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PVRIG (CD112R) is a co-inhibitory receptor belonging to the Poliovirus Receptor (PVR) family, expressed on Natural Killer (NK) cells and T cells. It binds to its ligand (CD112, also known as PVRL2 or Nectin-2), thereby inhibiting the response of T cells and NK cells against cancer. Consequently, CD112R holds significant potential in cancer immunotherapy as a novel target for immune checkpoint inhibition.

CD112 (Cluster of Differentiation 112)—also referred to as Nectin-2 or PVR-related protein 2 (PVRL2)—is a member of the Nectin family and is associated with tumor angiogenesis, growth, and metastasis. CD112 comprises an extracellular region containing three immunoglobulin (Ig)-like domains (two IgC domains and one distal IgV domain), a cytoplasmic tail, and a transmembrane region. CD112 is readily detectable on antigen-presenting cells or tumor cells; high-level expression of CD112 is correlated with tumor progression and poor prognosis in the majority of cancer patients.

The binding of CD112R to its ligand recruits SHP1 and SHP2, thereby triggering the inhibition of TCR/CD28 signaling within T cells. PVRIG can also exert immunosuppressive effects by competitively binding to the ligand shared with CD226, thereby blocking the stimulatory signals transmitted by CD226. This principle of immunosuppression is analogous to that employed by TIGIT—another member of the same receptor family—and it is plausible that these two receptors may exert synergistic effects in mediating immunosuppression.

Serving as target cells for the PVRIG (CD112R) Effector Reporter Cells, the CD112 TCR Activator CHO cells effectively recapitulate the in vivo signal transduction processes of PVRIG (CD112R); the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the CHO-K1 Human CD112/TCR Activator Cell Model

Figure 2. Recombinant CD112/TCR Activator/CHO constitutively expressing CD112.

Figure 3. Dose Response of PVRIG Blocking Antibody in PVRIG(CD112R) Effector Reporter Cell(C7) with CD112/TCR Activator CHO(C1).

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 CD112/TCR Activator 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.