishi1,
  2. Izumi Maki1,
  3. Maiko Sugizaki1,
  4. Kenichiro Mihara2,
  5. Sachiko Okamoto1 and
  6. Junichi Mineno1
  1. 1Takara Bio Inc., Kusatsu, Shiga, Japan
  2. 2Fujita Health University, Toyoake, Aichi, Japan


Background CAR-T cell therapy has shown highly effective clinical results in several diseases, but further improvement is necessary to target a wider range of antigens and tumors. In particular, excessive activation of CAR-T cells leads to cell exhaustion and reduction of naive/memory T cells’ population, which are important for long-term immune response. Therefore, suppressing non-antigen-specific activity is necessary for CAR-T cell production. However, when targeting tumor-related antigens that are also expressed on T cells, CAR-T cells recognize the antigens on the T cells, resulting in fratricide, poor cell growth, differentiation, and exhaustion during cell production process.In this study, we investigated a method for producing CAR-T cells targeting CD38 antigen that is common to T cells and tumor cells. CD38 is a suitable target antigen for CAR-T cell therapy because it is highly expressed in lymphocyte malignant tumors including B-cell non-Hodgkin’s lymphoma and multiple myeloma. However, as it is also intermediately expressed in normal blood cells, unwanted activation of CAR-T cells may be caused.

Methods We tried to suppress the expression of CD38 in CAR-T cells by co-expressing CD38 siRNAs, and prevent activation during cell production by modifying the signal domain of anti-CD38-CAR to the newly developed JAK/STAT-CAR. JAK/STAT-CAR contains the intracellular domain of the IL-2 receptor β chain and the STAT3 binding motif, which have been shown to improve the proliferation of CAR-T cells and suppresses differentiation compared to conventional second-generation CAR-T cells.For further improvement, CAR-T cells were prepared in the presence of the tyrosine kinase inhibitor Dasatinib to suppress activation during the cell manufacturing process.

Results CD38 siRNA co-expressing CAR-T cells showed decreased expression of CD38 and exhaustion markers, and the further reduction of exhaustion marker expression was observed in JAK/STAT CAR-T cells. However, compared to CAR-T cells targeting other antigens, CD38-CAR-T cells tended to be more exhausted and differentiated. As Dasatinib treatment maintained a high proportion of naive/memory T cells and was able to suppress exhaustion, combination of these approaches (CD38 siRNA-expressing CD38-JAK/STAT CAR-T cells with Dasatinib treatment) showed long-term persistence of antitumor activity in in vitro re-challenge assay.

Conclusions CD38 siRNA co-expressing CD38-JAK/STAT CAR-T cells produced in the presence of a tyrosine kinase inhibitor are expected to be suppressed excessive activation and maintain long-term antigen-specific activity. This approach is also expected to be applied to other CAR-T cell therapies targeting tumor-related antigens expressed on T cells.

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