Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Lentiviral vector mediated delivery and expression of sTNFR-Fc . (A) Schematic maps of the lentiviral transfer plasmids, pHR-hTNFR-Fc-eGFP and pHR-Fc-eGFP. LTR: long terminal repeat; ψ: packaging signal; SA, SD: splice donor, splice acceptor; CMV: cytomegalovirus promoter; hTNFR-Fc: codon-optimized gene encoding the extracellular domain of the human TNF receptor type 2, fused to the hinge domain from IgG1 and the Fc domain from human IgG3; Fc: hinge domain from IgG1 and the Fc domain from human IgG3; IRES: internal ribosome entry site; GFP: green fluorescent protein.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Expressing

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Stable and high level secretion of sTNFR-Fc in transduced cells . (A) Immunoblot detection of sTNFR-Fc protein in transfected cells. Mock = supernatant from mock transfected 293T cells; Intracellular = transfected cell lysate; and Secreted = supernatant from the transfected 293T cells. (B) Immunoblot detection of sTNFR-Fc protein in transduced cells intracellularly (cell lysate) and extracellularly (supernatant). HTB-T = transduced HTB-11 cells; CHME-T2 = CHME-5 cells transduced twice with the vector; CHME-T1 = CHME-5 cells transduced once with the vector. (C) Stable expression of sTNFR-Fc protein in conditioned medium of transduced cells. Expression of sTNFR-Fc in supernatants from transduced cells was measured by ELISA; results shown at different cell passages represent mean values from three independent experiments and error bars denote the standard deviation. (D) Stable expression of GFP in lentivirus vector-transduced cells. Transduced cells were passed in vitro and the percentage of GFP positive cells was determined every 5 passages by calculating the percentage of GFP+ cells within the culture using an inverted fluorescent microscope (Nikon Eclipse TE2000-U) with a digital camera attachment. Data presented here represent mean values from at least three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, In Vitro, Microscopy

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Specific binding of expressed sTNFR-Fc to TNF-α by dot immunoblot assay . One microgram of standard recombinant human TNF-α protein was spotted onto nitrocellular membrane (NCM) and then blocked and incubated with supernatants from normal and transduced cells. After three washes with TBST, a goat-anti-human IgG Fc-HRP conjugate was applied and incubated at RT for 1 h. Specific binding was visualized by color deposition on the NCM following incubation with diaminobenzidine (DAB) substrate. Lanes: HTB-NT = supernatant from non-transduced HTB-11 cells and CHME-NT = supernatant from non-transduced CHME-5 cells used as negative controls; HTB-T = supernatant collected from transduced HTB-11 cells and CHME-T2 = supernatant from transduced CHME-5 cells; and C + = purified recombinant sTNFR-Fc protein as a positive control.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Binding Assay, Western Blot, Recombinant, Membrane, Incubation, Purification, Positive Control

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Functional antagonization of sTNFR-Fc against TNF-α . As described in materials and methods, TNF-α sensitive L929 cells were treated with TNF-α alone (80 ng/mL) or with TNF-α plus culture supernatants from vector-transduced cells; purified recombinant sTNFR-Fc protein (160 ng/mL) was used as a positive control. After incubation for 24 h, cell viability was then evaluated by MTT assay. Viability was significantly higher for the cells treated with conditioned medium from transduced cells expressing hTNFR-Fc (** p < 0.01 for HTB-T; *p < 0.05 for CHME-T) when compared to cultures that received TNF-α alone, or TNF-α plus culture supernatants from parental cells (CHME-N and HTB-N). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Functional Assay, Plasmid Preparation, Purification, Recombinant, Positive Control, Incubation, MTT Assay, Expressing, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: sTNFR-Fc mediated protection of neuronal cells from TNF-α, HIV-Tat and gp120 . (A) Non-transduced human neuronal cells, HTB-11, were treated with TNF-α alone (80 ng/mL), or with TNF-α plus culture supernatants (1:10 dilution) from vector-transduced cells (as indicated). Cells were incubated at 37°C for 3 days, and viability of the cells was determined by the trypan blue exclusion assay. sTNFR-Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the hTNFR-Fc encoding vector; Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the vector encoding human Fc only; rTNFR = purified commercial recombinant TNFR-Fc protein (160 ng/mL), used as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (B) Vector transduced human HTB-11 cells were exposed to TNF-α as described in 7A. HTB-11 cells transduced either with the sTNFR-Fc or Fc encoding vector were used in this experiment, along with non-transduced cells as controls. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (C) sTNFR-Fc protects primary rat neurons against HIV-1 Tat-mediated toxicity. Medium from vector-transduced or parental HTB-11 cells was collected at day 10, diluted and mixed with 500 nM HIV-1 Tat protein, prior to addition to primary rat neurons. Following 24 hours incubation at 37°C, these test cultures along with the control were analyzed by TUNEL assay. Cell death was significantly reduced in neuronal cultures that were treated with Tat plus conditioned medium from HTB-11 cells transduced with the sTNRF-Fc encoding lentiviral vector (HTB-T 10d) versus cells treated with Tat plus conditioned medium from parental HTB-11 cells (HTB 10d) (*p < 0.01). NT, normal neuronal cells received no Tat, and -, neuronal cells exposed to Tat as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (D) sTNFR-Fc mediated neuronal protection of human HTB-11 cells against HIV-1 gp120 toxicity. Conditioned media from hTNFR-Fc or Fc vector transduced HTB-11 cells were collected, diluted and mixed with 100 ng/mL (gp120A) or 250 ng/mL (gp120B) HIV-1 gp120 protein, with or without HIV-1 Tat, prior to addition to HTB-11 cells. Following 3 days incubation at 37°C, cell viability of these cultures, together with control cultures, was determined by trypan blue exclusion assay (***p < 0.001). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Incubation, Trypan Blue Exclusion Assay, Transduction, Purification, Recombinant, Positive Control, Standard Deviation, TUNEL Assay

