Abstract 1795: Tumor-targeting Salmonella A1-R selectively delivers recombinant methioninase and inhibits syngeneic-cancer mouse models
Authors: Kubota Yutaro , Aoki Yusuke , Masaki Noriyuki , Obara Koya , Morinaga Sei , Mizuta Kohei , Sato Motokazu , Zhao Ming , Han Qinghong , Michael Bouvet , Tsunoda Takuya , Hoffman Robert M. 
Published: March 2024

Abstract
Background: Methionine addiction, termed the Hoffman effect, is a general hallmark of cancer. Methionine restriction (MR) using an MR diet or recombinant methioninase (rMETase) is effective for all types of cancer. However, methionine restriction also inhibits the activity of CD8-positive T-lymphocytes. Therefore, we hypothesized that methionine depletion in the tumor only may be more effective for cancer therapy. We previously developed Salmonella A1-R, which selectively targets and kills tumors. In the present study, we established rMETase-producing Salmonella A1-R, by transfer of the Pseudomonas putida methioninase gene, to target and inhibit syngeneic cancer mouse models.
Methods: A plasmid containing the Pseudomonas putida methioninase gene was extracted from rMETase-producing E. coli and inserted into Salmonella A1-R using electroporation. The rMETase-producing Salmonella A1-R (A1-R-rMETase) infected several cancer cell lines in vitro, including HT29, PC-3, MDA-MB-435, and Lewis Lung Carcinoma (LLC). LLC was chosen for in vivo studies as it supported extensive growth of A1-R-rMETase. We determined 108 A1-R-rMETase was a safe dosage in C57BL/6 mice. LLC cells (106) were injected in male C57BL/6 mice aged 4-6 weeks subcutaneously. After tumor growth, 18 mice were divided into three groups of 6. One group was injected with phosphate-buffered saline (PBS) via the tail vein, twice per week as a control. Another group was injected with 108 Salmonella A1-R via the tail vein, twice per week. Another group was injected with 108 A1-R-rMETase via the tail vein, twice per week for two weeks. Tumor size was measured with calipers three times per week for 3 weeks. On day 22, tumor methionine level was measured using HPLC in the PBS control and the mice injected with A1-R-rMETase.
Results: The mean LLC tumor size of each group on day 22 was as follows: The PBS control: 741.5 mm3; mice injected with A1-R: 566.3 mm3; and mice injected with A1-R-rMETase: 198.8 mm3. Turkey’s multiple comparisons test showed a significant difference between the PBS control and mice injected with A1-R-rMETase (p&lt;0.0001) and between mice injected with A1-R and mice injected with A1-R-rMETase (p=0.0036). However, the PBS control and the mice injected with A1-R did not show a significant difference (p=0.1794). The mice injected with A1-R-rMETase showed a significantly lower mean methionine level than mice injected with PBS (5.9 nM/mg protein vs. 11.1 nM/mg protein, p=0.0095, Mann Whitney test).
Conclusion: Tumor-targeting Salmonella A1-R modified to express the Pseudomonas putida methioninase gene (A1-R-rMETase), inhibited LLC tumor growth in a syngeneic mouse model and reduced the methionine level in the tumor. A1-R-rMETase combines the tumor targeting and killing capability of A1-R itself and restriction methionine selectively in tumors.
Citation Format: Yutaro Kubota, Yusuke Aoki, Noriyuki Masaki, Koya Obara, Sei Morinaga, Kohei Mizuta, Motokazu Sato, Ming Zhao, Qinghong Han, Bouvet Michael, Takuya Tsunoda, Robert M. Hoffman. Tumor-targeting Salmonella A1-R selectively delivers recombinant methioninase and inhibits syngeneic-cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1795.