Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich) , Zürich, Switzerland.
MAbs. 2021 Jan-Dec;13(1):1868066. doi: 10.1080/19420862.2020.1868066.
LIGHT is a member of the tumor necrosis factor superfamily, which has been claimed to mediate anti-tumor activity on the basis of cancer cures observed in immunocompetent mice bearing transgenic LIGHT-expressing tumors. The preclinical development of a LIGHT-based therapeutic has been hindered by the lack of functional stability exhibited by this protein. Here, we describe the cloning, expression, and characterization of five antibody-LIGHT fusion proteins, directed against the alternatively spliced extra domain A of fibronectin, a conserved tumor-associated antigen. Among the five tested formats, only the sequential fusion of the F8 antibody in single-chain diabody format, followed by the LIGHT homotrimer expressed as a single polypeptide, yielded a protein (termed "F8-LIGHT") that was not prone to aggregation. A quantitative biodistribution analysis in tumor-bearing mice, using radio-iodinated protein preparations, confirmed that F8-LIGHT was able to preferentially accumulate at the tumor site, with a tumor-to-blood ratio of ca. five to one 24 hours after intravenous administration. Tumor therapy experiments, performed in two murine tumor models (CT26 and WEHI-164), featuring different levels of lymphocyte infiltration into the neoplastic mass, revealed that F8-LIGHT could significantly reduce tumor-cell growth and was more potent than a similar fusion protein (KSF-LIGHT), directed against hen egg lysozyme and serving as negative control of irrelevant specificity in the mouse. At a mechanistic level, the activity of F8-LIGHT was mainly due to an intratumoral expansion of natural killer cells, whereas there was no evidence of expansion of CD8 + T cells, neither in the tumor, nor in draining lymph nodes. : CTLA-4: Cytotoxic T-lymphocytes-associated protein 4; EGFR: Epidermal growth factor receptor; HVEM: Herpesvirus entry mediator; IFNγ: Interferon-gamma; LIGHT: Lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes; LTβR: Lymphotoxin beta receptor; NF-κB: Nuclear factor "kappa-light-chain-enhancer" of activated B cells; NK: Natural killer cells; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; TNF: Tumor necrosis factor.
LIGHT 是肿瘤坏死因子超家族的成员，基于在携带转 LIGHT 表达肿瘤的免疫功能正常的小鼠中观察到的癌症治愈，据称其介导抗肿瘤活性。由于这种蛋白质表现出缺乏功能稳定性，因此基于 LIGHT 的治疗方法的临床前开发受到了阻碍。在这里，我们描述了五个针对纤维连接蛋白的选择性剪接外显子 A 的抗体-LIGHT 融合蛋白的克隆、表达和特性，纤维连接蛋白是一种保守的肿瘤相关抗原。在测试的五种形式中，只有以单链二价体形式连续融合 F8 抗体，然后再表达为单个多肽的 LIGHT 三聚体，才能产生不易聚集的蛋白质（称为“F8-LIGHT”）。使用放射性碘标记的蛋白质制剂在荷瘤小鼠中的定量生物分布分析证实，F8-LIGHT 能够优先在肿瘤部位积聚，静脉给药后 24 小时肿瘤与血液的比值约为五比一。在两个具有不同淋巴细胞浸润肿瘤块程度的小鼠肿瘤模型（CT26 和 WEHI-164）中进行的肿瘤治疗实验表明，F8-LIGHT 能够显著抑制肿瘤细胞生长，并且比针对鸡卵溶菌酶的类似融合蛋白（KSF-LIGHT）更有效，KSF-LIGHT 作为小鼠中无关特异性的阴性对照。在机制水平上，F8-LIGHT 的活性主要归因于肿瘤内自然杀伤细胞的扩增，而在肿瘤或引流淋巴结中均没有 CD8+T 细胞扩增的证据。 CTLA-4：细胞毒性 T 淋巴细胞相关蛋白 4；EGFR：表皮生长因子受体；HVEM：单纯疱疹病毒进入介质；IFNγ：干扰素-γ；LIGHT：淋巴毒素，诱导表达并与单纯疱疹病毒糖蛋白 D 竞争结合到 T 淋巴细胞上表达的单纯疱疹病毒进入介质；LTβR：淋巴毒素β受体；NF-κB：激活 B 细胞的“κ 轻链增强子”核因子；NK：自然杀伤细胞；PD-1：程序性细胞死亡蛋白 1；PD-L1：程序性死亡配体 1；TNF：肿瘤坏死因子。
