The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.
The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.
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DOI：
10.1371/journal.pone.0148366
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Hupp TR
中科院分区：
文献类型：
--
作者：
Jain S;Aresu L;Comazzi S;Shi J;Worrall E;Clayton J;Humphries W;Hemmington S;Davis P;Murray E;Limeneh AA;Ball K;Ruckova E;Muller P;Vojtesek B;Fahraeus R;Argyle D;Hupp TR
单抗是治疗人类疾病的主要药物，但由于缺乏临床相关的验证模型，其使用受到限制。零星的犬类肿瘤模仿了一些人类同类肿瘤的特征。发展犬类免疫疗法可以成为模拟人类疾病反应的一种方法。利妥昔单抗是一种用于治疗人类血液系统恶性肿瘤的先驱药物。针对犬类CD20的生物模拟物才刚刚由生物技术行业开发出来。为了建立犬-人比较模型系统，我们开发了一种新型的抗CD20单抗(NCD1.2)，可以与人和犬的CD20结合。NCD1.2具有亚纳摩尔Kd，由八位组红色结合试验定义。利用流式细胞仪，NCD1.2与临床来源的犬细胞结合，包括外周血中的B细胞和不同组织类型的B细胞淋巴瘤。犬组织免疫组织化学染色显示，NCD1.2与弥漫性大B细胞淋巴瘤、边缘带淋巴瘤和其他犬B细胞淋巴瘤的膜定位细胞结合。我们从杂交瘤中克隆了NCD1.2的重链和轻链，以确定是否可以获得活性支架作为未来的生物工具。用简并引物克隆杂交瘤细胞的VH和VL基因，并将其包装成单链(ScFv)，构建噬菌体展示文库。令人惊讶的是，我们从对CD20具有生物活性的杂交瘤中分离到两株scFv(scFv-3和scFv-7)。两株单链抗体具有相同的VH基因、不同的VL基因和相同的CDR3s，表明NCD1.2杂交瘤细胞至少编码了两个轻链mRNAs。将scFv-3和scFv-7克隆到哺乳动物载体中，在CHO细胞中分泌，抗体对重组CD20蛋白或多肽具有生物活性。将scFv-3和scFv-7克隆到ADEPT-CPG2生物偶联载体中，在细菌系统中表达时保持了生物活性。这些数据确定了一种重组的抗CD20单链抗体，它可能形成一个有用的工具，用于比较医学中生物偶联导向的抗CD20免疫疗法的评估。
Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.
抗体 - 羧肽酶G2结合物与苯酚芥末前药的抗肿瘤作用。
影响因子：
8.8
作者：
Blakey, D C;Davies, D H;Dowell, R I;East, S J;Burke, P J;Sharma, S K;Springer, C J;Mauger, A B;Melton, R G
通讯作者：
Melton, R G