DataSheet_1_Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display.pdf

To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies’ enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified.

为筛选用于治疗包括多发性骨髓瘤（multiple myeloma, MM）在内的浆细胞疾病的新型抗体，本研究以患者来源的恶性浆细胞免疫小鼠，构建了单链可变片段（single-chain Fragment variable, scFv）抗体库。为富集结合骨髓瘤抗原的抗体，采用噬菌体展示技术开展细胞淘选实验：先用健康供者白细胞对免疫抗体库进行吸附消减，随后通过两轮连续淘选，以L-363浆细胞系完成抗体筛选。通过下一代测序（next-generation sequencing, NGS）监测淘选过程中抗体的富集动态，最终筛选得到多个潜在候选抗体。最初共获得41种源自不同B细胞克隆的独特scFv抗体，其中9种可强效结合骨髓瘤细胞，且仅弱结合外周血单个核细胞（peripheral blood mononuclear cells, PBMC），研究人员对其开展了功能表征。利用稳定转染并表达单一骨髓瘤相关抗原的中国仓鼠卵巢细胞进行实验，结果显示：2种抗体分别靶向结合CD38与细胞间黏附分子1（intercellular adhesion molecule-1, ICAM-1），另有7种抗体所靶向的抗原尚未被明确鉴定。为评估新型抗体的治疗潜力，在首次概念验证研究中，研究人员将结合CD38的scFv噬菌体抗体改造为嵌合IgG1抗体。进一步分析表明，#5-CD38-IgG1与达雷妥尤单抗、艾沙妥昔单抗共享重叠表位，且其抗骨髓瘤活性与这两款临床获批的CD38单抗相当，具备强效抗肿瘤活性。上述结果证实，通过噬菌体展示与深度测序技术，可筛选得到具备多发性骨髓瘤免疫治疗潜力的新型抗体。