41BB agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor. 41BB agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor

Background: Ionizing radiation (IR) is a double-edge sword for immunotherapy as it may cause both immunosuppressive and immunostimulatory effects. The interactions of IR with the tumor microenvironment (TME) is a key factor for this balance. Fibroblast activation protein (FAP) is expressed on the surface of CAFs in many cancer types and its presence is associated with poor immune response to immune checkpoint blockade in patients. We hypothesize that IR increases FAP expression in CAFs, therefore the combination of IR with targeted immunomodulators such as an agonistic FAP-4-1BBL bispecific antibody fusion protein could enhance the immune-mediated antitumoral effect of these treatments given in combination. Methods: Murine transplantable TS/A tumor cells line was used to investigate increases in FAP expression in tumors after irradiation by IHQ and RT-PCR. We treated bilateral tumor-bearing mice in which only one of the lesions was locally irradiated (2 × 6 Gy) given alone or in combination with a systemic administration of the FAP-4-1BBL bispecific construct. Tumor sizes were followed over time and in the cellular composition microenvironment (TME) was assessed by immunochemistry and multiplex tissue immunofluorescence. Selective depletions of immune cell populations were used to delineate the immune system requirements for efficacy. Antibody of interest was labeled and tracked its distribution by CT. Colorectal carcinoma samples were also validated. Results: Irradiation of TS/A+CAF tumors showed clear increases of FAP expression levels after local irradiation. The suboptimal radiotherapy regimen in combination with FAP targeted 4-1BBL worked to attain primary tumor control as well as partial abscopal effect. Immune landscape analysis showed an effector and proinflammatory phenotype with an increased infiltration of immune cells. The antitumoral mechanism relied on CD8+ T cells, IFN-I, IFN-γ and CD137 expression. Biodistribution studies of bispecific antibody performed indicated enhanced tumor targeting after tumor irradiation. Human sample results confirmed enhanced immune infiltration and FAP expression after radiotherapy treatment. Conclusion: Our data provides a proof- of- concept and mechanistic insights pertaining the therapeutic efficacy of bispecific FAP-41BBL combined with local radiotherapy. Overall design: As our treatment was the combination of radiotherapy with muFAP-4-1BBL, we had 4 experimental groups to consider: vechile, radiotherapy, muFAP-4-1BBL and RT+muFAP-41BBL. We performed RNAseq of tumor in each condition to see differences in gene expression.

背景：电离辐射（Ionizing Radiation, IR）是免疫治疗的一把双刃剑，既可引发免疫抑制效应，也可产生免疫激活效应。其与肿瘤微环境（Tumor Microenvironment, TME）的相互作用是决定这两种效应平衡的关键因素。成纤维细胞激活蛋白（Fibroblast Activation Protein, FAP）在多种癌症类型的癌相关成纤维细胞（Cancer-Associated Fibroblasts, CAFs）表面表达，其表达水平与患者对免疫检查点阻断治疗的应答不佳相关。我们提出假说：电离辐射可上调癌相关成纤维细胞中的FAP表达，因此将电离辐射与靶向免疫调节剂（如激动型FAP-4-1BBL双特异性抗体融合蛋白）联合使用，可增强二者联合给药时免疫介导的抗肿瘤效应。方法：本研究使用小鼠可移植TS/A肿瘤细胞系，通过免疫组化（IHQ）与逆转录聚合酶链反应（RT-PCR）探究辐照后肿瘤内FAP表达的上调情况。我们构建双侧荷瘤小鼠模型，仅对其中一侧肿瘤进行局部照射（2次6Gy剂量），单独照射或联合全身给予FAP-4-1BBL双特异性构建体。定期监测肿瘤体积变化，并通过免疫组化与多重组织免疫荧光评估肿瘤微环境的细胞组成。采用免疫细胞群体选择性清除实验明确疗效所需的免疫系统组分。将目标抗体标记后，通过计算机断层扫描（Computed Tomography, CT）追踪其体内分布。同时对结直肠癌样本进行了验证。结果：对TS/A+CAF肿瘤进行局部辐照后，FAP表达水平显著上调。次优放疗方案联合靶向FAP的4-1BBL治疗，不仅实现了原发肿瘤的控制，还产生了部分远隔效应。免疫微环境分析显示，肿瘤呈现效应性促炎表型，免疫细胞浸润水平升高。抗肿瘤机制依赖于CD8+T细胞、I型干扰素、γ干扰素以及CD137的表达。双特异性抗体的生物分布研究表明，肿瘤辐照后其肿瘤靶向性增强。人体样本结果证实，放疗治疗后肿瘤的免疫浸润与FAP表达均出现上调。结论：本研究数据为双特异性FAP-41BBL联合局部放疗的治疗功效提供了概念验证与机制见解。整体实验设计：由于本研究的治疗方案为放疗联合小鼠源FAP-4-1BBL（muFAP-4-1BBL），共设置4个实验组：载体对照组、单纯放疗组、单纯muFAP-4-1BBL组以及放疗+muFAP-41BBL联合组。对各处理条件下的肿瘤样本进行RNA测序，以分析基因表达差异。