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Melanoma-associated antigen 4 (MAGEA4, also known as MAGE-A4 or MAGE4) is a tumor/testis antigen belonging to the MAGE-A protein family subtype. It exhibits high expression in various cancerous tissues—including esophageal, head and neck, lung, bladder, and gastric cancers, as well as melanoma, breast cancer, and colorectal cancer—as well as in the testes and placenta, while showing low expression in other normal tissues. MAGEA4 represents a potential target for cancer diagnosis and therapy. It plays a crucial role in the development and progression of various malignancies by participating in the regulation of cell growth, the cell cycle, and cell death through the expression of p53-related genes, and by maintaining DNA replication fidelity via translesion synthesis (TLS). Furthermore, MAGE-A proteins can be processed intracellularly into antigenic peptides, which then form complexes with Class I HLA molecules. These complexes are subsequently presented to CD8+ T cells via Class I MHC molecules, thereby eliciting a tumor-specific immune response in cancer patients.

The HLA-A*0201-MAGEA4 complex is a composite structure formed between a Class I Human Leukocyte Antigen (HLA) molecule and a MAGEA4 antigenic peptide; it is a member of the Class I Major Histocompatibility Complex (MHC) family. This complex is frequently utilized in T-cell receptor (TCR) therapy to activate T cells, enabling them to recognize and eliminate tumor cells—a principle illustrated in the figure below.

Figure 1. Schematic Diagram of the CHO-K1 Human MAGE-A4(GVYDGREHTV) HLA0201 Cell Model

Figure 2. Dose Response of IMC-C103C in NFAT-Luc Jurkat(C7C17) with or without MAGE-A4(GVYDGREHTV) HLA0201 CHO.

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  CHO-K1 Human MAGE-A4(GVYDGREHTV) HLA0201 Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.