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Anti-cancer therapies targeting EGFR family, including monoclonal antibodies and kinase inhibitors, are the state of art for solid tumors such as colorectal and non-small cell lung cancers. T cell redirecting bispecific antibodies are a promising approach for cancer immune therapy, whereas their application is limited to hematological malignancy due to adverse events caused by the systemic exposure to highly immunologically active molecule. To avoid the immunological adverse events, we have created a recombinant Bifidobacterium (APS002) that specifically colonizes in solid tumor and continuously secretes T cell redirecting antibody. The antibody was designed to form a diabody based on the sequence of dual specific antibody for EGFR/HER3 and CD3. The diabody (termed DBERB1) comprises the equal amount of two polypeptides, anti-h CD3 (VL) - anti-EGFR/hHER3 (VH) and anti-hEGFR/hHER3 (VL) - anti-hCD3 (VH), respectively. DBERB1 bound to human EGFR and HER3, and human CD3. DBERB1 demonstrated potent cytotoxic activities against EGFR- and HER3-expressing human colon cancer HCT116 cells in the presence of human peripheral blood mononuclear cells (hPBMC) with EC50 values of 3 - 9 × 10−12 M. When APS002 was i.v. dosed to HCT116 tumor-bearing SCID mice inoculated with hPBMC, APS002 colonized only in the tumor and DBERB1 was detected in the tumors but not in the plasma. The growth of HCT116 tumors was significantly suppressed by APS002 but not by Bifidobacterium transfected with control vector. In conclusion, our study in the mouse model indicates that APS002 is a novel and an extremely potent anti-cancer agent distributed only in the tumor. We are currently preparing for pre-clinical studies for the future clinical trials. Citation Format: Satoshi Kobayashi, Koichiro Shioya, Yuji Seki, Tomio Matsumura, Yasuyoshi Kanari, Yuko Shimatani, Hikaru Nakazawa, Mitsuo Umetsu, Shiro Kataoka, Takaaki Nakamura. Anti-tumor activity of Bifidobacterium secreting dual specific T cell redirecting antibody against EGFR/HER3-expressing cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 689.

Agonistic antibodies against immune checkpoint molecules, such as 4-1BB, reportedly demonstrate antitumor effects at the early clinical phase, whereas immune-related adverse events hamper further development. A single chain variable fragment (scFv), which is expected to have better penetration into tissues, is clinically being investigated, however, poor distribution into tumor tissues due to rapid clearance is a serious issue. In order to improve drug delivery and decrease adverse events, we have developed in situ Delivery and Production System (i-DPS) by genetically engineering a non-pathogenic anaerobic Bifidobacterium. We previously reported that the modified Bifidobacteria localized and proliferated only in the hypoxic area of the tumor engrafted in the mouse post i.v. injection, thus producing anti-tumor molecules persistently and selectively at the tumor site (AACR2010). Here we present i-DPS with agonistic anti-murine 4-1BB scFv. We created a recombinant Bifidobacterium producing and secreting anti-murine 4-1BB scFv. The anti-murine 4-1BB scFv increased IFN-γ production by 171% over the control group (PBS(-)) in the mouse splenocytes activated by ant-CD3 antibody in vitro. Anti-tumor effects of Bifidobacteria producing the anti-murine 4-1BB scFv were demonstrated in the syngeneic model of CT-26 in Balb/c mouse (61.7% tumor growth inhibition (TGI) at 1×109 cfu/mouse, i.v. on day 14). We further evaluated the anti-tumor effects of i-DPS for anti-murine 4-1BB scFv (1×109 cfu/mouse, i.v.) in combination with PD-1 antibody (i.p.), and confirmed that the combination therapy significantly suppressed the tumor growth on day 21 (TGI: combination, 60.7%; anti-murine 4-1BB scFv, 38.8%; anti-mPD-1 mAb, 8.9%). In conclusion, i-DPS for anti-4-1BB scFv will provide a new promising modality for the immune-therapy targeting hypoxic solid tumors. Citation Format: Tomio Matsumura, Koichiro Shioya, Yasuyoshi Kanari, Yuko Shimatani, Shiro Kataoka, Shun'ichiro Taniguchi, Takaaki Nakamura. Cancer immunotherapy with agonistic anti-4-1BB scFv producing and secreting Bifidobacterium in syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2735.

Introduction:FF-10502-01 is a synthetic pyrimidine nucleoside analogue structurally similar to gemcitabine (gem) with a substitution of sulfur for oxygen in the pentose ring. The anti-tumor effects of FF-10502-01 were studied in solid tumor cell lines and human tumor xenograft models. Methods:10 human solid tumor cell lines were studied and included the following: 4 pancreatic (PANC) (BxPC-3, SUIT-2, Capan-1 and MIA PaCa-2), colon (HCT116), lung (NCI-H460), ovary (SK-OV-3), prostate (LNCap.FGC), breast (MDA-MB231) and mantle cell lymphoma (MCL) (Jeko-1). The effect of FF-10502-01 on DNA synthesis versus gem was evaluated in a PANC cell line (Capan-1). Tumor growth suppression was evaluated in human PANC xenograft models, SUIT-2 and Capan-1. FF-10502-01 or gem was administered at 240 or 480 mg/kg once a week starting 7d after orthotopic transplantation for 18 weeks, n=20/grp. Animals were followed for 128d after transplantation. Capan-1 was transplanted SC into nude mice (n=8/grp). FF-10502-01 or gem was administered at 120, 240, 360 or 480 mg/kg once weekly x 4 weeks 7d after transplantation. Tumor growth inhibitory effects were evaluated for 27d after starting treatment. FF-10502-01 single-dose pharmacokinetics (PK) were investigated in the mouse, rat and dog. Results:FF-10502-01 demonstrated in vitro activity against the PANC, colon, lung, ovarian, prostate and MCL cell lines, with IC50s of 30 – 330 nM. The activity was 1.5 – 15-fold lower than gem, however, FF-10502-01 demonstrated a 23-fold higher inhibition of DNA α-polymerase-mediated synthesis over gem. In the SUIT-2 orthotropic implant model, both gem and FF-10502-01 improved survival over vehicle controls. Med survival was not reached for the 480 mg/kg gem grp or either the 240 or 480 mg/kg FF-10502-01 grps. The survival rate was 5%, 25%, 75% for the vehicle, 240 and 480 mg/kg gem grps (p PK following single bolus IV doses indicated a similar PK profile to other pyrimidine nucleoside analogues, including gem. Conclusions:FF-10502-01 is a new pyrimidine nucleoside analogue with demonstrated preclinical efficacy against solid tumors and a potentially more tolerable safety profile than gem. Citation Format: Shinji Mima, Hiroki Nishikawa, Shinichi Watanabe, Tamami Higuchi, Takeaki Suzuki, Hiroyuki Iwamura, Chihaya Kakinuma, Takaaki Nakamura, Yasuhiro Shimada. In vitro and in vivo evaluation of FF-10502-01, a new pyrimidine nucleoside analogue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5127. doi:10.1158/1538-7445.AM2017-5127