Journal: Cell reports. Medicine

Article Title: Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

doi: 10.1016/j.xcrm.2023.101134

Figure Lengend Snippet: Figure 3. SARS-CoV-2 IgG and neutralizing antibody responses (A and B) Serum IgG titers against wild-type SARS- CoV-2 RBD protein (A) and pseudovirus neutralizing antibody titers (ID50) against SARS-CoV-2 variants (B) for non-elderly (left side) and elderly (right side) participants (n = 10 per dose group, and n = 6 for the placebo group). Participants received one injection of VLPCOV-01 (0.3, 1.0, or 3.0 mg), 30 mg BNT162b2, or placebo on day 1 (week 0). Logarithmic values are reported as geometric mean titers for serum IgG and neutralizing antibody against pseudovirus. Bars indicate 95% CIs. (C) The correlation between serum neutralizing antibody titers against pseudovirus Wuhan (wild type) and IgG antibody titers against SARS-CoV-2 RBD protein following booster vaccination. Pearson’s product-moment correlation coefficient and p value were calculated following log trans- formation of source data (r = 0.950, p < 0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-human CD154-FITC (clone: TRAP1) BD Biosciences cat# 555699; RRID: AB_396049 Mouse anti-human CD3-BUV615 (clone: SP34-2) BD Biosciences cat# 751249; RRID: AB_2875266 Mouse anti-human CD4-PE-Cy5.5 (clone: S3.5) Thermo Ficher Scientific cat# MHCD0418; RRID: AB_10376013 Mouse anti-human CD8-BUV563 (clone: RPA-T8) BD Biosciences cat# 612914; RRID: AB_2870200 Mouse anti-human CD27-PE-Cy5 (clone: 1A4CD27) Beckman coulter cat# 6607107 Mouse anti-human CD45RO-BUV805 (clone: UCHL1) BD Biosciences cat# 748367; RRID: AB_2872786 Mouse anti-human IFN-g-BV786 (clone: 4S.B3) BioLegend cat# 502542; RRID: AB_2563882 Mouse anti-human TNF-BV650 (clone: MAb11) BioLegend cat# 502938; RRID: AB_2562741 Rat anti-human IL-13-BV421 (clone: JES10-5A2) BD Biosciences cat# 563580; RRID: AB_2738290 Mouse anti-human IL-21-Ax647 (clone: 3A3-N21) BD Biosciences cat# 560493; RRID: AB_1645421 Mouse anti-human IL-4-PE-Cy7 (clone:8D4-8) BD Biosciences cat# 560672; RRID: AB_1727547 Mouse anti-human IL-17A-BV605 (clone: BL168) BioLegend cat# 512326; RRID: AB_2563887 Rat anti-human IL-2-BUV737 (clone: MQ117H12) BD Biosciences cat# 612836 Mouse anti-human CD107A-BV711 (clone: H4A3) BioLegend cat# 328640; RRID: AB_2565840 Mouse anti-human MIP1b-Alexa700 (clone: D21-1351) BD Biosciences cat# 561278; RRID: AB_10612008 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG1 (AS35) ACROBiosystems cat# SAD-S35 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG2 (AS35) ACROBiosystems cat# SAD-S66 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG3 (AS35) ACROBiosystems cat# SAD-S67 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG4 (AS35) ACROBiosystems cat# SAD-S68 Mouse anti-Human IgG1 Fc Secondary Antibody, HRP Thermo Fisher Scientific cat# MH1715; RRID: AB_2539710 Mouse Anti-Human IgG2 Fc-BIOT HP6002 SouthernBiotech cat# 9070-08; RRID: AB_2796638 Mouse Anti-Human IgG3 Hinge-BIOT HP6050 SouthernBiotech cat# 9210-08; RRID: AB_2796700 Mouse Anti-Human IgG4 Fc-BIOT HP6025 SouthernBiotech cat# 9200-08; RRID: AB_2796692 Biological samples Human PBMCs This study This study Chemicals, peptides, and recombinant proteins Benzonase Nuclease, Purity >90% MERCK Millipore cat# 70746 (Continued on next page) Cell Reports Medicine 4, 101134, August 15, 2023 e1