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 binds to human TNFR2 and cynomolgus TNFR2 selectively. (A) Binding assay of AN3025 to human TNFR2 on plate by ELISA (EC 50 = 0.052nM). (B) Binding assay of AN3025 to human TNFR1 on plate by ELISA. (C) Binding assay of AN3025 to cynomolgus TNFR2 on plate by ELISA (EC 50 = 0.053nM). (D) Binding assay of AN3025 to mouse TNFR2 on plate by ELISA. (E) Binding assay of AN3025 to rat TNFR2 on plate by ELISA. The ELISA assays were tested in duplicates. Values were expressed as Mean ± SEM.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 inhibits TNFα-TNFR2 mediated cell death of hTNFR2 overexpressing Jurkat cell and enhances T effector cell function in Tregs/Teff co-culture assay. (A) Expression of human TNFR2 on wildtype Jurkat cells. (B) Viability of wildtype Jurkat cells after stimulation with titrated human TNFα. (C) Expression of human TNFR2 on established hTNFR2 overexpressing Jurkat single cell clone. (D) Viability of hTNFR2 overexpressing Jurkat cells after stimulation with titrated human TNFα. (E) Viability of hTNFR2 overexpressing Jurkat cells after stimulation with constant 0.5ng/mL human TNFα in the presence of titrated AN3025 or titrated human IgG 1 , κ. (F) Competitive ELISA binding assay of AN3025 with biotinylated human TNFα to human TNFR2 on plate. (G, H) Human iTregs were co-cultured with CFSE-labeled autologous CD4 + Teff cells for 4 days. (G) CFSE lo T effector cell percentage was quantified by flow cytometry. (H) IFNγ in the supernatants was detected by ELISA assay. Values were expressed as Mean ± SEM. **P < 0.01; ***P < 0.001.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Cell Function Assay, Co-culture Assay, Expressing, Competitive ELISA, Binding Assay, Cell Culture, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 significantly inhibits MC38 tumor growth as a monotherapy in hTNFR2 mouse model. (A–E) Murine colon cancer MC38 cells (5E5) were implanted subcutaneously into homozygous TNFR2 humanized mice (female). Mice were grouped when tumor volume reached approximately 100 mm 3 . (A, B) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg or 1mg/kg every 4 days intraperitoneally for 5 doses in total. (A) Tumor volume measurement during the treatment (n=8 each group). (B) Body weight record during the treatment (n=8 each group). (C) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg every 3 days intraperitoneally for 3 doses in total. Tregs (CD45 + CD3 + CD4 + Foxp3 + ) frequency in total CD4 + T cells in the MC38 tumors was quantified by flow cytometry (n=6 each group). (D, E) MC38 tumor bearing TNFR2 humanized mice were treated intraperitoneally with 10mg/kg AN3025 alone or combination of 10mg/kg AN3025, 0.3mg/mouse anti-mouse CD4 depletion antibody and 0.3mg/mouse anti-mouse CD8 depletion antibody every 4 days for 5 doses in total. (D) Tumor volume measurement during the treatment (n=8 each group). (E) Body weight record during the treatment (n=8 each group). Values were expressed as Mean ± SEM. **P < 0.01; ***P < 0.001.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 enhances anti-tumor efficacy of mouse PD-1 antibody in combination study. (A–H) Murine colon cancer MC38 cells (5E5) were implanted subcutaneously into homozygous TNFR2 humanized mice (female). Mice were grouped when tumor volume reached approximately 100 mm 3 . (A–F) MC38 tumor bearing TNFR2 humanized mice were treated with 10 mg/kg AN3025 or 10mg/kg mouse PD-1 antibody (mPD-1 Ab) every 3 days intraperitoneally for 7 doses in total. (A) Tumor volume measurement during the treatment (n=8 each group). (B) Body weight record during the treatment (n=8 each group). (C) Representative immunohistochemistry images of CD8 staining on MC38 tumors. (D) Quantifications of CD8 + T cells per mm 2 by immunohistochemistry (n=3 each group). (E) Representative immunohistochemistry images of CD4 staining on MC38 tumors. (F) Quantifications of CD4 + T cells per mm 2 by immunohistochemistry (n=3 each group). (G, H) MC38 tumor bearing TNFR2 humanized mice were treated with 3mg/kg AN3025 or 3mg/kg mPD-1 Ab or combination of AN3025 and mPD-1 Ab every 3 days intraperitoneally for 7 doses in total. (G) Tumor volume measurement during the treatment (n=8 each group). (H) Body weight record during the treatment (n=8 each group). Values were expressed as Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Immunohistochemistry, Staining