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Tumor cells can evade recognition and killing by the host immune system through immune checkpoint receptors. Therefore, blocking these immune checkpoint receptors may represent a broadly effective tumor immunotherapy approach. Currently, although anti-PD-1/PD-L1 antibodies are relatively mature, similar to anti-CTLA4 antibodies, patient overall response rates remain low due to the presence of resistance, making the search for new tumor immunotherapy targets urgent.

  
Programmed Cell Death Protein 1 (PD-1), a receptor expressed on activated T cells, binds to its ligands PD-L1 and PD-L2 to negatively regulate immune responses. PD-1 ligands are present in most cancers, and the PD-1:PD-L1/2 interaction inhibits T cell activity, allowing cancer cells to escape immune surveillance. The PD-1/PD-L1 signaling pathway is an important component of tumor immune suppression, capable of inhibiting T lymphocyte activation, enhancing tumor cell immune tolerance, and thus enabling tumor immune escape. Binding of PD-1 to PD-L1 weakens T cell-mediated immune surveillance, leading to impaired immune responses and even T cell apoptosis. PD-1/PD-L1 inhibitors can relieve immune suppression of anti-tumor T cells, resulting in T cell proliferation, infiltration into the tumor microenvironment, and induction of anti-tumor responses. The PD-1:PD-L1/2 pathway also participates in regulating autoimmune responses, making these proteins promising therapeutic targets for various cancers as well as multiple sclerosis, arthritis, lupus, and type 1 diabetes.

  
4-1BB [also known as CD137 or TNF receptor superfamily member 9 (TNFRSF9)] is a glycosylated type I membrane protein belonging to the TNFR superfamily, composed of four extracellular cysteine-rich pseudo-repetitive domains. The cytoplasmic region of 4-1BB contains TNF receptor-associated factor (TRAF) binding motifs. 4-1BB expression is primarily found on the surface of activated cytotoxic CD8+ T cells and helper CD4+ T cells. Upon activation, 4-1BB expression can be induced in NK cells, B cells, monocytes, and dendritic cells. As an inducible co-stimulatory molecule, 4-1BB enhances T cell responses to antigens. When upregulated and trimerized on T cells by 4-1BBL, 4-1BB recruits TRAF adaptor proteins to the cytoplasmic TRAF binding motif, thereby initiating co-stimulatory signaling. The co-stimulatory activity of 4-1BB has the potential to complement and enhance the anti-tumor activity of PD-1/PD-L1 blockade by improving the function and survival of long-term stimulated CD8+ T cells in the tumor microenvironment and expanding T cell clonality and overall prevalence. Tumor antigen-specific T cells express both PD-1 and 4-1BB, providing further theoretical basis for co-targeting these pathways to amplify tumor-specific T cell responses. Bispecific antibody (bsAb) technology offers the opportunity to generate immunotherapeutic agents with superior or novel properties compared to individual mAbs or their mixtures.

The PD1 4-1BB Dual Effector Reporter Cell reporter gene drug target model effectively simulates the signal transduction process of PD1 and 4-1BB in vivo, as illustrated in the figure below.

Figure 1. Schematic diagram of the PD1 4-1BB Dual Effector Reporter Cell model.

Figure 2. Recombinant PD1 41BB Dual Effector Reporter Cell stably expressing PD-1 and 41BB.

Figure 3. Dose Response of Blocking Antibodies in PD-1 4-1BB Dual Effector Reporter Cells (C64) With PD-L1 aAPC Cells.

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.