Techniques: Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells

doi: 10.1136/jitc-2023-008460

Figure Lengend Snippet: Characterization of NKG2D BiTE activity. (A) Representative flow cytometry histogram plots after human glioblastoma cells U87 were stained for total surface NKG2DLs using biotinylated recombinant human NKG2D-Fc and streptavidin-PE secondary (NKG2D-Fc-Bio-Strep-PE), biotinylated human IgG1 isotype (IgG1-Fc-Bio-Strep-PE), or streptavidin-PE secondary only (Strep-PE). (B) Schematic representation of NKG2D BiTE and Control BiTE. The ectodomain of human NKG2D receptor (amino acids 78–216) was used for the NKG2D BiTE. For the Control BiTE, amino acid residues in the stirrup loop (B5-B5’ loop) of NKG2D were mutated to abrogate ligand binding. NKG2D/NKG2Db5 ectodomains were linked to an anti-CD3ε single chain variable fragment (scFv) and a second NKG2D/NKG2Db5 ectodomain by flexible glycine-serine linkers. BiTEs contained N-terminal human immunoglobulin signal peptide and C-terminal decahistidine tag for mammalian secretion and protein detection. Diagram created with BioRender.com. (C) U87 cells were treated with 1 nM NKG2D BiTE, 1 nM Control BiTE, 1 μL CD3/CD28 Dynabeads or left untreated and co-cultured with peripheral blood mononuclear cell-derived T cells at an effector:target ratio of 5:1 for 72 hours before assessing expression of CD25, (D) CD69, (E) granzyme B, (F) perforin, (G) CD107a or lysosomal associated membrane protein-1 and (H) intracellular interferon (IFN-γ) on both CD4 + (red) and CD8 + (blue) T cells by flow cytometry. Data represents the mean percentage of positive T cells of triplicate repeats±SD. (I) U87 cell viability was assessed from the same experiments using a viability dye by flow cytometry. U87 cell viability was assessed either as untreated cells in monoculture, untreated co-cultures with T cells, co-cultures treated with 1 nM NKG2D BiTE or 1 nM Control BiTE. Data represents the mean percentage of live U87 cells of triplicate repeats±SD. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post hoc analysis for (C)–(H) and an one-way ANOVA followed by Dunnett’s post hoc test for (I). Significance was assessed versus untreated cells within the relevant group or versus U87 cells alone in (I) (ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001). ANOVA, analysis of variance; BiTE, bispecific T-cell engager; NKG2D, natural killer group 2 member D; NKG2DLs, NKG2D ligands.

Article Snippet: As an isotype control, recombinant human IgG1 (rhIgG1-Fc, (R&D systems)) was also biotinylated.

Techniques: Activity Assay, Flow Cytometry, Staining, Recombinant, Control, Ligand Binding Assay, Cell Culture, Derivative Assay, Expressing, Membrane

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 1. SDS-PAGE (12.5% gel), showing purity of the SAP and soluble L-selectin preparations. In Western blots, SAP was equally detected by anti-SAP (lane 5) or L-selectin (lane 6) antibodies. From left to right: (1) Low-molecular-weight mar- kers, silver stained on blot; (2) SAP (100 ng/lane), silver stained on blot; (3) low-molecular-weight markers, silver stained in gel; (4) SAP (400 ng/lane), silver stained in gel; (5) SAP (800 ng/ lane), immunodetection with anti-SAP mAb CBL305; (6) SAP (1000 ng/lane), immunodetection with human L-selectin-IgG1 chimera in the presence of 5 mM Ca2+ followed by biotinylated mouse anti-human IgG antibody (Fc-specific); (7) soluble L- selectin (3000 ng/lane), silver stained in gel; (8) soluble L- selectin (1000 ng/lane), immunodetection with anti-human L- selectin mAb Dreg200.

