LIGHT protein suppresses tumor growth by augmentation of immune response

Creation of a LIGHT mutant with the capacity to evade the decoy receptor for cancer therapy

“…LIGHT protein was generated by truncation of the N-terminal 65 amino acids, which removes the cytoplasmic and transmembrane domains. We have confirmed that non-glycosylated LIGHT expressed in E. coli had the same bioactivity and receptor binding capacity compared to LIGHT expressed in mammalian cells, although LIGHT has a single N-linked glycosylation site (Asn102) [12]. So, in this study, we manufactured LIGHT mutants in E. coli as previously described [12].…”

“…Therefore, we expect that LIGHT might have the potential for superior therapeutic efficiency in immunotherapy for various cancers, and also the potential for marked anti-tumor activity in restraining metastasis and recrudescence. In fact, we and others have also demonstrated the usefulness of LIGHT for cancer immunotherapy [7,[9][10][11][12][13]. Therefore, LIGHT has recently attracted a great deal of attention as a superior agent for cytokine-based cancer immunotherapy for the eradication of not only local, but also disseminated, metastases.…”

“…Production of LIGHT protein by using E. coli BL21(DE3) (Stratagene) was performed as described previously [12]. Briefly, BL21(DE3) cells harboring the expression plasmid pET15b-LIGHTs (amino acids Gly66-Val240) were incubated in Terrific Broth including 1 mM isopropyl b-D-1-thiogalactopyranoside, and the resultant inclusion bodies were washed, solubilized, and reduced by the methods previously described [12].…”

“…Briefly, BL21(DE3) cells harboring the expression plasmid pET15b-LIGHTs (amino acids Gly66-Val240) were incubated in Terrific Broth including 1 mM isopropyl b-D-1-thiogalactopyranoside, and the resultant inclusion bodies were washed, solubilized, and reduced by the methods previously described [12]. Then, the solubilized LIGHT was refolded by 100-fold dilution in a refolding buffer for 36 h at 4 C. After dialysis against a buffer containing 20 mM Tris-HCl (pH 7.4) and 100 mM urea, active trimeric LIGHT proteins were purified by passage through a HiPrep Sephacryl S-100 HR column (GE Healthcare) following ion-exchange chromatography (Q Sepharose Fast Flow; GE Healthcare).…”

“…Human LTbR, HVEM, or DcR3-Fc chimera (R&D Systems) was diluted to 50 mg/mL in 10 mM sodium acetate (pH 4.5) and immobilized onto a CM5 sensor chip by using an amine coupling kit (GE Healthcare) as described [12]. During the association phase, LIGHTs diluted in HBS-EP running buffer (GE Healthcare) to 35, 17.5, 8.8, 4.4, or 2.2 nM were passed over the immobilized receptors for 2 min at a flow rate of 20 mL/min.…”