Abstract 296: Antigen-independent delivery of 4-1BB agonist to the tumor microenvironment improves immune response while reducing hepatotoxicity
Authors: Hyun Hyesun , Sun Bo , Montgomery Stephanie A. , Griffin Teresa , Yan Juanzhu , Wielgus Albert , Wang Yue , Zhang Tian , Cheng Jianjun , Wang Andrew Z. 
Published: June 2022

Abstract
Background: Clinical progress of α4-1BB has been impeded by hepatotoxicity. Current research is focused on improving targeted delivery of α4-1BB to the tumor using tumor-specific markers. However, targeted delivery of 4-1BB agonists to tumors faces challenges due to a lack of tumor biomarkers and effector T cells within tumors. To overcome these challenges, we developed antigen-independent targeting approach based on unnatural sugar-mediated metabolic glycoengineering and bio-orthogonal click chemistry to deliver α4-1BB to tumors. We utilized nanoparticles that can deliver a sugar analogue containing click-chemistry moiety to the tumor microenvironment.
Method: The Ac4ManNAz was loaded in mPEG-PLGA nanoparticles (MazNP), and the loading efficiency was determined by HPLC. DBCO-functionalized α4-1BB (DBCO-α4-1BB) was synthesized via amine-NHS coupling reaction. The tumor inhibition efficiency was assessed on mice bearing different tumor models. Mice were either inoculated subcutaneously on the left flank (75,000 B16F10 cells) or injected on the left fourth mammary fat pad (100,000 4T1 cells). Mice were given MazNPs (IV) on day 5, 6, 10, and 11, and DBCO-α4-1BB (IV) and αPD-1 (IP) on day 8 and 13. T lymphocytes were analyzed by fluorescent IF staining. Serum liver enzyme was examined to evaluate hepatotoxicity of treatments. Liver pathology was assessed by H&E and CD8+ IHC staining.
Results: The αPD1 plus DBCO-α4-1BB with MazNPs showed a 36.4% cure rate, compared to 0% of αPD1 plus DBCO-α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz in B16F10 melanoma model. We then re-challenged the cured mice with 200,000 B16F10 cells and 50% of cured mice survived without any further treatment. In 4T1 breast cancer model, the survival data analyzed using the log-rank test showed that the median survival of αPD1 plus DBCO-α4-1BB with MazNP was increased by 71%, compared to the PBS, and 26% αPD1 plus α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz. We showed that the involvement of NK and CD8+ T cells in the antitumor efficacy of αPD1 plus DBCO-α4-1BB with MazNP. Notably, a massive CD8+ T cell infiltration in the liver was observed in both αPD1 plus α4-1BB (29.8 ± 18.2%) and αPD1 plus DBCO-α4-1BB with Ac4ManNAz (22.1 ± 10.9), significantly higher than PBS control (0.9 ± 0.6%), while the percentage of CD8+ T cells in the MazNP treatment (5.4 ± 5.8%) was five times lower. Hepatotoxicity was further confirmed by serum liver enzyme analysis that ALT and AST levels were substantially elevated by αPD1 plus α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz, as compared to PBS control group, while the MazNP-treated mice had normal serum ALT and AST levels except for one.
Conclusion: Our findings demonstrated that antigen-independent targeted delivery of α4-1BB can improve anti-tumor immune responses while reducing hepatotoxicity.
Citation Format: Hyesun Hyun, Bo Sun, Stephanie A. Montgomery, Teresa Griffin, Juanzhu Yan, Albert Wielgus, Yue Wang, Tian Zhang, Jianjun Cheng, Andrew Z. Wang. Antigen-independent delivery of 4-1BB agonist to the tumor microenvironment improves immune response while reducing hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 296.