建立雙參數報告系統以有效篩選高功能性T細胞受體以應用於T細胞免疫療法

Abstract

T細胞受體工程性T細胞 (engineered TCR-T cell)是一種應用於過繼性T細胞療法(adoptive T-cell therapy)的改造T細胞，對於治療人類癌症具有巨大的潛力。將患者周邊血中分離出自體CD8+ T細胞，在體外將細胞進行培養放大後，透過基因編輯讓細胞表達對腫瘤具有特異性的T細胞受體，最後再重新輸注回患者體內，以更有效率地清除腫瘤細胞。為了提升治療效率，測量這些T細胞受體工程性T細胞之效用功能的好壞至關重要。而其中，T細胞受體的功能親和力 (functional avidity)就是決定T細胞功能的關鍵因素之一，它反映了T細胞對不同濃度的抗原之反應能力。因此，為了能夠有效地評估T細胞受體的功能親和力，我們利用缺乏內源性T細胞受體的Jurkat細胞株開發了一個雙參數報告系統 (dual-parameter reporter system)。 這個系統能夠藉由報告基因的表達，分析在T 細胞活化中十分重要的轉錄因子NFAT和NF-κB的活性，來測量T細胞活化的程度。通過將具有腫瘤特異性的TCR轉導到這些報告細胞中，我們可以在較繁雜的T細胞功能測試前，就先快速地篩選出具有高功能親和力的候選T細胞受體。此外，由於此系統能夠同時測量NFAT和NF-κB的活性，相較於評估單一的T細胞活化路徑，更能夠提高功能親和力的可靠性和參考價值，並且可以深入比較兩個路徑與T細胞功能之間的關聯性。這個報告系統能夠加速用於癌症治療的T細胞受體工程性T細胞的篩選。透過測量候選T細胞受體的功能親和力，我們可以優先篩選出可能具有理想之效用功能的T細胞受體工程性T細胞，以此提高免疫細胞療法的效率。

T cell receptor (TCR)-engineered T cells (TCR-T) represent a promising approach for adoptive T cell immunotherapy (ACT) and hold great potential in the treatment of human cancer. Autologous CD8+ T cells can be isolated from patients' peripheral blood, expanded in vitro, modified to express tumor-specific TCRs, and finally reinfused into patients’ body to improve antitumor ability. To optimize this treatment strategy, it is crucial to evaluate the efficacy of T cell effector functions. One key factor in determining T cell functionality is the functional avidity of the TCR, which reflects the T cell's response to varying antigen levels. To efficiently assess TCR functional avidity, we have developed a dual parameter reporter system utilizing the Jurkat cell line devoid of endogenous TCRs. This system allows for the analysis of NFAT and NF-κB activities, two critical transcription factors required for T cell activation. By transducing tumor-specific TCRs into these reporter cells, we can effectively select TCR candidates with enhanced effector functions before conducting functional assays on tumor-specific TCR-T cells. Additionally, the system enables simultaneous measurement of the expression levels of NFAT and NF-κB, enhancing the reliability and reference value of the measured functional avidity. Furthermore, it enables a comparative analysis of the two TCR activation signaling pathways and the effector functions of engineered TCR-T cells, providing valuable insights into their interplay. Ultimately, this system holds the potential to expedite the development of effective T cell-based immunotherapies for cancer treatment.