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303 PiggyBac transposon-mediated HER2-CAR-T cells exert anti-tumor efficacy against cholangiocarcinoma
  1. Koichiro Shioya1,
  2. Motoya Mie2,
  3. Yuji Mishima3,
  4. Tomio Matsumura1,
  5. Yuta Ohira1,
  6. Miyuki Tanaka4,
  7. Shigeki Yagyu5,
  8. Yozo Nakazawa4 and
  9. Lilin Zhang1
  1. 1BrightPath Biotherapeutics Co., Ltd., Tokyo, Japan
  2. 2BrightPath Biotherapeutics Co., Ltd., Chiyoda-ku, Japan
  3. 3BrightPath Biotherapeutics Co., Ltd., Kawasaki, Kanagawa, Japan
  4. 4Shinshu University School of Medicine, Matsumoto, Japan
  5. 5Shinshu University Innovative Research and Liaison Organization, Matsumoto, Japan
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Cholangiocarcinoma (CCA) is an aggressive cancer often diagnosed at a late stage, posing challenges in finding effective treatments for most patients and results in poor prognosis. HER2 overexpression has been reported in cholangiocarcinoma, making it an attractive target for the treatment. We have developed a HER2-CAR-T cells (BP2301) characterized by CAR-T cells comprising abundance of stem cell memory-like T cells (Tscm). In the present study, we evaluated the anti-tumor efficacy of BP2301 using a CCA xenograft model with patient-derived cell transplantation.

Methods BP2301 were manufactured by simultaneously transfecting HER2-CAR and PB transposase expression vectors into peripheral blood mononuclear cells (PBMC) using electroporation. BP2301 was expanded with UV-inactivated antigen presenting autologous PBMC expressing HER2, CD80 and 4–1BBL. The efficacy of BP2301 in treating CCA was evaluated using Sik01, a CCA patient-derived cell line with moderate HER2 expression that did not respond to Trastuzumab emtansine (T-DM1). In vitro killing activity of BP2301 for Sik01 tumor cells was evaluated by xCELLigence. To evaluate in vivo anti-tumor efficacy, BP2301 was administered intravenously into Sik01 tumor xenograft model. The blood and tumors from these mice were collected and analyzed for pharmacokinetics and biodistribution of BP2301 by flowcytometric analysis or immunohistochemistry.

Results In vitro study demonstrated that BP2301 exhibited killing activity against Sik01 cells. A single administration of BP2301 eradicated Sik01 tumor in 2 out of 5 mice, and these mice rejected Sik01 cell rechallenge. The pharmacokinetics of BP2301 was analyzed by flowcytometry and we found that BP2301 expanded in the mice and reached to the highest cell number on day 14 post-administration. In the mice whose tumors completely regressed and rejected the second tumor rechallenge, BP2301 could be detected until 71 days after dosing. Pathological and immunohistochemistry examinations revealed extensive infiltration of human CD8 positive cells into Sik01 tumor and tumor destruction after BP2301 administration.

Conclusions In this study, we have demonstrated the potent anti-tumor efficacy in a patient derived CCA cell transplantation model. Our findings indicate that the antitumor activity of BP2301 is correlated with its long-term persistence after adoptive transfer. Additionally, the study revealed that BP2301 exhibits remarkable homing and expansion abilities in the tumor site. Currently, a phase 1 clinical trial is ongoing to evaluate the safety and efficacy of BP2301 in HER2-expressing sarcoma and gynecologic malignancies. Our results about anti-tumor efficacy against CCA provide support for the future development of BP2301 as a potential treatment option for CCA patients with HER2 expression.

Acknowledgements The authors are grateful to Dr. Seiji Okada, professor of Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection and Graduate School of Medical Science, Kumamoto University, for providing Sik01 cells for this study.

Ethics Approval All animal experiments were performed following approval (#AIA230035–001) by the Institutional Animal Care and Use Committee of Central Institute for Experimental Animals (Matsumoto, Japan).

All human materials were handled following approval (#BP20190830) by the Medical and Genomic Research Institutional Review Board of BrightPath biotherapeutics Co., Ltd. (Tokyo, Japan).

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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