嵌合抗原受体工程 T 细胞 (CAR-T) 在各种临床试验中进行了研究，用于治疗血液恶性肿瘤以外的癌症实体。一个主要障碍是鉴定在肿瘤上高表达但在健康细胞上不表达的靶抗原，因为“靶内/肿瘤外”细胞毒性通常是无法忍受的。大约 90% 的癌症和白血病呈 Thomsen-Friedenreich 碳水化合物抗原 CD176 阳性，该抗原与肿瘤进展、转移和治疗耐药相关。相比之下，由于碳水化合物链或唾液酸化的延长，CD176 无法与健康细胞上的配体结合。因此，预计相应的 CD176-CAR-T 不会产生“靶向/脱肿瘤”细胞毒性，并且抗原逃逸的可能性较低。使用抗 CD176 单克隆抗体 (mAb) Nemod-TF2，评估了 CD176 的存在多种健康或癌性组织和细胞。为了靶向 CD176，我们生成了两种不同的间隔区长度不同的第二代 CD176-CAR 构建体。它们对 CD176 的特异性在报告细胞以及原代 CD8 T 细胞中进行了测试，这些细胞与 CD176 肿瘤细胞系作为 CD176 血液和实体癌症实体的模型共培养，以及通过霍乱弧菌神经氨酸酶 (VCN) 揭示健康细胞上的 CD176 后） 治疗。随后，对两种 CD176-CAR 启动靶标特异性 T 细胞信号传导和激活、细胞因子释放以及细胞毒性的能力进行了彻底检查。在原发性肿瘤组织以及细胞系中检测到了 CD176 的特异性表达。相应的血液和实体癌症实体。 CD176-CAR 在识别 CD176 癌细胞系和未掩蔽的 CD176 后介导 T 细胞信号传导（NF-κB 激活）和 T 细胞激活（CD69、CD137 表达），从而短间隔区实现了卓越的靶标识别。重要的是，它们还释放效应分子（例如干扰素-γ、颗粒酶 B 和穿孔素），介导针对 CD176 癌细胞的细胞毒性，并在重复抗原刺激后保持功能。此处，CD176L-CAR-T 表现出稍高的增殖和介质释放能力。由于两种 CD176-CAR-T 都不对 CD176 对照细胞发生反应，因此它们的反应被证明是目标特异性的。基因工程 CD176-CAR-T 特异性识别在癌细胞上广泛表达的 CD176。由于 CD176 在大多数健康细胞上被掩盖，因此该抗原和相应的 CAR-T 代表了一种治疗各种血液和实体癌的有前途的方法，同时避免“靶点/肿瘤外”细胞毒性。版权所有 © 2023 Dragon, Beermann,乌姆兰、博尼法修斯、马林科尼科、鲁尔、凯勒、盖勒特、韦斯、迈尔-海恩、齐默尔曼、里斯、托尔、博特尔、梅克-科尔霍夫、山本、布拉斯克、尚巴赫、胡斯特、胡德切克和艾兹-维斯珀。

Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts.Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity.Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific.Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.Copyright © 2023 Dragon, Beermann, Umland, Bonifacius, Malinconico, Ruhl, Kehler, Gellert, Weiß, Mayer-Hain, Zimmermann, Riese, Thol, Beutel, Maecker-Kolhoff, Yamamoto, Blasczyk, Schambach, Hust, Hudecek and Eiz-Vesper.