Table_1_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.xlsx

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.

ErbB家族受体酪氨酸激酶（receptor tyrosine kinase, RTK）是胰腺癌治疗中小分子药物与抗体类疗法的首要靶点。然而当前针对该肿瘤的治疗方案仍未达最优，其局限包括疗效不足、耐药性及毒性反应。本研究依托新型BiXAb™四价结构平台，通过合理设计表位组合，制备了针对表皮生长因子受体（epidermal growth factor receptor, EGFR）、人表皮生长因子受体2（human epidermal growth factor receptor 2, HER2）与人表皮生长因子受体3（human epidermal growth factor receptor 3, HER3）的双特异性抗体。随后我们对这些双特异性抗体开展筛选，并与亲本单克隆抗体及抗体配对组合进行性能对比。本次筛选的检测指标涵盖：与同源受体的结合能力（单特异性与双特异性）、细胞内磷酸化信号通路活性、细胞增殖水平、细胞凋亡情况及受体表达量，同时还包含免疫系统介导效应检测，即抗体依赖性细胞介导的细胞毒性（antibody-dependent cell-mediated cytotoxicity, ADCC）与补体依赖性细胞毒性（complement-dependent cytotoxicity, CDC）。在本次测试的30种BiXAbs™中，我们选定3Patri-1Cetu-Fc、3Patri-1Matu-Fc与3Patri-2Trastu-Fc作为核心候选分子。在胰腺癌临床前小鼠模型中，针对EGFR与HER2或HER3的这三款高效双特异性抗体的体内实验结果显示，其可在致密肿瘤组织中实现深度渗透，并可显著抑制肿瘤生长。本研究采用的半理性/半经验研究策略，整合了多种免疫检测手段以对比预选抗体、抗体组合与双特异性抗体的性能，这是首次针对胰腺癌中ErbB家族靶点筛选高效双特异性抗体的探索性研究。