基因轉殖大腸桿菌具有感測腫瘤微環境暨藥物輸送能力之探討分析

Evaluation of Engineered Escherichia coli as a Tumor Sensing and Drug Delivery Device for Enhancing Tumor Immunotherapy

龔穎之(Ying-Chih Kung)

指導教授 ： 吳亘承

國立臺灣大學/生命科學院/生化科技學系/碩士(2021年)

https://doi.org/10.6342/NTU202100409

摘要

免疫檢查點抑制法 (immune checkpoint inhibition) 是現今癌症免疫治療中具有發展潛力的療法，其可反轉癌細胞經由免疫檢查點抑制T細胞活化之過程，進而激活免疫系統消除癌細胞。然而免疫檢查點抑制法仍有許多限制，例如：(1) 抑制劑藥物為大分子蛋白質或抗體，無法有效地深入固體腫瘤深處；(2) 免疫功能過度激活導致全身性自體免疫疾病。因此，如何有效地將免疫檢查點抑制藥物運送至腫瘤微環境是一個待克服的問題。在本研究中，我們利用基因工程改造的微生物載體作為免疫檢查點抑制劑的傳輸系統，以增進免疫檢查點抑制法的成效。我們希望以合成生物學之方法建構出具有 (1) 辨認腫瘤微環境中的免疫調控訊息interferon γ (IFN-γ) 的能力與 (2) 受IFN-γ偵測訊號調控免疫檢查點抑制劑的生合成之腸道益生菌 E. coli Nissle 1917 (EcN)。我們成功讓微生物生產並外泌出免疫檢查點抑制劑，且抑制劑在濃度約52 μM的情形下，能夠有效地與癌細胞表面上的免疫檢查點蛋白質結合。然而，我們目前建構的IFN-γ感測系統尚無法有效開啟下游目標基因，推測可能是IFN-γ產生的壓力訊號不足，仍需更進一步探討與調整基因迴路以達到偵測效果。綜合來說，本研究所建構的微生物載體系統能外泌免疫檢查點抑制劑藥物，利用合成生物學的優勢，未來能與其他技術合併，幫助新一代抗癌藥物的發展。

關鍵字

癌症免疫療法 ； 免疫檢查點 ； IFN-γ ； 細菌療法 ； Nissle 1917

並列摘要

Immune checkpoint inhibition has great potential for development in cancer immunotherapy nowadays. Immune checkpoint inhibitors revert the T cell inhibitory signaling generated by cancer cells through immune checkpoint pathway and reactivated subsequent anti-tumor immune response. Still, there are some concerns in immune checkpoint inhibition therapy. For example, inhibitors like antibodies could hardly penetrate into the core of solid tumors. Also, uncontrolled activation of the immune system causes systematic immune-related adverse events, like cytokine release syndrome (CRS). Therefore, how to effectively deliver immune checkpoint inhibitors to tumor microenvironments remains a big challenge. With the intrinsic anti-tumor property of anaerobic bacteria, bacteria-mediated tumor therapy (BMTT) has made tremendous progress in cancer therapeutic studies recently. In this research, we utilize genetically engineered microorganisms as drug delivery vectors to enhance the efficiency of immune checkpoint inhibition therapy. With the strategy of synthetic biology, we aim to engineer a novel probiotic vehicle with the ability of sensing interferon γ (IFN-γ), a signaling cytokine released by the immune system against tumors, and synthesizing a common immune checkpoint inhibitor, PDL1 blocker, upon sensing the immune systems in the tumor microenvironments. We successfully modified the probiotic E. coli strain Nissle 1917 (EcN) to produce and secrete out anti-PDL1 peptide, TPP. TPP peptide effectively blocked immune checkpoint protein on surface of tumor cells at the concentration of 52 μM. However, the IFN-γ sensing system currently constructed in the research briefly showed no significant detection ability. More characterizations and genetic circuit reprogramming will be implemented to tune up the sensing capability. In summary, we successfully constructed the secretory system of immune checkpoint inhibitor in bacteria. We envision this system is able to be integrated with other synthetic biology modalities developing a new generation anti-cancer therapy in the future.

並列關鍵字

cancer immunotherapy ； immune checkpoint ； IFN-γ ； BMTT ； EcN

參考文獻

1. Hanahan, D. and Weinberg R., Hallmarks of Cancer: The Next Generation. Cell, 2011. 144(5): p. 646-674.

2. Swann, J.B. and M.J. Smyth, Immune surveillance of tumors. Journal of Clinical Investigation, 2007. 117(5): p. 1137-1146.

3. Gerber, D.E., Targeted therapies: a new generation of cancer treatments. American Family Physician, 2008. 77(3): p. 311-319.

4. Serrano, P., et al., Clinical Development and Initial Approval of Novel Immune Checkpoint Inhibitors in Oncology: Insights From a Global Regulatory Perspective. Clinical Pharmacology and Therapeutics, 2019. 105(3): p. 582-597.

5. Ishida, Y., et al., Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. The EMBO Journal, 1992. 11(11): p. 3887-3895.