The SHM16 monoclonal antibody reacts with EPHA2, a transmembrane receptor glycoprotein with widespread expression in epithelial cells. Owing to its cell type-specific expression, EphA2 was originally named epithelial cell kinase (eck), but subsequent experiments revealed its expression in many solid tumors, wherein its overexpression correlates with malignancy and a poor prognosis. Among its eight different interacting ephrin A-family ligands, EphA2 exhibits an overt preference for ephrin A1. Interestingly, ephrin A1’s expression pattern is generally observed to be attenuated in EphA2-overexpressing aggressive tumors. EphA2-ephrin A1 signaling regulates proliferation, survival, migration, morphology, cell-to-cell repulsion, and adhesion during embryonic development and controls the processes of angiogenesis and tumorigenesis. The SHM16 antibody, generated by immunizing mice with EphA2-positive human melanoma cells, is reported to interact with an EphA2 epitope differing from that affecting ephrin A1 binding to EphA2, and it doesn’t affect EphA2 interaction with ephrin A1 on the cell surface. The internalization of SHM16 through EphA2 has been shown to inhibit the proliferative and metastatic behavior of melanoma cells in vitro. An immunotoxin generated by conjugating the SHM16 antibody with saporin (an intracellular cytotoxin) is reported to exhibit dose-dependent growth inhibition and cytotoxicity in EphA2-positive melanoma cells in vitro, while the immunotoxin did not affect EphA2-negative cells. The SHM16 antibody has been used for characterization of human head and neck cancer cell lines using mass cytometry in Optimized Multicolor Immunofluorescence Panel-45, i.e., OMIP-45. Like most of the EphA-targeted monoclonal antibodies, SHM16 exhibits an agonist effect, and is reported to activate the EphA2 signaling pathway by mimicking the anti-oncogenic effect of ephrin-A1 on melanoma cell lines.
Tweedell RE, Tao D, Hamerly T, Robinson TM, Larsen S, Grønning AGB, Norris AM, King JG, Law HCH, Baumbach J, Bergmann-Leitner ES, Dinglasan RR. (2019). "The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3" Front Microbiol . PubMed
In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3-5% and early development of P. falciparum exoerythrocytic forms-a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.
in vitro stimulation of EphA2 signaling, Immunoprecipitation, Flow Cytometry, Functional Assays, Agonistic Antibodies
Sakamoto A, Kato K, Hasegawa T, Ikeda S. (2018). "An Agonistic Antibody to EPHA2 Exhibits Antitumor Effects on Human Melanoma Cells" Anticancer Res 38(6):3273-3282. PubMed
Background/aim: EPH receptor A2 (EPHA2) is highly expressed in aggressive types of human cancer, and is expected to be an excellent target molecule for antibody treatments. In this study, we investigated the therapeutic potential of antibody to EPHA2 against melanoma in vitro. Materials and methods: We generated three monoclonal antibodies (mAbs) to EPHA2 and examined cell-surface expression by flow cytometry. To investigate the ability to inhibit tumor cell migration therapy with mAbs to EPHA2, we performed a wound scratch assay and invasion assay. We investigated the therapeutic effects of immunotoxins consisting of toxin-conjugated EPHA2 mAbs. Results: All human melanoma cell lines studied expressed EPHA2. Like natural ligand ephrin-A1, one of EPHA2 mAbs, SHM16, inhibited metastatic behavior of cells, such as migration and invasion. In addition, drastic growth inhibition and cytotoxicity were found using immunotoxin-conjugated SHM16. Conclusion: These observations indicate a promising role for EPHA2 as a target in antibody treatments for melanoma, and demonstrate the potential therapeutic effects of an agonistic antibody to EPHA2.
Flow Cytometry
Brodie TM, Tosevski V, Medová M. (2018). "OMIP-045: Characterizing human head and neck tumors and cancer cell lines with mass cytometry" Cytometry A 93(4):406-410. PubMed
