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Microbes & Immunity A novel anti-EphB2 monoclonal antibody for flow cytometry

cell types. 2,6,9 Through this bidirectional signaling, the Eph 2.2. Preparation of cell lines
system regulates tissue development, homeostasis, and LS174T (human colorectal cancer), LN229 (human
regeneration; dysregulation of this system is implicated glioblastoma), Chinese hamster ovary (CHO)-K1, and
in various diseases, including cancer. 3,4,10-21 Hence, P3X63Ag8U.1 (P3U1) cell lines were obtained from the
monoclonal antibody (mAb)-based tumor therapies have American Type Culture Collection (USA).
been developed for certain Eph receptors. 10,22-29
pCMV6-myc-DDK vector with EphB2 (Catalog No.:
Dysregulation of the Eph system is observed in both RC223882, Accession No.: NM_004442) was purchased
tumor cells and tumor microenvironment. The Eph system from OriGene Technologies, Inc. (USA). The EphB2
19
plays distinct roles in tumor development, functioning as plasmids were transfected into CHO-K1 and LN229 cells
both tumor promoters and suppressors depending on the using a Neon transfection system (Thermo Fisher
cellular context. Ephrin type-B receptor 2 (EphB2) is Scientific Inc., USA). Stable transfectants were established
19
overexpressed in several tumors, such as glioblastoma, through cell sorting using the 2H9 conjugated with RB545
30
32
31
breast cancer, hepatocellular carcinoma, and malignant and a cell sorter (SH800; Sony Corp., Japan), followed by
mesothelioma, correlating with poor clinical outcomes. cultivation in a medium containing 0.5 mg/mL of G418
21
In these tumors, EphB2 promotes migration and invasion (Nacalai Tesque, Inc., Japan).
through forward signaling. 33,34
Other Eph receptor cDNAs, including EphA1
In contrast, the expression of EphB2 is downregulated in (Catalog No.: RC213689, Accession No.: NM_005232),
certain tumors, such as colorectal cancer. In the intestinal EphA4 (Catalog No.: RC211230, Accession No.:
35
epithelium, EphB receptors facilitate the proliferation of NM_004438), EphA5 (Catalog No.: RC213206, Accession
stem and progenitor cells. Notably, intestinal epithelial No.: NM_004439), EphA6 (Catalog No.: RC223510,
36
cell migration is impaired in mice lacking EphB2 and Accession No.: NM_001080448), EphA7 (Catalog
EphB3. Without EphB signaling, there is approximately No.: RC226293, Accession No.: NM_004440), EphA8
a 50% reduction in the number of proliferating cells. (Catalog No.: RC220352, Accession No.: NM_020526),
36
Furthermore, EphB receptor expression is elevated in EphA10 (Catalog No.: RC218374, Accession No.:
intestinal adenomas, but functions as a tumor suppressor NM_001099439), EphB1 (Catalog No.: RC214301,
37
by inhibiting invasive growth. EphB signaling promotes Accession No.: NM_004441), and EphB6 (Catalog No.:
adherens junction formation in colorectal cancer cells, RC229404, Accession No.: NM_004445), were purchased
thereby suppressing cancer progression by inhibiting from OriGene Technologies, Inc. (USA). EphA2 (Catalog
invasive growth. Loss of EphB2 expression occurs during No.: HGY095959, Accession No.: NM_004431), EphA3
38
the progression to carcinoma and initiation of invasive (Catalog No.: HGY053437, Accession No.: NM_005233),
growth. 39 and EphB3 (Catalog No.: HGX039581, Accession No.:

To evaluate the expression of EphB2 and target EphB2- NM_004443) cDNAs were purchased from RIKEN DNA
positive cancer cells, the development of mAbs against Bank (Japan).
EphB2 is essential. Previous studies have developed anti- EphA2 and EphB3 cDNAs were cloned into a pCAGzeo
receptor tyrosine kinase mAbs against the human epidermal vector (FUJIFILM Wako Pure Chemical Corporation,
growth factor receptor (EGFR) (clone EMab-17), HER2 Japan), EphB6 cDNA was cloned into a pCMV6 vector,
40
(H Mab-19), and HER3 (H Mab-17) using the Cell- EphA1 cDNA was cloned into a pCAGzeo-ssnPA vector,
42
41
2
3
Based Immunization and Screening (CBIS) method. The while EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,
CBIS method involves immunizing antigen-overexpressed EphA10, and EphB1 cDNAs were cloned into a pCAGzeo_
cells followed by high-throughput hybridoma screening ssnPA16 vector.
using flow cytometry. This study reports the development
of the novel anti-EphB2 mAbs using the CBIS method. The plasmids were transfected into CHO-K1 cells and
stable transfectants were established through staining with
2. Materials and methods mAbs: an anti-EphA2 mAb (clone SHM16; BioLegend,
USA), an anti-EphB3 mAb (clone 647354; R&D Systems
2.1. Antibodies Inc., USA), an anti-EphB6 mAb (clone T49-25; BioLegend,
™
OptiBuild RB545 mouse anti-human EphB2 mAb USA), and an anti-PA tag mAb (clone NZ-1 for EphA2,
(clone 2H9; mouse IgG , kappa) was purchased from BD EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10,
1
Bioscience (USA). Alexa Fluor 488-conjugated anti-mouse and EphB1), followed by sorting using SH800. After
IgG was purchased from Cell Signaling Technology, Inc. sorting, the cells were cultivated in a medium containing
(USA). 0.5 mg/mL of Zeocin (InvivoGen, USA) or 0.5 mg/mL of

Volume 2 Issue 3 (2025) 169 doi: 10.36922/mi.5728