靶向EGFR和CD47的新型双功能抗体融合蛋白的抗肿瘤作用及效应机制研究

Bifunctional antibodies, which are designed to simultaneously target tumor specific antigen and immune checkpoint, have great potential for the treatment of cancer. Recently, CD47 blockade has been as another immune checkpoint therapy for cancer. The anti-EGFR monoclonal antibodies are established agents in the treatment of mCRC with overexpressed EGFR. Despite the clinical efficacy, 35%–40% of patients are unresponsive to the antibodies. We previously engineered a novel bifunctional antibody fusion protein (denoted as Bi-SP) simultaneously targeting CD47 and EGFR for the first time. Bi-SP has the potential to enhance anti-tumor immune responses by specifically targeting EGFR-overexpressing tumor cells as well as activating macrophages and CD8+ T cells. In the present study, we will investigate the phagocytosis-stimulation potency of Bi-SP against tumors in vitro and in NOD-SCID mice engrafted with human tumors. In addition, NOD-SCID mice reconstituted with human immune system will be established to evaluate the activation of CD8+ T Cells. To elucidate the anti-tumor mechanism of action of Bi-SP, we will assess the innate immune responses such as tumor associated macrophages (TAMs) polarization and activation upon treatment with Bi-SP in tumor xenografts. Furthermore, anti-tumor adaptive immune responses including intra-tumoral infiltration of CD8+ T cells and induced production of IFN-γ will be investigated in murine models with a functional human immune system. In conclusion, CD47 antagonist that activates innate and adaptive immunity in combination with selective targeting of EGFR+ cancer cells will be investigated as a potential powerful therapeutic regimen against EGFR-overexpressing cancer.

开发可同时靶向肿瘤特异性抗原和免疫检查点的双功能抗体是肿瘤生物治疗中最具发展潜力的策略之一。最新研究表明，CD47亦是一个重要的免疫检查点。抗EGFR抗体已用于EGFR阳性的转移性结肠癌的临床治疗，但仍有35%-40%的患者对其不反应。在本研究前期工作中，我们首次构建了靶向EGFR和CD47的双功能抗体融合蛋白Bi-SP，以期其在特异性靶向过表达EGFR肿瘤细胞的同时激活肿瘤微环境中巨噬细胞和CD8+ T细胞，以达到增强抗肿瘤免疫反应的目的。本研究中，我们将利用体外吞噬试验并建立两种肿瘤异种移植小鼠模型评估Bi-SP的抗肿瘤效果，进一步通过研究其对肿瘤微环境中肿瘤相关巨噬细胞分化和激活的作用以及对CD8+ T细胞的激活等方面来阐释其作用机制。本课题旨在研究将新型抗EGFR抗体和CD47拮抗剂结合制成双功能抗体的抗肿瘤效果并阐明其效应机制，以期为过表达EGFR肿瘤的生物治疗提供一个新策略。

人表皮生长因子受体EGFR是一种过表达于多种肿瘤细胞表面的肿瘤特异性抗原，靶向EGFR的抗体已经在临床上广泛用于转移性结肠癌及非小细胞肺癌的治疗。CD47分子是一个最近发现的重要免疫检查点，在大部分肿瘤细胞中过表达，又可称为整合素相关蛋白。它表达于细胞表面，可结合巨噬细胞、DC细胞等表面的信号调节蛋白-α (Signal-Regulatory Protein α, SIRPα)进而磷酸化SIRPα 的胞质内免疫受体酪氨酸抑制性基序，招募磷酸酶SHP-1 后向其发出“ 别吃我(Don’t eat me)”的抑制性信号。在本项目的工作中，我们首先成功表达了靶向EGFR和CD47的双功能抗体融合蛋白Bi-SP，经SDS-PAGE检测其纯度达到了70%以上。接下来，我们通过共聚焦荧光显微镜观察到Bi-SP处理显著增强了巨噬细胞对表皮癌A431细胞吞噬的能力。此外，我们在NOD-SCID异种移植荷瘤小鼠上也观察到了Bi-SP较强的体内抗肿瘤效果。更重要的是，与单用抗EGFR抗体或高亲和力SIRPα突变体相比，Bi-SP的抗肿瘤效果显著增强，这可能与Bi-SP在小鼠体内招募了更多的巨噬细胞和免疫效应细胞相关。令人惊奇地是，我们还发现Bi-SP在高剂量或低剂量条件下均显示了更低的红细胞毒性。接下来，我们构建了靶向EGFR的新型免疫毒素Ptoxin (PT)，其核心序列是新型抗EGFR抗体可变区融合铜绿假单胞菌外毒素PE38的序列。PT在食管癌细胞KYSE-150和KYSE-450的体外试验及小鼠异种移植肿瘤模型上均显示了较强的抗肿瘤活性；同时我们发现PT处理食管癌细胞还可以有效诱导ROS积累并促进细胞凋亡。经过进一步分析其效应机制，我们发现这可能和Nrf2/Keap1抗氧化信号通路被抑制相关。综上，本研究通过构建双功能抗体融合蛋白和新型免疫毒素实现靶向治疗和免疫治疗的有效结合，探索了提高抗体类药物抗肿瘤治疗效果的可行方案，以期为将来应用于临床治疗奠定理论基础。