Journal: Frontiers in bioengineering and biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: FIGURE 3 GS542–CD40L fusions are expressed and bind both CD40 and PDL1. Culture medium of HeLa cells infected/transfected by plasmids encoding the indicated GS542–CD40L fusions (also named sdAb–CD40L) were analyzed for transgene expression by immunoblot using an HRP-conjugated anti- FLAG tag monoclonal antibody (A); the ability of the expressed fusion to bind simultaneously CD40 and PDL1 proteins by ELISA (B). SdAb–CD40L and sdAb–link–CD40L refer to the fusion of GS542 sdAb directly to the CD40L ectodomain or through a (GGGS)3 linker, respectively.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Transfection, Expressing, Western Blot, FLAG-tag, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in bioengineering and biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: FIGURE 4 GS542–linker–TNFSF fusions are potent TNFRSF agonists in the presence of PDL1+ cells only. Culture medium of HeLa cells either infected by the indicated recombinant virus (B) or infected/transfected by indicated plasmids (A, C) encoding different GS542–TNFSF fusions were analyzed for their CD40 agonist activity using InvivoGen HEK-blue CD40L cells (A, B) or for their 4-1BB agonist activity using Promega 4-1BB effector cells (C). SdAb–CD40L and sdAb–link–CD40L refer to the fusion of GS542 sdAb directly to the CD40L ectodomain or through a (GGGS)3 linker, respectively. Accordingly, VV–sdAb–link–CD40L is a vaccinia virus encoding the sdAb–link–CD40L construct. SdAb–link–4-1BBL refers to the fusion of GS542 sdAb to the 4-1BBL ectodomain through a (GGGS)3 linker.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Recombinant, Virus, Transfection, Activity Assay, Construct

Journal: Frontiers in bioengineering and biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: FIGURE 5 The CD40 agonist activity of sdAb–linker–CD40L is inhibited by coincubation with a PDL1 blocking antibody, and sdAb–linker–CD40L has a PD1/ PDL1 blocking activity. CD40 agonist activity of culture supernatants of VV–sdAb–linker–CD40L-infected cells was analyzed, as shown in Figure 4, in the presence or absence of 100 μg/mL of PDL1 blocking antibody (avelumab). In the case of the addition of avelumab, or of its isotype control, only undiluted supernatants were tested (A). The PD1/PDL1 blocking activity of the culture medium of A549 cells infected by the mentioned virus was measured, as shown in Figure 2D (B). VV–sdAb–link–CD40L is a vaccinia virus encoding the sdAb–link–CD40L construct. VV–scDimeric: VV encoding single-chain dimeric sdAb GS542; VV–monomeric: VV encoding monomeric sdAb GS542; and VV–avelumab: VV encoding avelumab.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Activity Assay, Blocking Assay, Infection, Control, Virus, Construct

Journal: Frontiers in bioengineering and biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: FIGURE 6 SP-D–CD40L fusions are all expressed at approximately the same level and bind CD40 in vitro. The culture medium of HeLa cells infected/ transfected by plasmids encoding the indicated SP-D–CD40L fusions were analyzed for transgene expression by immunoblot, in both non-reducing and reducing conditions, and using an HRP-conjugated anti-FLAG tag monoclonal antibody (A); the ability of the expressed fusions to bind immobilized CD40 was assessed by ELISA (B). Blue arrows, closed, and open red arrowheads indicate monomers, trimers, and multimers, respectively (A). Full length is the fusion of the entire human SP-D deleted of the CRD and fused to the human CD40L ectodomain. 12R COL is the fusion of oligomeric domains of human SPD containing only 12 collagen repeats and fused to the CD40L ectodomain. The 12R COL link is the same construct as previously described but with a (GGGS)3 linker between the C-terminus of the collagen domain and the N-terminus of the CD40L ectodomain. OR COL and OR COL link are the same constructions described previously except that they are lacking any collagen domain (zero collagen repeat). CD40L is a construction encoding the human CD40L ectodomain. For details on constructions, refer to Table 1 and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: In Vitro, Infection, Transfection, Expressing, Western Blot, FLAG-tag, Enzyme-linked Immunosorbent Assay, Construct

Journal: Frontiers in bioengineering and biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: FIGURE 7 SP-D–CD40L fusions with shortened collagen domains are potent CD40 agonists. Culture medium of HeLa cells either infected by the indicated recombinant virus (B) or infected/transfected by indicated plasmids (A) encoding the different SP-D–CD40L fusions was analyzed for their CD40 agonist activity using InvivoGen HEK-blue CD40L cells (A, B). Full length is the fusion of the entire human SP-D deleted of the CRD and fused to the human CD40L ectodomain. 12R COL is the fusion of oligomeric domains of human SPD containing only 12 collagen repeats and fused to the CD40L ectodomain. 12R COL link is the same construct as described previously but with a (GGGS)3 linker between the C-terminus of the collagen domain and the N-terminus of the CD40L ectodomain. OR COL and OR COL link are the same constructions described previously except that they are lacking any collagen domain (zero collagen Repeat). CD40L is a construction encoding the human CD40L ectodomain. For details on constructions, refer to Table 1 and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Recombinant, Virus, Transfection, Activity Assay, Construct

Journal: Frontiers in bioengineering and biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: FIGURE 8 SP-D–4-1BBL fusion is expressed at approximately the same level as 4-1BBL but is a much better 4-1BB agonist. Culture medium of HeLa cells infected/transfected (no recombinant virus was generated) by plasmids encoding SP-D–4-1BBL or –4-1BBL was analyzed for transgene expression by immunoblot using an HRP-conjugated anti-FLAG tag monoclonal antibody (A) and their 4-1BB agonist activity using Promega 4-1BB reporter cells (B). 12R COL–4-1BBL is the fusion of oligomeric domains of human SPD containing only 12 collagen repeats and fused to the 4-1BBL ectodomain. 4- 1BBL is a construction encoding human CD40L ectodomain. Irr. refers to a plasmid coding an irrelevant protein. For details on constructions, refer to Table 1 and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Transfection, Recombinant, Virus, Generated, Expressing, Western Blot, FLAG-tag, Activity Assay, Plasmid Preparation