Immunopharmacology and Targeted Therapy Laboratory, Department of Experimental Therapeutics, Unit 1950, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA.
Present address: Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, Liaoning, China.
J Exp Clin Cancer Res. 2019 Jul 30;38(1):332. doi: 10.1186/s13046-019-1333-6.
Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade.
We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct's antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo.
GrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined.
GrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.
近年来，旨在增强 T 细胞和 B 细胞介导的杀伤肿瘤细胞的免疫治疗方法在临床上取得了成功，这表明其在广泛的肿瘤类型中具有巨大的治疗潜力。T 细胞和 B 细胞对靶细胞进行复杂识别后，丝氨酸蛋白酶颗粒酶 B（GrB）递送至肿瘤细胞，导致细胞毒性损伤，从而引发特征明确的多模式凋亡级联反应。
我们设计了一种重组融合构建体 GrB-Fc-4D5，由与人源化抗 HER2 scFv 融合的活性 GrB 组成，用于识别肿瘤细胞并模拟 T 细胞和 B 细胞治疗递呈 GrB。我们评估了该构建体的抗原结合特异性和 GrB 酶活性，以及对靶细胞和对照细胞的体外细胞毒性和内化作用。我们还评估了体内的药代动力学和毒理学参数。
GrB-Fc-4D5 对 SKBR3、MCF7 和 MDA-MB-231 等 HER2 阳性细胞具有高度细胞毒性，IC 值分别为 56、99 和 27 nM，且对一组无论 PI-9 抑制剂内源性表达水平如何的 HER2+细胞系均具有细胞毒性。与 Kadcyla 同期进行的研究表明，该构建体对几乎所有测试细胞的体外活性相似。GrB-Fc-4D5 能快速内化至靶细胞 SKOV3 中，在接触后 1 小时内迅速将 GrB 递送至细胞质隔室。与相对较高的分子量（160 kDa）一致，该构建体在小鼠体内的终末半衰期为 39.2 小时。在 BALB/c 小鼠中进行的毒性研究表明，SGPT、SGOT 或血清 LDH 无统计学显著变化。对治疗小鼠组织的组织病理学分析显示，所有检查的组织均无与药物相关的变化。
GrB-Fc-4D5 显示出优异的、特异性的细胞毒性，且在正常、抗原阴性的小鼠模型中无显著毒性。该构建体是针对 HER2 表达肿瘤的新方法，是进一步开发的优秀候选物。
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