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The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. The manufacturing process of CAR-T cells should be optimized to prevent early T cell exhaustion during expansion. Activation of T cells by monoclonal antibodies is a critical step for T cell expansion, which may sometimes induce excess stimulation and exhaustion of T cells. Given that piggyBac transposon (PB)-based gene transfer could circumvent the conventional pre-activation of T cells, we established a manufacturing method of PB-mediated HER2-specific CAR-T cells (PB-HER2-CAR-T cells) that maintains their memory phenotype without early T cell exhaustion. Through stimulation of CAR-transduced T cells with autologous peripheral blood mononuclear cell-derived feeder cells expressing both truncated HER2, CD80, and 4-1BBL proteins, we could effectively propagate memory-rich, PD-1-negative PB-HER2-CAR-T cells. PB-HER2-CAR-T cells demonstrated sustained antitumor efficacy in vitro and debulked the HER2-positive tumors in vivo . Mice treated with PB-HER2-CAR-T cells rejected the second tumor establishment owing to the in vivo expansion of PB-HER2-CAR-T cells. Our simple and effective manufacturing process using PB system and genetically modified donor-derived feeder cells is a promising strategy for the use of PB-CAR-T cell therapy., Competing Interests: S.H., M.K., and T.S. are employees of Brightpath Biotherapeutics, Co., Ltd. The other authors declare no competing interests., (© 2021 The Author(s).)

Ephrin type-B receptor 4 (EPHB4), expressed in tumors including rhabdomyosarcoma, is a suitable target for chimeric antigen receptor (CAR)-T cells. Ligand-independent activation of EPHB4 causes cell proliferation and malignant transformation in rhabdomyosarcoma, whereas ligand-dependent stimulation of EPHB4 induces apoptosis in rhabdomyosarcoma. Therefore, we hypothesized that ligand-based, EPHB4-specific CAR-T cells may kill rhabdomyosarcoma cells without stimulating downstream cell proliferation mechanisms. We developed novel CAR-T cells by targeting EPHB4 via EPHRIN B2, a natural ligand of EPHB4. The generation of EPHB4-CAR-T cells via piggyBac (PB) transposon-based gene transfer resulted in sufficient T cell expansion and CAR positivity (78.5% ± 5.9%). PB-EPHB4-CAR-T cells displayed a dominant stem cell memory fraction (59.4% ± 7.2%) as well as low PD-1 expression (0.60% ± 0.21%) after 14 days of expansion. The PB-EPHB4-CAR-T cells inhibited EPHB4-positive tumor cells without activating cell proliferation downstream of EPHB4, even after multiple tumor re-challenges and suppressed tumor growth in xenograft-bearing mice. Therefore, PB-EPHB4-CAR-T cells possess a memory-rich fraction without early T cell exhaustion and show potential as promising therapeutic agents for treating rhabdomyosarcoma and other EPHB4-positive tumors., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The importance of echocardiography is generally known for acute coronary syndrome. However, assessing cardiac biomarker elevation is important together with echocardiography as poor images are obtained in some cases and patients may show unstable angina or angina on effort without asynergy. We examined the usefulness of high sensitive troponin I (hs-cTnI) in 60 patients from October 2014. We performed hs-cTnI and echocardiography in 10 cases (acute myocardial infarction: 8, unstable angina: 1, angina on effort: 1) among those patients. In the 8 acute myocardial infarction cases, asynergy was noted in all cases on echocardiography, but CK, CK-MB, and H-FABP cardiac biomarkers showed mixed negativity and positivity. Also, the unstable angina and angina on effort did not show asynergy but hs-cTnI was positive, the case of unstable angina showed elevation over time, but the case of angina on effort did not show elevation with time. We suggest that echocardiography and hs-cTnI are useful for acute myocardial infarction, although care may be necessary when assessing ACS patients with no detected asynergy in echocardiographs or non-ACS patients with acutely elevated hs-cTnI). In addition, it was suggested that confirmation of a change over time of hs-cTnI is necessary in angina patients.