为增强T细胞工程抗体（TCEs）的治疗指数，我们设计了经过遮盖的精准活化的TCEs（XPAT蛋白），以肿瘤抗原（人表皮生长因子受体2（HER2）或表皮生长因子受体（EGFR））和CD3为靶点。无结构的XTEN多肽遮盖物夹在TCE的N和C端之间，并被设计成可以被肿瘤微环境中的蛋白酶释放。体外实验表明，未遮盖的HER2-XPAT（uTCE）表现出强效细胞毒性，XTEN多肽遮盖提供多达4个数量级的保护。体内实验表明，HER2-XPAT蛋白能够诱导蛋白酶依赖性的抗肿瘤活性，并且在健康组织中具有蛋白酶稳定性。在非人灵长类动物中，HER2-XPAT蛋白具有强大的安全边际（最大耐受浓度比uTCE高400倍以上）。HER2-XPAT蛋白的裂解在健康和疾病人类及非人灵长类动物的血浆样本中都很低且相似，支持该稳定性向患者的转化。EGFR-XPAT蛋白确认了XPAT技术在更广泛表达在健康组织中的肿瘤靶点上的实用性。© 2023. 作者。

To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking providing up to 4-log-fold protection. In vivo, HER2-XPAT protein induces protease-dependent antitumor activity and is proteolytically stable in healthy tissues. In non-human primates, HER2-XPAT protein demonstrates a strong safety margin (>400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT protein cleavage is low and similar in plasma samples from healthy and diseased humans and non-human primates, supporting translatability of stability to patients. EGFR-XPAT protein confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.© 2023. The Author(s).