Part:BBa_K5073029

IL-12：Secretion of IL-12

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

To overcome the inhibitory effects of the tumor microenvironment (TME) in HCC, we incorporated a sequence regulating IL-12 secretion into the GPC3-CAR construct, enabling the GPC3-CAR molecule to secrete a certain amount of IL-12.IL-12is a pro-inflammatory cytokine with potent tumor-suppressor activity and a promising candidate for combinatorial immunotherapy [1] . It can directly support the sustained cytotoxic activity of T cells, enhance antigen presentation, attenuate antigen-negative escape, and remodel endogenous immunosuppressive cells within the TME [2] . We experimentally demonstrated that HEK-293T cells transfected with GPC3-CAR were able to secrete IL-12 in the presence of AFP. However, HEK-293T cells that were not transfected with the GPC3-CAR concule or in the absence of AFP failed to express IL-12, validating the functional effectiveness of this design.

Detection of the GPC3-CAR function

We verified the expression of GPC3-CAR and the response of AFP promoter. Firstly, the secretion of IL-12 from co-cultured HEK-293T cells using ELISA., and the results showed that HEK-293T cells transfected with GPC3-CAR were able to efficiently secrete IL-12 under the presence of AFP (Fig1.A-B)

Fig1. A. Schematic diagram of the co-culture experimental groups; B. IL-12 detection standard curve ( y = 0.0022x + 0.4139, R² = 0.9747);

References

[1] Tugues, S., Burkhard, S. H., Ohs, I., Vrohlings, M., Nussbaum, K., Vom Berg, J., Kulig, P., & Becher, B. (2015). New insights into IL-12-mediated tumor suppression. Cell death and differentiation, 22(2), 237–246.

[2] Kerkar, S. P., Goldszmid, R. S., Muranski, P., Chinnasamy, D., Yu, Z., Reger, R. N., Leonardi, A. J., Morgan, R. A., Wang, E., Marincola, F. M., Trinchieri, G., Rosenberg, S. A., & Restifo, N. P. (2011). IL-12 triggers a programmatic change in dysfunctional myeloid-derived cells within mouse tumors. The Journal of clinical investigation, 121(12), 4746–4757.