Ea₈Mab-9: A novel monoclonal antibody against erythropoietin-producing hepatocellular receptor A8 for flow cytometry

Abstract

Erythropoietin-producing hepatocellular receptor A8 (EphA8) is a type I transmembrane protein that belongs to the largest erythropoietin-producing hepatocellular (Eph) family among receptor tyrosine kinases. By binding to its membrane-bound ephrin-A or ephrin-B ligands on adjacent cells, Eph receptors form complexes and mediate bidirectional signaling activities, triggering cell-cell adhesion and repulsion. Increased expression of EphA8 correlates with poor prognosis in some types of cancer. Therefore, developing sensitive monoclonal antibodies (mAbs) for EphA8 has been desired for treatment, diagnosis, and further basic research. In particular, there are no anti-EphA8 mAbs that can be used for flow cytometry. A novel, specific, and sensitive anti-human EphA8 mAb, which applies to flow cytometry, clone Ea₈Mab-9 (mouse immunoglobulin G1, kappa), was established using the Cell-Based Immunization and Screening method. Ea₈Mab-9 reacted with EphA8-overexpressed Chinese hamster ovary-K1 cells (CHO/EphA8) and EphA8-overexpressed LN229 glioblastoma cells (LN229/EphA8) in flow cytometry. Notably, Ea₈Mab-9 did not recognize other members of the Eph receptor family. Furthermore, Ea₈Mab-9 demonstrated a high binding affinity for CHO/EphA8 and LN229/EphA8, with dissociation constants of 1.3 × 10-9 M and 1.6 × 10-9 M, respectively. The reaction of Ea₈Mab-9 with CHO/EphA8 was completely blocked by a recombinant EphA8 protein. Ea₈Mab-9 could be useful for analyzing the EphA8-related biological responses using flow cytometry, owing to its high affinity and specificity.

Keywords

EphA8 / Cell-Based Immunization and Screening method / Monoclonal antibody / Flow cytometry

References

[1] Tuzi NL, Gullick WJ. Eph, the largest known family of putative growth factor receptors. Br J Cancer. 1994; 69(3):417-421. doi: 10.1038/bjc.1994.77

[2] Pasquale EB. Eph receptors and Ephrins in cancer progression. Nat Rev Cancer. 2024; 24(1):5-27. doi: 10.1038/s41568-023-00634-x

[3] Pasquale EB. Eph-ephrin bidirectional signaling in physiology and disease. Cell. 2008; 133(1):38-52. doi: 10.1016/j.cell.2008.03.011

[4] Hruska M, Dalva MB. Ephrin regulation of synapse formation, function and plasticity. Mol Cell Neurosci. 2012; 50(1):35-44. doi: 10.1016/j.mcn.2012.03.004

[5] Drescher U. The Eph family in the patterning of neural development. Curr Biol. 1997; 7(12): R799-R807. doi: 10.1016/s0960-9822(06)00409-x

[6] Yang D, Jin C, Ma H, et al. EphrinB2/EphB4 pathway in postnatal angiogenesis: A potential therapeutic target for ischemic cardiovascular disease. Angiogenesis. 2016; 19(3):297-309. doi: 10.1007/s10456-016-9514-9

[7] Darling TK, Lamb TJ. Emerging roles for Eph receptors and ephrin ligands in immunity. Front Immunol. 2019;10:1473. doi: 10.3389/fimmu.2019.01473

[8] Herath NI, Boyd AW. The role of Eph receptors and ephrin ligands in colorectal cancer. Int J Cancer. 2010; 126(9):2003-2011. doi: 10.1002/ijc.25147

[9] Chan J, Watt VM. eek and erk, new members of the Eph subclass of receptor protein-tyrosine kinases. Oncogene. 1991; 6(6):1057-1061.

[10] Park S, Sánchez MP. The eek receptor, a member of the Eph family of tyrosine protein kinases, can be activated by three different Eph family ligands. Oncogene. 1997; 14(5):533-542. doi: 10.1038/sj.onc.1200857

[11] Choi S, Park S. Phosphorylation at Tyr-838 in the kinase domain of EphA8 modulates Fyn binding to the Tyr- 615 site by enhancing tyrosine kinase activity. Oncogene. 1999; 18(39):5413-5422. doi: 10.1038/sj.onc.1202917

[12] Gu C, Park S. The EphA8 receptor regulates integrin activity through p110gamma phosphatidylinositol-3 kinase in a tyrosine kinase activity-independent manner. Mol Cell Biol. 2001; 21(14):4579-4597. doi: 10.1128/mcb.21.14.4579-4597.2001

[13] Wang SD, Rath P, Lal B, et al. EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase. Oncogene. 2012; 31(50):5132-5143. doi: 10.1038/onc.2012.16

[14] Bhatia S, Bukkapatnam S, Van Court B, et al. The effects of ephrinB2 signaling on proliferation and invasion in glioblastoma multiforme. Mol Carcinog. 2020; 59(9):1064-1075. doi: 10.1002/mc.23237

[15] Kim Y, Park E, Noh H, Park S. Expression of EphA8-Fc in transgenic mouse embryos induces apoptosis of neural epithelial cells during brain development. Dev Neurobiol. 2013; 73(9):702-712. doi: 10.1002/dneu.22092

[16] Park S, Frisén J, Barbacid M. Aberrant axonal projections in mice lacking EphA8 (Eek) tyrosine protein kinase receptors. EMBO J. 1997; 16(11):3106-3114. doi: 10.1093/emboj/16.11.3106

[17] Gu C, Shim S, Shin J, et al. The EphA8 receptor induces sustained MAP kinase activation to promote neurite outgrowth in neuronal cells. Oncogene. 2005; 24(26):4243-4256. doi: 10.1038/sj.onc.1208584

