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Development of piggyBac transposon-mediated HER2-CAR-T cells for the treatment of solid tumors
1*11123431**
Koichiro Shioya , Tomio Matsumura , Yuta Ohira , Naomi Komatsuzaki , Manaka Shinagawa , Miyuki Tanaka , Shigeki Yagyu , Yozo Nakazawa , Lilin Zhang

1; BrightPath Biotherapeutics Co., Ltd., Tokyo, Japan, 2;Department of Obstetrics and Gynecology, Shinshu University School of Medicine, Matsumoto, Japan,3;Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan. 4;Center for Advanced Research of Gene and Cell Therapy, Shinshu
University, Matsumoto, Japan. *Contact 1: shioya_k@brightpathbio.com, **Contact 2: zhang_l@brightpathbio.com

4. BP2301 exhibited memory-like phenotype 1)

Introduction
Although chimeric antigen receptor (CAR)-T
therapies have achieved remarkable success inthe treatment of hematologic malignancies, theoutcome for patients with solid tumors remainspoor. There are several reasons behind this,including exhaustion of CAR-T cell, poor homing
and penetration in the tumor, and the lack ofpersistence in the immunosuppressive tumor
microenvironment. To solve these problems, wehave developed HER2-CAR-T cells (BP2301)
using the piggyBac (PB) transposon-based genetransfer system.
1. piggyBac Transposon System

2. CAR structure

6. BP2301 eradicated rhabdomyosarcoma andrejected re-challenged tumor in xenograft model 1)

8. Study design for Phase 1 clinical trial(BP2301-001)
3 + 3 Dose-escalation Design (N=12)
2.7 × 106
cells/kg

8.3 × 105
cells/kg

Lymphodepletion:
3 days regimen (Flu 25 mg/m2 + Cy 250 mg/m2)➢Primary objective :・ Safety and tolerability➢Secondary objective : ・ Expansion and persistence of BP2301
・ Efficacy of BP2301
HER2-CAR was detected by recombinant human ErbB2/HER2-Fc chimera
protein with a goat anti-human IgG Fc fragment-specific antibody. Antibodies ofCD4, CD8, CD45RA, CCR7, PD-1, TIM-3, LAG3 and CD3 were used for thecharacterization of HER2-CAR+ cells.

5. BP2301 showed persistent cytotoxicity
against HER2+ sarcoma in a serial killingassay 1)Anti-tumor effect of BP2301 was evaluated in SJCRH30-FFluc xenograft model.
NSG mice were inoculated with 1 × 106 SJCRH30-FFluc cells on day 0. Vehicle,
PB-CD19-CAR-T cells or BP2301 ( 6 × 106 CAR+ T cells) were i.v. administered one
week after inoculation. Tumor burden was measured as bioluminescence signal
intensity (BLI) and presented as total flux (p/s). On day 28, 1 × 106 SJCRH30-FFluccells were re-inoculated onto the mice for tumor re-challenge experiment. Rechallenged tumors are indicated by yellow arrowheads.

7. BP2301 eradicated inoculated tumor in anovarian cancer xenograft modelA➢Key inclusion criteria➢Key exclusion criteria

・Recurrent or advanced
Osteosarcoma, Soft tissue
sarcoma, Gynecologicalcancer
・5 to 65 years old・KPS ≥ 50%
・Adequate organ function
・HER2 expression > 1+・Active infection
・Central nervous systemdiseases・Significant autoimmune
diseases・PregnancyConclusion➢ A non-viral GMP manufacturing process of
BP2301 has been optimized using piggyBac
transposon system with AP cells.
B

3. BP2301 GMP manufacturing process➢ BP2301 exhibits stem cell memory phenotype
and no significant T-cell exhaustion markers.

➢ BP2301 showed potent anti-tumor activity invitro and in vivo.
A) in vitro cytotoxicity analysis of BP2301 was evaluated using sarcoma celllines. Tumor cells were seeded on xCelligence E-plates 16 at 0.5-2 × 104
cells/well for 18-24h and then BP2301 or CD19-CAR-T cells (negative control)
were added at an E:T ratio of 1:1, then real-time impedance was measured for
72 h and presented as the normalized cell index using an xCELLigence RTCA
DP system.
B) in vitro anti-tumor activity of BP2301 was evaluated in a serial killing assay.
BP 2301 was incubated with U-2OS at an E:T ratio of 1:1 for 72 h, and thenwas collected, incubated with U-2OS again for the next round of killing. Cellimpedance was measured for 72h in each round by an xCELLigence RTCADP system.

A) in vitro cytotoxicity analysis of BP2301 was evaluated using gynecologicalcancer lines. The cancer cells were incubated with BP2301 at an E:T ratio of 1:1for killing assay based on cell impedance. T cells derived from same donor wereused as negative control.
B) Anti-tumor effect of BP2301 was evaluated in SK-OV-3 xenograft model. NSG
mice were inoculated with 2 × 106 SK-OV-3 cells on day 0. Vehicle or BP2301 (2× 106 CAR+ T cells ) were i.v. administered on day 11, 14 and 18, respectively(red arrows).

➢ A Phase 1 clinical trial for patients withrecurrent or advanced osteosarcoma, soft
tissue sarcoma and gynecological cancer isscheduled in the second quarter of 2022.
References
1) Nakamura K., et al. Autologous antigen-presenting cells efficiently expand piggyBac
transposon CAR-T cells with predominant memory phenotype, Mol Ther Methods Clin
Dev. 21 315-324, 2021.