Article Snippet: For measurement of mAb 24 binding, 5 105 lymphocytes in 200 lL HBSS (0.6 mM Ca2+, 0.2 mMMg2+, 0.125% human serum albumin) were pre-incubated at 22 C for 10 min with f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 15 lg/mLmAb 24 (kindly provided by Dr. Nancy Hogg and Dr. AlisonMcDowall, Cancer Research UK, London, UK) or murine IgG1 control (R&D Systems) and 15 lg/mL FITC-conjugated F(ab0)2 goat anti-mouse IgG (H+L, Fc-directed) (Dianova, Hamburg, Germany) and then for a further 1 min with or without 11.4 lg/mL Fab fragments of the anti-L-selectin mAb Dreg55 and Dreg200.

Techniques: SDS Page, Western Blot, Molecular Weight, Staining, Immunodetection

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 2. SDS-PAGE (7.5% gel), showing purity of the human L- selectin-IgG1 chimera. In Western blots, the human L-selectin- IgG1 chimera was equally detected by anti-L-selectin antibody (lane 5), anti-human IgG Fc antibody (lane 6) or SAP (lane 7). From left to right: (1) High-molecular-weight markers, silver stained on blot; (2) human L-selectin-IgG1 chimera (100 ng/ lane), silver stained on blot; (3) high-molecular-weight mar- kers, silver stained in gel; (4) human L-selectin-IgG1 chimera (400 ng/lane), silver stained in gel; (5) human L-selectin-IgG1 chimera (13 ng/lane), immunodetection with biotinylated anti- human L-selectin mAb Dreg200; (6) human L-selectin-IgG1 chimera (25 ng/lane), immunodetection with biotinylated mouse anti-human IgG antibody (Fc-specific); (7) human L- selectin-IgG1 chimera (200 ng/lane), immunodetection with biotinylated SAP in the presence of 5 mM Ca2+.

Article Snippet: For measurement of mAb 24 binding, 5 105 lymphocytes in 200 lL HBSS (0.6 mM Ca2+, 0.2 mMMg2+, 0.125% human serum albumin) were pre-incubated at 22 C for 10 min with f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 15 lg/mLmAb 24 (kindly provided by Dr. Nancy Hogg and Dr. AlisonMcDowall, Cancer Research UK, London, UK) or murine IgG1 control (R&D Systems) and 15 lg/mL FITC-conjugated F(ab0)2 goat anti-mouse IgG (H+L, Fc-directed) (Dianova, Hamburg, Germany) and then for a further 1 min with or without 11.4 lg/mL Fab fragments of the anti-L-selectin mAb Dreg55 and Dreg200.

Techniques: SDS Page, Western Blot, High Molecular Weight, Staining, Immunodetection

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 3. Binding of various concentrations of biotinylated SAP or anti-L-selectin mAb Dreg200 to L-, P- or E-selectin-IgG chimeras coated on microtiter wells. Bound SAP or antibody was detected luminometrically after reaction with ExtrAvidin- alkaline phosphatase (RLU, relative light units). Prior treatment of biotinylated SAP with sialidase or N-glycosidase did not result in altered binding curves. Virtually no binding of biotinylated SAP was observed to uncoated wells or to wells coated with any selectin chimera in the absence of Ca2+ (EDTA). Means SD of four independent experiments.

Article Snippet: For measurement of mAb 24 binding, 5 105 lymphocytes in 200 lL HBSS (0.6 mM Ca2+, 0.2 mMMg2+, 0.125% human serum albumin) were pre-incubated at 22 C for 10 min with f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 15 lg/mLmAb 24 (kindly provided by Dr. Nancy Hogg and Dr. AlisonMcDowall, Cancer Research UK, London, UK) or murine IgG1 control (R&D Systems) and 15 lg/mL FITC-conjugated F(ab0)2 goat anti-mouse IgG (H+L, Fc-directed) (Dianova, Hamburg, Germany) and then for a further 1 min with or without 11.4 lg/mL Fab fragments of the anti-L-selectin mAb Dreg55 and Dreg200.

