Zhou Hong, Mohamedali Khalid A, Gonzalez-Angulo Ana Maria, Cao Yu, Migliorini Mary, Cheung Lawrence H, LoBello Janine, Lei Xiudong, Qi Yuan, Hittelman Walter N, Winkles Jeffrey A, Tran Nhan L, Rosenblum Michael G
Department of Experimental Therapeutics, M.D. Anderson Cancer Center, Houston, Texas.
Department of Breast Medical Oncology, MD Anderson Cancer Center, Houston, Texas. Department of Systems Biology, MD Anderson Cancer Center, Houston, Texas.
Mol Cancer Ther. 2014 Nov;13(11):2688-705. doi: 10.1158/1535-7163.MCT-14-0346. Epub 2014 Sep 19.
The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)-containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome c-related proapoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared with vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. The Cancer Genome Atlas analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P < 0.0001) compared with hormone receptor-positive breast cancer, and in basal-like 2 tumors (P = 0.01) compared with other TNBC molecular subtypes. IHC analysis of a 101 patient TNBC tumor microarray showed that 55 of 101 (54%) of tumors stained positive for Fn14, suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully human, GrB-containing fusion constructs may form the basis for a new class of novel, potent, and highly effective constructs for targeted therapeutic applications.
细胞因子肿瘤坏死因子样弱凋亡诱导因子（TWEAK）及其受体Fn14，已成为癌症治疗中具有潜在价值的靶点。构建了含颗粒酶B（GrB）的靶向Fn14的构建体，其靶向部分为Fn14配体TWEAK（GrB-TWEAK）或抗Fn14人源化单链抗体（GrB-Fc-IT4）。两种构建体对一组表达Fn14的人肿瘤细胞，包括三阴性乳腺癌（TNBC）细胞系，均表现出高亲和力和选择性细胞毒性。靶细胞中GrB抑制剂PI-9的细胞表达对任一构建体的细胞毒性作用均无影响。多药耐药蛋白1（MDR1）的细胞表达对融合构建体无交叉耐药性。GrB-TWEAK和GrB-Fc-IT4激活细胞内半胱天冬酶级联反应和细胞色素c相关的促凋亡途径，这与GrB在靶细胞中的已知细胞内功能一致。用GrB-TWEAK治疗已建立HT-29异种移植瘤的小鼠，与单独使用载体相比，肿瘤生长受到显著抑制（P < 0.05）。当给携带原位MDA-MB-231（TNBC）肿瘤异种移植瘤的小鼠施用GrB-TWEAK和GrB-Fc-IT4时，二者均显示出显著的肿瘤生长抑制作用。癌症基因组图谱分析显示，与激素受体阳性乳腺癌相比，Fn14 mRNA表达在TNBC和HER2阳性疾病中显著更高（P < 0.0001），与其他TNBC分子亚型相比，在基底样2肿瘤中显著更高（P = 0.01）。对101例患者的TNBC肿瘤微阵列进行免疫组化分析显示，101例肿瘤中有55例（占比54%）Fn14染色呈阳性，这表明这可能是精准治疗方法的一个极佳潜在靶点。使用完全人源化的含GrB融合构建体靶向Fn14，可能为一类新型、强效且高效的靶向治疗应用构建体奠定基础。
