Hitomi Shimizu

Abstract A23: Anti-PD-1 antibody scFv producing recombinant Bifidobacterium exerts antitumor effect in a larger fraction of the treated mice compared to full-length anti-PD-1 antibody

Anti-PD-1 therapy has improved therapeutic outcomes of patients in multiple cancer types. However, the therapy has demonstrated clinical benefits in only a small fraction of patients. The reason of the limited response in clinical practice is not fully understood. In an aim to improve anti-cancer drug delivery and potency, we have been developing in situ delivery and production system (i-DPS) by modifying a non-pathogenic anaerobic bacterium, Bifidobacterium, which localizes and proliferates only in the hypoxic environment like solid tumors after intravenous administration, produces anticancer proteins, enzymes or other pharmacologically active molecules selectively at the tumor site. Here we present anti-human PD-1 antibody scFv producing i-DPS in cancer immunotherapy, which could be specifically delivered to and amplified only at the hypoxic sites of solid tumors. A series of in vitro assays has been performed to confirm the stable expression and secretion of anti-human PD-1 scFv by recombinant Bifidobacterium, the binding inhibition of PD-1/PD-L1 interaction and elevated IFN gamma production in mixed lymphocyte culture by anti-human PD-1 scFv secreted from recombinant Bifidobacterium. Anti-murine PD-1 scFv producing Bifidobacterium as surrogate systemically administered to the syngeneic mice model demonstrated significant tumor growth inhibition. Of particular interest, the suppression of tumor growth was observed in a larger fraction of the treated mice while the control anti-PD-1 antibody showed the effect on only a few mice. The analysis of tumor infiltrating lymphocytes and myeloid cells will be presented as well. Taken together, i-DPS for anti-PD-1 antibody provides a new promising immune-therapeutic modality to target hypoxic solid tumors and also provides a unique insight for antibody drug delivery in cancer immunotherapy. Citation Format: Koichiro Shioya, Li Wang, Tomio Matsumura, Hitomi Shimizu, Yasuyoshi Kanari, Yuji Seki, Yuko Shimatani, Shun’ichiro Taniguchi, Shiro Kataoka. Anti-PD-1 antibody scFv producing recombinant Bifidobacterium exerts antitumor effect in a larger fraction of the treated mice compared to full-length anti-PD-1 antibody [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A23.

Abstract A29: Anti-CTLA-4 antibody scFv producing recombinant Bifidobacterium secretes CTLA-4 blocker specifically inside hypoxic tumor and suppresses tumor growth in syngeneic mice model

Success of immune checkpoint antibody drugs has provided broad perspective in cancer immunotherapy. Anti-PD-1 antibody and anti-CTLA-4 antibody showed notable efficacy and the combination complimentarily enhanced antitumor benefit. Nevertheless, either single-agent therapy or combination therapy is facing immune-related adverse events (irAEs), which is major cause of treatment discontinuation. Also, still large fraction of cancer patients doesn9t respond to the antibody immunotherapy. Since non-specific systemic activation of normal immune system by the therapy is one of major reasons to cause the above problems, approaches to target the hypoxic condition of tumors may help increasing the drug9s tumor-targeting specificity, which results in minimizing the irAEs and improving the response rate in patients. In an aim to improve anti-cancer drug delivery efficacy, we have been developing in situ Delivery and Production System ( i -DPS) by modifying a non-pathogenic anaerobic bacterium, Bifidobacterium , which localizes and proliferates only in the hypoxic environment like solid tumors after intravenous administration, produces anticancer proteins, enzymes or other pharmacologically active molecules selectively at the tumor site. Here we present anti-human CTLA-4 antibody scFv producing i -DPS in cancer immunotherapy, which could be specifically delivered to and amplified only at the hypoxic sites of solid tumors. A series of in vitro assays has been performed to confirm the stable expression and secretion of anti-hCTLA-4 scFv by recombinant Bifidobacterium , the binding affinity of purified anti-hCTLA-4 scFv and the IC50 competing with hCTLA-4/hCD80 or hCD86 interaction as well. Anti-murine CTLA-4 scFv producing Bifidobacterium as surrogate systemically administered to the syngeneic mice model demonstrated significant tumor growth inhibition. Moreover, the combination therapy of anti-mCTLA-4 scFv producing Bifidobacterium and anti-murine PD-1 antibody was more effective than each of monotherapy. It is noteworthy that scFv was only detected in tumor tissue but not in blood proved by immunoprecipitation assay. Altogether, i -DPS for anti-CTLA-4 antibody provide a new promising immune-therapeutic modality to target hypoxic solid tumors and also provide a unique insight for antibody drug delivery in cancer immunotherapy. Citation Format: Koichiro Shioya, Shiro Kataoka, Li Wang, Tomio Matsumura, Hitomi Shimizu, Yasuyoshi Kanari, Yuji Seki, Yuko Shimatani, Minoru Fujimori, Shun9ichiro Taniguchi. Anti-CTLA-4 antibody scFv producing recombinant Bifidobacterium secretes CTLA-4 blocker specifically inside hypoxic tumor and suppresses tumor growth in syngeneic mice model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A29.

