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Glycoprotein 100 (GP100) is a transmembrane glycoprotein primarily expressed by melanocytes. It is aberrantly overexpressed in over 90% of melanomas, establishing itself as a significant tumor-associated antigen. HLA-A*02:01 is a Class I Human Leukocyte Antigen (HLA) molecule that occurs with high frequency within the human population; its peptide-binding groove specifically recognizes 9–10 amino acid peptides containing hydrophobic anchor residues. When these two components interact, peptide fragments generated from the proteasomal degradation of the GP100 protein are transported to the endoplasmic reticulum, where they are embedded within the antigen-binding groove of HLA-A*02:01 to form a stable complex, which is then presented on the surface of tumor cells. This complex can be specifically recognized by the T-cell receptors (TCRs) of CD8⁺ T cells. Upon binding to the central region of the peptide and the HLA α-helical structure, the TCR synergizes with CD8 co-receptor signaling to activate cytotoxic T-cell pathways (triggering the release of perforin, Granzyme B, and inflammatory cytokines), ultimately mediating the targeted killing of melanoma cells. This principle serves as the molecular foundation for various immunotherapies, including tumor vaccines and bispecific antibodies (such as Tebentafusp). By activating anti-tumor immunity through precise antigen presentation and TCR recognition, the GP100/HLA-A*02:01 complex stands as a classic therapeutic target for melanoma.

GP100(YLEPGPVTA)/HLA-A*02:01 CHO: Based on the CHO-K1 cell line, this cell line stably expresses the complex comprising the GP100(YLEPGPVTA) peptide and the HLA-A*02:01 molecule.

The HLA-A*02:01–GP100 complex is a composite structure formed by a Class I Human Leukocyte Antigen (HLA) molecule and a GP100 antigen peptide; it is a member of the Class I Major Histocompatibility Complex (MHC) family. This complex is frequently utilized in T-cell receptor (TCR) therapies to activate T cells, thereby enabling them to recognize and eliminate tumor cells. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the CHO-K1 Human GP100(YLEP GPVTA) HLA0201 Cell Model

Figure 2. Dose Response of Tebentafusp (IMCgp100) in NFAT-Luc Jurkat(C7C17) with or without GP100(YLEPGPVTA) 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 GP100(YLEP GPVTA) 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.