TGF-beta trap
A TGF-, trap technology, applied in the field of TGF-β trap, can solve the problems of loss of immune regulation, increased inflammatory response, decreased wound healing, etc.
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[0119] Materials and methods:
[0120] TGF-β reporter cell line. TGF-β-responsive stable cell lines were created by transfecting HEK-293T cells with pGL4.28 (Promega), a TGF-β-responsive element containing Driven luciferase expression plasmid. Transfected cells were selected using hygromycin for two months.
[0121] transfection. TGF-β trap constructs were transiently transfected into the TGF-β reporter 293T cell line with Lipofectamine (Thermo Fisher) using the recommended protocol followed by overnight incubation. These cells were then stimulated for 18 hours with TGF-β1, TGF-β2 or mouse TGF-β1 (Cell Signaling Technology) at the concentrations indicated in the figures. Following stimulation, responses were measured using the Luciferase Assay System (Promega) according to the recommended protocol.
[0122] IgG titer measurement:TGF-β Trap IgG fusion titers were measured using a Protein A biosensor on a ForteBio Octet Red96 instrument. HEK-293T cells were transfected...
example 1
[0123] Example 1: Inhibition of TGF-β Responses by Constitutively Active TGF-β Trap Constructs
[0124] 293T cell line stably expressing TGF-β-inducible luciferase was transfected with a CMV-driven expression construct comparing Sushi domain (Sushi), unmodified hinge (AltH), with or without TGFBRII (SNAP) Fc domain (Fc) of Fc with modified hinge ((C27S)H), Fc with or without TGFBRII (trap) (SEQ ID NO: 15, 17, 19 and 21; with corresponding SEQ ID NO : DNA sequences of 14, 16, 18 and 20). Cells were incubated overnight, washed and stimulated with TGF-β at the indicated concentrations. The resulting luciferase activity was measured after 18 hours. Figure 1A with 1B The data shown in indicate that these trap constructs inhibit TGF-β at low levels (0-1 ng / ml), but only constructs with modified hinges are effective at high concentrations.
example 2
[0125] Example 2: Inhibition of TGF-β Responses by TGF-β Inducible Trap Constructs
[0126] A 293T cell line stably expressing TGF-β-induced luciferase was transfected with a TGF-β-responsive element (TGFBRE)-driven expression construct to compare Sushi domain (Sushi), unmodified hinge (AltH), with or Fc domain (Fc) without TGFBRII (trap) compared to modified hinge ((C27S)H), Fc with or without TGFBRII (trap) (SEQ ID NO: 7, 9, 11 and 13; DNA sequences having corresponding SEQ ID NO: 6, 8, 10 and 12). Cells were incubated overnight, washed and stimulated with TGF-β at the indicated concentrations. The resulting luciferase activity was measured after 18 hours and the data are shown in Figure 2A with 2B middle. The original construct was unable to block TGF-β activity in an inducible form, whereas the modified construct was effective at low concentrations (0.1ng / ml) and still exhibited neutralization at moderate to high concentrations (1-10ng / ml) active.
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