Abstract 282: Novel delivery system to bring immune checkpoint antibodies to tumor microenvironment

Introduction: Cancer immunotherapy by blocking immune checkpoint (ICP) pathways with antibodies, such as anti PD-1 antibodies (Nivolumab, Pembrolizumab) and anti CTLA-4 antibody (Ipilimumab), has been reported to be clinically effective for several solid tumors. However, when the antibodies are systemically administered, they exhibit immune-related adverse events (ir-AE), that is, serious autoimmune toxicities, probably because their immunosuppressive function through binding to ICP molecules cannot discriminate normal tissues from solid cancers. If it is possible to selectively block immune-suppressive pathways only at the tumor site, both more anti-tumor effects and less ir-AE will be expected. In this context, we have been trying to establish engineered Bifidobacterium, which is nonpathogenic anaerobic bacteria and selectively colonizes in the hypoxic tumors, to express and secrete scFvs for antibodies against ICP molecules. Materials and Methods: We transformed Bifidobacterium with expression vector encoding a single chain fragment of the variable region (scFv) for anti-murine PD-1 (mPD-1) antibody. The biological activity of the product was examined by binding ELISA, competitive ELISA and cell binding by FACS. The anti-tumor activity was assessed by i.v. administration with the engineered Bifidobactrerium to mice bearing murine colorectal tumor (CT-26). Results: First, we succeeded in establishing a Bifidobactrerium strain producing and secreting anti-mPD-1 scFv. Binding of the recombinant scFv to mPD-1 was observed by ELISA and FACS, and its inhibition ability against mPD-1/mPD-L1 interaction was also confirmed in the system of ELISA. Furthermore, tumor growth of CT-26 formed in the syngenic mice was markedly suppressed by i.v. treatment with the engineered Bifidobactrerium producing anti-mPD-1 scFv. The antitumor effect was comparable with that obtained by a commercially available anti-mPD-1 antibody that was i.t. injected. As expected, both the scFv and the recombinant bacteria were specifically detected at the tumor sites by immunohistochemistry and colony plating assay, respectively, 7 days after i.v. injection of the recombinant Bifidobacterium to tumor bearing mice. Conclusion: Thus, the engineered Bifidobacterium modified to produce and secrete scFv against ICP molecules will provide a new promising immune-therapeutic modality to target hypoxic solid tumors. The clinical application of the present study will be prospectively expected by referring the ongoing clinical trial of APS001F, a recombinant Bifidobacterium modified to express cytosine deaminase that converts 5-FC, a pro-drug to 5-FU specifically in tumor lesions. Citation Format: Koichiro Shioya, Koichi Koseki, Tomio Matsumura, Hitomi Shimizu, Yuko Shimatani, Shun9ichiro Taniguchi. Novel delivery system to bring immune checkpoint antibodies to tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 282. doi:10.1158/1538-7445.AM2015-282

Abstract A110: Tumor targeting therapy of solid tumors by engineered B. longum secreting human TNF-alpha.

Tumor necrosis factor alpha (TNF-α) is a multifunctional cytokine playing key roles in apoptosis and cell survival as well as in inflammation and immunity. Since the human TNF-α (hTNF-α) gene was cloned in 1984, a range of clinical trials using recombinant hTNF-α (rhTNF-α) has been set up against a variety of solid tumors, but most of the studies failed because of its instability in the body and severe adverse events. The current use of rhTNF-α in cancer is for the regional treatment of locally advanced solid tumors, and it has been demonstrated that rhTNF-α, by isolated limb perfusion setting, acts synergistically with cytostatic drugs on soft tissue sarcomas and metastatic melanomas, resulting in excellent clinical responses. Thus, TNF-α can be expected the great antitumor activity without adverse events when delivered only to the tumor sites. We previously reported that a strain of nonpathogenic and anaerobic bacteria, Bifidobacterium longum (B. longum), selectively localized to solid tumors and proliferated within these sites after the systemic administration. In this study, we developed engineered B. longum (3BTNF) transformed with the plasmid encoding secreting hTNF-α, and examined the characteristics of the strain from the point of views of molecular cell biology and antitumor efficacy. The engineered strain of B. longum, 3BTNF cells secreted high amount of the protein into the culture medium, and the molecular size was 17 kDa which was the same as that of natural hTNF-α. The crude protein showed a cytotoxic activity against tumor cell lines in vitro, and the activity was neutralized by the goat anti-hTNF-α. The protein was confirmed to induce apoptosis in tumor cell lines with co-existence of adriamycin (ADM) through the caspase-3-dependent pathway. In order to demonstrate the antitumor effect, it was examined that 3BTNF was administered intravenously twice weekly for 4 times (Days 1, 4, 8 and 11) with supplement of maltose into nude mice bearing human breast carcinoma KPL-1 (n=6). A significant antitumor effect was observed, i.e., tumor growth inhibition (T/C%) was 48.3% on Day 22 (p=0.021). When the 3BTNF treatment was combined with ADM (Days 0 and 7), a marked effect was obtained, i.e., T/C 12.2% (p=0.0001) in 3BTNF plus ADM group or 27.1% in ADM alone group, respectively. Against another human cancer cell line, colorectal carcinoma HCT 116 cell, obvious antitumor effects were also observed by the treatment with 3BTNF alone and in combination with chemotherapy. A meaningful amount of hTNF- (mean, 1.3 ± 1.5 ng/g tumor) was detected in the tumor tissue in HCT 116 bearing mice, concomitantly detected with a large number of live 3BTNF (mean, 1.2E+7 ± 2.1E+7 cfu/g tumor) on Day 25. In contrast, hTNF-α in the serum was not detected. No serious side effect was noted after 3BTNF administration in these mice. Taken together, these findings indicate that the engineered B. longum secreting hTNF-α has attractive therapeutic potential as a new candidate of antitumor modality targeting hypoxic solid tumors. A part of this study was supported by the grant from NEDO (New Energy and Industrial Technology Development Organization). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A110.