[18] Cui Z, Liu C, Wang X, Xiang Y. A pan-cancer analysis of EphA family gene expression and its association with prognosis, tumor microenvironment, and therapeutic targets. Front Oncol. 2024;14:1378087. doi: 10.3389/fonc.2024.1378087

[19] Sahoo AR, Buck M. Structural and functional insights into the transmembrane domain association of Eph receptors. Int J Mol Sci. 2021; 22(16):8593. doi: 10.3390/ijms22168593

[20] Pergaris A, Danas E, Goutas D, Sykaras AG, Soranidis A, Theocharis S. The clinical impact of the EPH/ ephrin system in cancer: Unwinding the thread. Int J Mol Sci. 2021; 22(16):8412. doi: 10.3390/ijms22168412

[21] Janes PW, Vail ME, Gan HK, Scott AM. Antibody targeting of Eph receptors in cancer. Pharmaceuticals (Basel). 2020; 13(5):88. doi: 10.3390/ph13050088

[22] Liu L, Wang X, Ge W. EphA8 is a prognostic factor for oral tongue squamous cell carcinoma. Med Sci Monit. 2018; 24:7213-7222. doi: 10.12659/msm.910909

[23] Liu X, Xu Y, Jin Q, et al. EphA8 is a prognostic marker for epithelial ovarian cancer. Oncotarget. 2016; 7(15):20801-20809. doi: 10.18632/oncotarget.8018

[24] Wang Y, Zhou N, Li P, et al. EphA 8 acts as an oncogene and contributes to poor prognosis in gastric cancer via regulation of ADAM10. J Cell Physiol. 2019; 234(11):20408-20419. doi: 10.1002/jcp.28642

[25] Wang GH, Ni K, Gu C, et al. EphA8 inhibits cell apoptosis via AKT signaling and is associated with poor prognosis in breast cancer. Oncol Rep. 2021; 46(2):183. doi: 10.3892/or.2021.8134

[26] Lucero M, Thind J, Sandoval J, Senaati S, Jimenez B, Kandpal RP. Stem-like cells from invasive breast carcinoma cell line MDA-MB-231 express a distinct set of Eph receptors and ephrin ligands. Cancer Genomics Proteomics. 2020; 17(6):729-738. doi: 10.21873/cgp.20227

[27] Ren W, Chen S, Liu G, Wang X, Ye H, Xi Y. TUSC7 acts as a tumor suppressor in colorectal cancer. Am J Transl Res. 2017; 9(9):4026-4035.

[28] Yan Y, Wang Q, Yan XL, et al. miR-10a controls glioma migration and invasion through regulating epithelial-mesenchymal transition via EphA8. FEBS Lett. 2015; 589(6):756-765. doi: 10.1016/j.febslet.2015.02.005

[29] Satofuka H, Suzuki H, Tanaka T, Li G, Kaneko MK, Kato Y. Development of an anti-human EphA2 monoclonal antibody Ea2Mab-7 for multiple applications. Biochem Biophys Rep. 2025;42:101998. doi: 10.1016/j.bbrep.2025.101998

[30] Ubukata R, Suzuki H, Hirose M, et al. Establishment of a highly sensitive and specific anti-EphB2 monoclonal antibody (Eb2Mab-12) for flow cytometry. MI. 2025:5728. doi: 10.36922/mi.5728

[31] Nanamiya R, Suzuki H, Kaneko MK, Kato Y. Development of an anti-EphB4 monoclonal antibody for multiple applications against breast cancers. Monoclon Antib Immunodiagn Immunother. 2023; 42(5):166-177. doi: 10.1089/mab.2023.0015

[32] Suzuki H, Ohishi T, Tanaka T, Kaneko MK, Kato Y. A cancer-specific monoclonal antibody against podocalyxin exerted antitumor activities in pancreatic cancer xenografts. Int J Mol Sci. 2023; 25(1):161. doi: 10.3390/ijms25010161

[33] Paya L, Rafat A, Talebi M, et al. The effect of tumor resection and radiotherapy on the expression of stem cell markers (CD44 and CD133) in patients with squamous cell carcinoma. Int J Hematol Oncol Stem Cell Res. 2024; 18(1):92-99. doi: 10.18502/ijhoscr.v18i1.14748

[34] Da Silva DD, Araldi RP, Belizario MR, Rocha WG, Maciel RMB, Cerutti JM. DLK 1 is associated with stemness phenotype in medullary thyroid carcinoma cell lines. Int J Mol Sci. 2024; 25(22):11924. doi: 10.3390/ijms252211924

[35] Zhou Y, Sakurai H. Emerging and diverse functions of the EphA2 noncanonical pathway in cancer progression. Biol Pharm Bull. 2017; 40(10):1616-1624. doi: 10.1248/bpb.b17-00446

[36] Hosking M, Shirinbak S, Omilusik K, et al. 268 development of FT825/ONO-8250: An off-the-shelf CAR-T cell with preferential HER2 targeting and engineered to enable multi-antigen targeting, improve trafficking, and overcome immunosuppression. J Immunother Cancer. 2023; 11(Suppl 1):A307-A307. doi: 10.1136/jitc-2023-SITC2023.0268

[37] Chalise L, Kato A, Ohno M, et al. Efficacy of cancer-specific anti-podoplanin CAR-T cells and oncolytic herpes virus G47Δ combination therapy against glioblastoma. Mol Ther Oncolytics. 2022; 26:265-274. doi: 10.1016/j.omto.2022.07.006

[38] Mishima Y, Okada S, Ishikawa A, et al. Development of chimeric antigen receptor T cells targeting cancer-expressing podocalyxin. Regen Ther. 2025; 28:292-300. doi: 10.1016/j.reth.2024.12.010