Page 159 - MI-2-4

Microbes & Immunity A novel anti-EphA8 monoclonal antibody

to EphA8 and EphA10. The EphB subfamily consists 10a, a promoter of cancer invasion, leads to increased
of five members, such as EphB1 to EphB4 and EphB6. EphA8 expression and suppression of cancer progression
Eight ephrin ligands have been identified, including in colorectal cancer and glioma. 27,28 Further research is
glycosylphosphatidylinositol-anchored ephrin A1 to necessary to clarify the role of EphA8 in either promoting
A5 and transmembrane ephrin B1 to B3. Following the or suppressing cancer-related functions.
binding to receptors, forward signaling is activated on the Various monoclonal antibodies (mAbs) against
receptor side, and reverse signaling is generated on the human Eph receptors, including EphA2, EphB2, and
29
30
3
ligand side, controlling various biological homeostasis. EphB4, have previously been developed by the Cell-
31
Eph receptors play critical roles in the nervous system Based Immunization and Screening (CBIS) method.
(EphA3, EphA4, EphA5, EphB1, EphB2, and EphB3), This method preserves the native structure of membrane
4,5
cardiovascular system (EphB4), immune system (EphA1, proteins during immunization and enables the efficient
6
EphA2, EphA3, EphA4, EphA7, EphA10, EphB1, EphB2, generation of antibodies that recognize modifications
EphB4, and EphB6), and gastrointestinal system (EphB2 and/or three-dimensional structures of the extracellular
7
and EphB3). 8 domains of membrane proteins. Since flow cytometry is
EphA8 complementary DNA (cDNA) was first isolated used for high-throughput screening in the CBIS method,
from a rat brain cDNA library and named eek (eph- and mAbs suitable for this application are prioritized. However,
elk-related kinase, EEK) in 1991. Another group cloned anti-EphA8 mAbs suitable for flow cytometry are not yet
9
the mouse EphA8 molecule in 1997. EphA8 is one of the available. Therefore, the establishment of anti-EphA8
10
members of the RTK family, and its regulatory mechanism mAbs is essential to support basic research and preclinical
is thought to be based on tyrosine kinase (TK) activity. studies related to cancer therapy.
Phosphorylation of Tyr-615 in the EphA8 juxtamembrane In this study, an anti-human EphA8 mAb (clone
domain mediates a strong association with the SH2 domain Ea Mab-9) suitable for flow cytometry was successfully
8
of Fyn, a member of the Src TKs. Phosphorylation established using the CBIS method.
11
of Tyr-838 in the EphA8 kinase domain modulates
Fyn binding to Tyr-615, resulting in attenuation of cell 2. Materials and methods
adhesion through cellular cytoskeletal modifications.
11
Interestingly, TK activity-independent functions of EphA8 2.1. Cell lines and stable transfectants
are also emerging. Ephrin A5-induced EphA8-integrin LN229, Chinese hamster ovary (CHO)-K1, and
interaction is promoted by phosphatidylinositol 3-kinase P3X63Ag8U.1 (P3U1) cells were obtained from the
in a TK-independent manner. Similar to representative American Type Culture Collection (USA). The cDNA
12
growth factor receptors such as epidermal growth factor encoding human EphA8 (Accession No. NM_020526;
receptor, Eph receptors play a role in cell proliferation Catalog No.: RC220352) was purchased from OriGene
primarily through forward signaling. 13,14 In addition to Technologies Inc. (USA). The open reading frame of
regulating cell-cell attachment and cell motility, EphA8 EphA8, excluding the signal sequence, was subcloned into
is involved in organ development and axon growth. 15,16 the pCAG-Ble vector (FUJIFILM Wako Pure Chemical
EphA8 induces caspase-dependent apoptotic cell death Corporation, Japan) with either an interleukin 2-signal
of ephrin A5-expressing neural epithelial cells during sequence and PA16 tag or a MAP16 tag at the N-terminus,
early brain development. Loss of EphA8 disrupts using the in-fusion HD Cloning Kit (Takara Bio Inc.,
15
axon guidance during mammalian nervous system Japan). The resulting plasmid was transfected into the cell
development. Furthermore, EphA8 facilitates neurite lines using the Neon Transfection System (Thermo Fisher
16
outgrowth by sustaining mitogen-activated protein kinase Scientific Inc., USA). Subsequently, LN229 and CHO-
activity in neuronal cells. 17 K1 cells stably overexpressing EphA8 with a deletion
EphA8 expression has also been reported to be of amino acids 1 – 27 and an N-terminal MAP16 tag
associated with cancer. 2,18-21 EphA8 upregulation is (hereafter described as LN229/EphA8 and CHO/EphA8,
observed in various cancers, including oral tongue respectively), as well as LN229 cells stably overexpressing
squamous cell carcinoma (OTSCC), ovarian cancer, EphA8 with the same deletion and an N-terminal PA16
22
23
gastric cancer, and breast cancer. EphA8 and ephrin A5 tag (hereafter described as LN229/PA16-EphA8), were
24
25
contribute to the invasiveness of stem cells isolated from established using a cell sorter (SH800, Sony Corp., Japan).
MDA-MB-231, a triple-negative invasive breast cancer cell cDNAs for various Eph receptors were obtained,
26
line. In contrast, tumor suppressor functions of EphA8 including EphA1 (Catalog No.: RC213689, Accession
have also been proposed. Reducing expression of miR- No.: NM_005232), EphA4 (Catalog No.: RC211230,
Volume 2 Issue 4 (2025) 151 doi: 10.36922/MI025060010