Techniques: Binding Assay

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 4. Impact of Dreg200 or Dreg55, murine IgG1 control, heparin, fucoidan, asialofetuin or a2-macroglobulin on the binding of biotinylated SAP (1 lg/mL in TBS containing 5 mM Ca2+) to wells coated with human L-selectin-IgG1 chimera. Means of three independent experiments (SD never exceeded 0.035 RLU).

Article Snippet: For measurement of mAb 24 binding, 5 105 lymphocytes in 200 lL HBSS (0.6 mM Ca2+, 0.2 mMMg2+, 0.125% human serum albumin) were pre-incubated at 22 C for 10 min with f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 15 lg/mLmAb 24 (kindly provided by Dr. Nancy Hogg and Dr. AlisonMcDowall, Cancer Research UK, London, UK) or murine IgG1 control (R&D Systems) and 15 lg/mL FITC-conjugated F(ab0)2 goat anti-mouse IgG (H+L, Fc-directed) (Dianova, Hamburg, Germany) and then for a further 1 min with or without 11.4 lg/mL Fab fragments of the anti-L-selectin mAb Dreg55 and Dreg200.

Techniques: Control, Binding Assay

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 7. SPR measurements demonstrating Ca2+-dependent binding of mouse L-selectin-IgG*protein G*gold conjugates to immobilized SAP. Reaction of mouse L-selectin-human IgG*protein G*gold conjugates (20 nm or 40 nm diameter) to SAP covalently attached to a Biacore CM5 chip (track 2), as opposed to BSA (track 1). The four cycles show the difference in RU between track 2 and track 1 at a flow rate of 40 lL/min. The left arrow of each cycle marks the injection at the start of the cycle. Cycle 1: 20-nm gold conjugate + 1 mM Ca2+; cycle 2: 20- nm gold conjugate + 3 mM EDTA; cycle 3: 40-nm gold conjugate + 1 mM Ca2+; cycle 4: 40-nm gold conjugate + 3 mM EDTA. The right arrows of the four cycles mark the start points of injections of EDTA buffer without conjugate to regenerate the chip. X axis: time (minutes).

Article Snippet: For measurement of mAb 24 binding, 5 105 lymphocytes in 200 lL HBSS (0.6 mM Ca2+, 0.2 mMMg2+, 0.125% human serum albumin) were pre-incubated at 22 C for 10 min with f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 15 lg/mLmAb 24 (kindly provided by Dr. Nancy Hogg and Dr. AlisonMcDowall, Cancer Research UK, London, UK) or murine IgG1 control (R&D Systems) and 15 lg/mL FITC-conjugated F(ab0)2 goat anti-mouse IgG (H+L, Fc-directed) (Dianova, Hamburg, Germany) and then for a further 1 min with or without 11.4 lg/mL Fab fragments of the anti-L-selectin mAb Dreg55 and Dreg200.

Techniques: Binding Assay, Injection

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 8. In flow chamber assays with an effective wall shear stress of 2.0–2.6 dyn/cm2 (representative for L-selectin-depen- dent adhesion), the number of adherent neutrophils per high power field (HPF) to confluent layers of TNF-a-stimulated (500 U/mL for 4 h at 37C; TEBU GmbH, Germany) or un- stimulated (control) human umbilical cord endothelial cells was counted with or without SAP (0.1 or 1 lmol/L) or the anti-L- selectin antibody Dreg200 (50 lg/mL) being present during the experiment (means SD; n = 3–9). The insert shows adhesion of neutrophils to TNF-a-activated endothelial cells in the presence of varying concentrations of Dreg 200 (black; 50, 10, or 1 lg/mL), murine IgG1 isotype (light grey; 50, 10, or 1 lg/mL) and fucoidan (dark grey; 50, 5, or 0.5 lg/mL).

