Development of a novel anti-erythropoietin-producing hepatocellular receptor B6 monoclonal antibody Eb6Mab-3 for flow cytometry

IF 2.3 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports Pub Date : 2025-02-21 DOI:10.1016/j.bbrep.2025.101960

Tomohiro Tanaka , Yu Kaneko , Haruto Yamamoto , Guanjie Li, Shiori Fujisawa, Hiroyuki Satofuka, Keisuke Shinoda, Takuya Nakamura, Mika K. Kaneko, Hiroyuki Suzuki, Yukinari Kato

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引用次数: 0

Abstract

Erythropoietin-producing hepatocellular receptor B6 (EphB6) is a member of the largest Eph subfamily of receptor tyrosine kinases. EphB6 is widely expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphB6 is involved in cancer pathology despite lacking kinase activity. Developing sensitive monoclonal antibodies (mAbs) for EphB6 has been desired for treatment, diagnosis, and further analysis of EphB6. This study established a novel specific and sensitive anti-human EphB6 mAb clone Eb₆Mab-3 (mouse IgG₁, kappa) by the Cell-Based Immunization and Screening (CBIS) method. In flow cytometry, Eb₆Mab-3 demonstrated reactivity with EphB6-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB6) and endogenously EphB6-expressing DLD-1 colorectal cancer cells. Cross-reactivity of Eb₆Mab-3 was not observed. Eb₆Mab-3 demonstrated a moderate binding affinity (dissociation constant; K_D) for CHO/EphB6 (K_D: 2.6 ± 1.0 × 10⁻⁸ M) and a high binding affinity for DLD-1 (K_D: 3.4 ± 1.3 × 10⁻⁹ M). Eb₆Mab-3 can detect EphB6 protein in CHO/EphB6 lysate in Western blot. Eb₆Mab-3, established by the CBIS method, could be valuable for analyzing the EphB6-associated cellular functions and has potential applications in diagnosis and treatment with specificity and high affinity for cancer cells.

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促红细胞生成素肝细胞受体 B6（EphB6）是最大的受体酪氨酸激酶 Eph 亚家族的成员。EphB6 广泛表达于各种组织，通过与其膜结合的ephrin 配体和其他受体相互作用来调节细胞的稳态。尽管 EphB6 缺乏激酶活性，但它与癌症病理有关。针对 EphB6 开发灵敏的单克隆抗体（mAbs）一直是治疗、诊断和进一步分析 EphB6 所需要的。本研究通过细胞免疫和筛选（CBIS）方法建立了一种新型特异性和敏感性抗人 EphB6 mAb 克隆 Eb6Mab-3（小鼠 IgG1，kappa）。在流式细胞术中，Eb6Mab-3 与过表达 EphB6 的中国仓鼠卵巢-K1 细胞（CHO/EphB6）和内源性表达 EphB6 的 DLD-1 大肠癌细胞有反应性。未观察到 Eb6Mab-3 的交叉反应。Eb6Mab-3 与 CHO/EphB6 的结合亲和力（解离常数；KD）适中（KD：2.6 ± 1.0 × 10-8 M），与 DLD-1 的结合亲和力较高（KD：3.4 ± 1.3 × 10-9 M）。在 Western 印迹中，Eb6Mab-3 可以检测 CHO/EphB6 裂解液中的 EphB6 蛋白。通过 CBIS 方法建立的 Eb6Mab-3 可用于分析与 EphB6 相关的细胞功能，其对癌细胞的特异性和高亲和力在诊断和治疗中具有潜在的应用价值。

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来源期刊

Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics

CiteScore

4.60

自引率

0.00%

发文量

191

审稿时长

59 days

期刊介绍： Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.