#303 PiggyBac transposon-mediated HER2-CAR-T cells exert anti-tumor efficacy against cholangiocarcinoma.
Koichiro Shioya1, Motoya Mie, Yuji Mishima, Tomio Matsumura, Yuta Ohira,, Miyuki Tanaka3, Shigeki Yagyu4, Yozo Nakazawa3, Lilin Zhang1**
1; BrightPath Biotherapeutics Co., Ltd., Tokyo, Japan, 2;Department for the Promotion of Regional Medicine, Shinshu University School of Medicine, Matsumoto, Japan. 3;Center
for Advanced Research of Gene and Cell Therapy, Shinshu University, Matsumoto, Japan. 4;Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.

#303
Introduction
Cholangiocarcinoma (CCA) is an aggressive
cancer often diagnosed at a late stage, posing
challenges in finding effective treatments for mostpatients and results in poor prognosis. HER2
overexpression
hasbeenreportedincholangiocarcinoma, making it an attractive target
for the treatment. We have developed a non-viral
HER2-CAR-T cells (BP2301) using PiggyBac
transposon characterized by CAR-T cells
comprising abundance of stem cell memory-like Tcells (Tscm). Currently, a phase 1 clinical trial isongoing to evaluate the safety and efficacy ofBP2301 in HER2-expressing sarcoma andgynecologic malignancies. In the present study,
we evaluated the anti-tumor efficacy of BP2301
using a CCA xenograft model with patient-derived
cell transplantation.
1. piggyBac Transposon System isadopted for transducing in BP2301.

4. Although CCA cells (Sik01 cells) are expressing
HER2, these cells do not respond T-DM1.

5. BP2301 has potent killing activity against
Sik01 cells.

A) HER2 protein on SK-OV-3 or Sik01 cell surface wasdetected by flowcytometric analysis using anti-HER2 antibody.
B) Effects on cell growth were tested in the culture of Sik01
cells adding 1 – 10000 ng/mL T-DM1 or trastuzumab.

In vitro killing activity of BP2301 was evaluated using
Sik01 cells. The cancer cells were co-cultured with
BP2301 at an E:T ratio of 1:1 - 4:1 for killing assay
based on the cell impedance.

8. The tumor was collapsed and infiltrated by a larger
amount of CD8 positive cells.

6. BP2301 eradicated tumor and rejected re-challenge tumor in Sik01 xenograft model.
NOG mice were inoculated with 5 × 106 Sik01
cells on day 0. HBSS (Vehicle) or 3.6× 106 HER2CAR T cells (BP2301) were administeredintravenously on day 41, respectively (red arrow).
BP2301 treated Sik01 tumor bearing mice werere-inoculated with 5 × 106 Sik01 cells on day 90.

2. CAR comprises FRP5,
CD28 and CD3ζ.

The tumors from the BP2301 treated mice were excised on day 7
and 14 after dosing. The specimens were immunostained with antihCD3, anti-hCD4 and anti-hCD8a.

Conclusion➢ BP2301 showed potent anti-tumor activity ina CCA xenograft model, and the anti-tumor
activity is correlated with long-term
persistence of BP2301 after adoptive transfer.
➢ BP2301 exhibits remarkable homing andexpansion abilities in the tumor site.

3. BP2301 GMP manufacturing process was optimized.

7. BP2301 was detected in the peripheral blood of CR mice for up to 71 days after dosing.

➢ Our results provide support for the future
development of BP2301 as a potential
treatment option for CCA patients with HER2expression.

BP2301 in blood

Cells/100 μL

300200Acknowledgement
1000020406080

The blood from the BP2301 treated mice were collected and analyzed forpharmacokinetics on day 7, 14, 48, 55 and 71 after dosing. BP2301 wasdetected by flowcytometric analysis. Red dots indicate samples from CR mice.

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

Days post-dosing*Contact 1: shioya_k@brightpathbio.com, **Contact 2: zhang_l@brightpathbio.com