Article Snippet: For measurement of mAb 24 binding, 5 105 lymphocytes in 200 lL HBSS (0.6 mM Ca2+, 0.2 mMMg2+, 0.125% human serum albumin) were pre-incubated at 22 C for 10 min with f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 15 lg/mLmAb 24 (kindly provided by Dr. Nancy Hogg and Dr. AlisonMcDowall, Cancer Research UK, London, UK) or murine IgG1 control (R&D Systems) and 15 lg/mL FITC-conjugated F(ab0)2 goat anti-mouse IgG (H+L, Fc-directed) (Dianova, Hamburg, Germany) and then for a further 1 min with or without 11.4 lg/mL Fab fragments of the anti-L-selectin mAb Dreg55 and Dreg200.

Techniques: Shear, Control

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 9. Up-regulation of the lymphocyte activation-depen- dent integrin neoepitope (mAb 24 epitope) by 10 min of incubation at 37C with 3 mM Mn2+ (positive control, top) or with 5 lg/mL SAP (bottom). Filled histograms: Staining at 0 min (identical to IgG1 negative control histograms at 0 and 10 min). Full-line histograms: Expression at 10 min. Dotted-line histo- gram: Partial inhibition by Dreg200 Fab.

Article Snippet: For measurement of mAb 24 binding, 5 105 lymphocytes in 200 lL HBSS (0.6 mM Ca2+, 0.2 mMMg2+, 0.125% human serum albumin) were pre-incubated at 22 C for 10 min with f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 15 lg/mLmAb 24 (kindly provided by Dr. Nancy Hogg and Dr. AlisonMcDowall, Cancer Research UK, London, UK) or murine IgG1 control (R&D Systems) and 15 lg/mL FITC-conjugated F(ab0)2 goat anti-mouse IgG (H+L, Fc-directed) (Dianova, Hamburg, Germany) and then for a further 1 min with or without 11.4 lg/mL Fab fragments of the anti-L-selectin mAb Dreg55 and Dreg200.

Techniques: Activation Assay, Incubation, Positive Control, Staining, Negative Control, Expressing, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.

doi: 10.4049/jimmunol.176.3.1889

Figure Lengend Snippet: FIGURE 7. Abs to IL-1 or IL-6 do not inhibit chemokine secretion in response to TWEAK. C57BL/6 mesangial cells were stimulated for 24 h with and without 100 ng/ml TWEAK and 5 g/ml monoclonal rat anti- mouse IL-1 and IL-6. Rat IgG is an IgG2b mAb that was the rat isotype- matched control. A, CCL2/MCP-1; B, CCL5/RANTES; C, CXCL1/KC. Shown here are the mean SD values for duplicate wells; similar results were found in several independent experiments.

Article Snippet: We found that there was significant binding to the surface of murine mesangial cells by hFc-TWEAK (Fig. 1B), but not by a CTLA4-Ig control fusion protein (R&D Systems) or a human IgG1 mAb (PHP010; Serotec), which share the same IgG1 Fc as hFc-TWEAK (data not shown).

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.

doi: 10.4049/jimmunol.176.3.1889

Figure Lengend Snippet: FIGURE 9. Murine anti-TWEAK mAbs prevent the induction of che- mokines in response to TWEAK stimulation of mesangial cells. The 2.P5.G9 and 2P2.D10 are two IgG2a mouse mAbs against murine TWEAK, derived by immunization of TWEAK knockout mice, as de- scribed in Materials and Methods. P1.17 is an isotype-matched control Ab. C57BL/6 mesangial cells were left untreated, or treated with TWEAK with and without these mAbs at a concentration of 5 g/ml for 24 h. Shown here are the mean SD values for duplicate wells; similar results were found in several independent experiments.

Article Snippet: We found that there was significant binding to the surface of murine mesangial cells by hFc-TWEAK (Fig. 1B), but not by a CTLA4-Ig control fusion protein (R&D Systems) or a human IgG1 mAb (PHP010; Serotec), which share the same IgG1 Fc as hFc-TWEAK (data not shown).

Techniques: Derivative Assay, Knock-Out, Control, Concentration Assay