Committed to providing comprehensive support for global drug development

PD-1 (Programmed Death Receptor-1), an immune checkpoint receptor found on the surface of T cells, typically functions to suppress excessive T-cell activity; however, cancer cells exploit this principle to evade immune attack, leading to T-cell "exhaustion." Conversely, IL-2—a key cytokine for T-cell proliferation and survival—promotes the expansion of effector T cells (Teff) and natural killer (NK) cells; yet, conventional IL-2 therapies also activate regulatory T cells (Treg), which may subsequently dampen the overall immune response.

By integrating PD-1 checkpoint blockade with the T-cell-enhancing effects of IL-2, novel therapies can be developed to "reawaken" exhausted T cells within the tumor microenvironment. This approach aims to generate a more potent and precise anti-cancer response, potentially with lower toxicity compared to the administration of either agent alone. These therapeutic molecules facilitate the targeted delivery of IL-2 directly to PD-1-expressing tumor-infiltrating lymphocytes (TILs), thereby augmenting their capacity to eliminate cancer cells.

The PD-1 & IL-2/IL-15 (hCD122/hCD132) Effector Reporter Cell model serves as an excellent *in vitro* surrogate for simulating the signal transduction pathways mediated by the PD-1 and IL-2 receptor heterodimers *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the Jurkat E6.1 Human PD-1&IL2/IL15(hCD122/hCD132) Effector Reporter Cell Model

Figure 2. Recombinant PD-1&IL2/IL15(hCD122/hCD132) Effector Reporter Cell stably expressing PD-1&CD122&CD132&CD25.



Figure 3. Recombinant PD-1(High Expression)&IL2/IL15(hCD122/hCD132) Effector Reporter Cell and PD-1(Medium
Expression)&IL2/IL15(hCD122/hCD132) Effector Reporter Cell and PD-1(Low Expression)&IL2/IL15(hCD122/hCD132) Effector Reporter Cell stably expressing PD-1.



Figure 4. Dose Response of IL2 (IL2Rβγ bias)&Anti-PD-1 Fusion in PD-1(High Expression) &IL2/IL15(hCD122/hCD132) Effector Reporter Cell(C9C14) vs IL2/IL15(hCD122/hCD132) Effector Reporter Cell(C9).



Figure 5. Dose Response of IL2 (lL2Rβγ bias)&Anti-PD-1 Fusion in PD-1&IL2/IL15(hCD122/hCD132) Effector Reporter Cell vs IL2/IL15(hCD122/hCD132) Effector Reporter Cell(C9).



Figure 6. Dose Response of Samples in PD-1(High Expression)&IL2/IL15(hCD122/hCD132) Effector Reporter Cell(C9C14).

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.