Abstract：
Cancer immunotherapy, where a patient\'s immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.
摘要：
癌症免疫疗法,病人的免疫系统被用来选择性地根除癌细胞，是癌症治疗的主导策略。然而,免疫检查点抑制剂(ICI)的成功受到所报告的全身和器官特异性毒性的阻碍,并且三分之二的患者是无应答者或随后获得批准的ICI耐药性.因此,投入了大量努力来发现旨在减少副作用和提高效力的新型靶向免疫疗法。一种方法是利用双特异性抗体的双重靶向特征，这使得它们在癌症免疫疗法中越来越受欢迎。然而,缺乏用于肿瘤对照非肿瘤环境中候选药物的活化排序的简单和预测性筛选方法。在这里，我们提出了一种基于细胞的试验，通过将B细胞与工程化的人胚肾293T细胞（HEK293T）共培养来模拟肿瘤微环境，呈现可控密度的血小板衍生生长因子受体β（PDGFRβ）。通过携带限制PDGFRβRNA翻译的调控元件的遗传构建体,建立了HEK293T细胞上具有三种不同表面蛋白水平的靶密度组。我们采用称为AffiMab的双特异性抗体-亲和体构建体作为模型,其能够结合癌细胞上的PDGFRβ和由B细胞表达的CD40。CD40介导的免疫细胞信号的特异性激活与两个最高的受体表达细胞系证明，水平2/3和水平4，而在低表达细胞系中低至无。受体调节的概念和所提出的共培养方案对于评估和开发用于免疫肿瘤学应用的新型双特异性抗体可能具有普遍的实用性。
