Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells
Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells
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DOI：
10.3389/fimmu.2018.02490
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发表时间：
2018-11-02
影响因子：
7.3
通讯作者：
Beerli, Roger R.
中科院分区：
文献类型：
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作者：
Hellmann, Ina;Waldmeier, Lorenz;Beerli, Roger R.
受体酪氨酸激酶样孤儿受体2 （ROR2）已被确定为多种癌症适应症中高度相关的肿瘤相关抗原，包括肾细胞癌和骨肉瘤，使其成为靶向癌症治疗的一个有吸引力的靶点。在这里，我们描述了从头发现完全的人类ror2特异性抗体和由此衍生的强效抗体药物偶联物（ADC），通过使用转单抗哺乳动物细胞IgG展示平台结合从人免疫球蛋白转基因动物的免疫文库中发现的抗体，并使用二级ADC试验进行内化抗体的功能筛选。该发现策略需要用癌症抗原ROR2免疫转基因小鼠，其中含有转基因的IgH和IgL链基因位点，以及有限数量的完整人V、D和J基因片段。随后，从免疫动物中恢复抗体库，通过转座子介导的祖B淋巴细胞显示（“转座子单抗显示”）表达和筛选它们作为全长人IgG文库，用于ROR2结合。通过单细胞分选分离的单个细胞“转座单抗”克隆，能够表达膜结合和分泌的人IgG，在抗体发现过程中直接筛选，不仅与人ROR2具有高亲和力结合，而且使用具有二级抗人IgG-毒素偶联物的细胞毒性试验具有adc功能。利用这一策略，我们鉴定并验证了12种具有纳米摩尔亲和力的全人源单克隆抗人ROR2抗体，这些抗体作为adc非常有效，可能是治疗人类癌症的有希望的候选者。在抗体发现的早期阶段筛选功能抗体和内化抗体，证明了基于哺乳动物细胞的转单抗显示平台在选择功能结合物方面的实用性，并作为提高治疗相关adc开发效率的有力工具。
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.