Data Sheet 1_An mRNA-based workflow validating neo-epitope presentation through HLA-I/peptide affinity purification.pdf

Presentation of human leukocyte antigen (HLA)-class I-restricted neo-epitopes is key for inducing an adaptive cytotoxic T-lymphocyte response against cancer. Validating presentation of these cancer-specific neo-epitopes following delivery to antigen-presenting cells (APCs) is critical to advance personalized therapeutic cancer vaccines. Current workflows for neo-epitope identification are often laborious and depend on computational deconvolution to determine the correct peptide sequence and its corresponding restriction element. We evaluated an mRNA-based workflow for more precise purification of HLA-I-peptide (pHLA)-complexes, facilitating peptide identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach uses mRNA encoding a specific HLA-I-molecule fused to a Twin-Strep-Tag (HLA-TST), allowing affinity-based purification and downstream analysis of pHLA-complexes. As a proof-of-concept, we co-electroporated mRNA encoding TST-HLA-A*02:01 and mRNA encoding an HLA-A*02:01-restricted epitope in HLA-A*02:01-negative APCs. We demonstrated successful purification and detection of the delivered epitope via LC-MS/MS. These findings highlight the potential of the mRNA-based workflow to verify neo-epitope presentation by APCs. Still, further investigation is necessary to fully understand the technical variables that can influence peptide identification by LC-MS/MS.

人类白细胞抗原 (HLA) I类限制性新表位的呈递，是诱导针对癌症的适应性细胞毒性T淋巴细胞应答的关键环节。将这类癌症特异性新表位递送至抗原呈递细胞 (APCs) 后验证其呈递情况，对于推进个性化治疗性癌症疫苗的研发至关重要。当前用于新表位鉴定的工作流程往往较为繁琐，且需依赖计算反卷积来确定正确的肽序列及其对应的限制性元件。我们评估了一种基于信使RNA (mRNA) 的工作流程，该流程可更精准地纯化HLA-I-肽 (pHLA) 复合物，从而通过液相色谱-串联质谱 (LC-MS/MS) 简化肽段鉴定流程。该方法使用编码与Twin-Strep标签 (Twin-Strep-Tag) 融合的特定HLA-I分子的mRNA，可实现亲和纯化以及pHLA复合物的下游分析。作为概念验证，我们将编码TST-HLA-A*02:01的mRNA与编码HLA-A*02:01限制性表位的mRNA共电穿孔至HLA-A*02:01阴性的抗原呈递细胞中。我们通过LC-MS/MS成功纯化并检测到了递送的表位。上述研究结果凸显了基于mRNA的工作流程在验证抗原呈递细胞呈递新表位方面的应用潜力。不过，仍需开展进一步研究以全面阐明可能影响LC-MS/MS肽段鉴定的各类技术变量。