High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules
High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules
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DOI：
10.1126/sciimmunol.aav0860
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发表时间：
2019-07-01
影响因子：
24.8
通讯作者：
Maurer, Dominik
中科院分区：
文献类型：
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作者：
Moritz, Andreas;Anjanappa, Raghavendra;Maurer, Dominik
主要组织相容性复合体 (MHC) I 类分子在细胞表面呈递短肽配体，供细胞毒性 CD8(+) T 细胞询问。呈递肿瘤相关肽（例如新抗原）的 MHC I 类复合物代表了目前正在开发的癌症免疫治疗方法的关键靶标，这使得它们对于疗效和安全性筛选非常重要。如果没有肽配体，MHC I 类复合物不稳定且快速衰变，使得用于分析目的的可溶性单体的生产需要大量劳动力。我们开发了一种二硫键稳定的 HLA-A*02:01 分子，无需肽即可稳定，但可以在一步加载过程中与所选配体形成肽-MHC 复合物 (pMHC)。我们说明了工程突变体和野生型分子在野生型或亲和力成熟的 T 细胞受体 (TCR) 亲和力方面的相似性，并提出了证实结合动力学测量的晶体结构。此外，我们还展示了一个高通量结合动力学测量平台，用于分析双特异性 TCR (bsTCR) 分子与二硫键稳定的 HLA-A*02:01 分子产生的多种 pMHC 文库的结合特性。我们证明 bsTCR 对 pMHC 的亲和力指示了体外功能，并生成 bsTCR 结合基序来识别人类蛋白质组中潜在的脱靶相互作用。这些发现展示了该平台和工程化 HLA-A*02:01 分子在 pMHC 靶向生物制剂新兴领域的潜力。
Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8(+) T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive. We have developed a disulfide-stabilized HLA-A*02:01 molecule that is stable without peptide but can form peptide-MHC complexes (pMHCs) with ligands of choice in a one-step loading procedure. We illustrate the similarity between the engineered mutant and the wild-type molecule with respect to affinity of wild-type or affinity-matured T cell receptors (TCRs) and present a crystal structure corroborating the binding kinetics measurements. In addition, we demonstrate a high-throughput binding kinetics measurement platform to analyze the binding characteristics of bispecific TCR (bsTCR) molecules against diverse pMHC libraries produced with the disulfide-stabilized HLA-A*02:01 molecule. We show that bsTCR affinities for pMHCs are indicative of in vitro function and generate a bsTCR binding motif to identify potential off-target interactions in the human proteome. These findings showcase the potential of the platform and the engineered HLA-A*02:01 molecule in the emerging field of pMHC-targeting biologics.