recombinant human msln  (Sino Biological)

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Biopanning of a phage library to identify mesothelin-binding peptides. (A) Schematic of biopanning. A phage library was incubated with parental HEK 293T cells; unbound phage was then incubated with HEK 293T cells transfected with a green fluorescent protein (GFP)-tagged mesothelin (MSLN) expression vector (MSLN-HEK 293T). Cell-bound phage was eluted and amplified for the next round. Created with BioRender.com . (B) MSLN-HEK 293T cells 48 h after transfection (green) and parental HEK 293T cells stained with an anti-MSLN antibody (red) and diamidino-2-phenylindole (DAPI) (blue). (C) Phage titer enrichment across screening rounds. pfu, plaque-forming units. (D) Phage-cell-binding ELISA showing binding of individual phage clones to parental versus MSLN-expressing HEK 293T cells. OD, optical density.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Incubation, Transfection, Expressing, Plasmid Preparation, Amplification, Staining, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN-expressing cells. (A) Parental and GFP-tagged (green) MSLN-expressing HEK 293T cells were incubated with tetramethylrhodamine-5-maleimide (TAMRA)-labeled MSLNpep or a control peptide (red) at 4 °C for 1 h and DAPI (blue). (B) Immunoblotting of MSLN expression in AsPC-1 and MIA PaCa-2 pancreatic tumor cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) AsPC-1 and MIA PaCa-2 cells were incubated with fluorescein isothiocyanate (FITC)–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red). Nuclei were stained with DAPI (blue), and images were merged. (D and E) AsPC-1 cells were treated with small interfering RNA (siRNA) against MSLN (100 and 200 nM) to knock down MSLN expression or with control (Ctrl) siRNA (200 nM). Cells were incubated with an anti-MSLN antibody (D) or 25 μM FITC–MSLNpep (E) and analyzed by flow cytometry. *** P < 0.001 by one-way ANOVA. (F) AsPC-1 cells were treated with MSLN-siRNA or Ctrl-siRNA (200 nM). After silencing, cells were incubated with 25 μM FITC–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red) (1:1,000). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Expressing, Incubation, Labeling, Control, Western Blot, Staining, Small Interfering RNA, Knockdown, Flow Cytometry

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN protein. (A) HEK 293T and AsPC-1 cell lysates (20, 200, and 400 μg) were incubated with biotin-labeled MSLNpep or control peptide (Ctrlpep) and pulled down with streptavidin-coated magnetic beads. Immunoblotting of the precipitates was performed using an anti-MSLN antibody. (B) Quantification of band intensity (%) for each pull-down relative to input in (A) using ImageJ software. Data are mean ± standard error from 3 independent experiments. *** P < 0.001 by one-way analysis of variance (ANOVA). (C) HEK 293 and AsPC-1 cells were incubated at 4 °C for 1 h with FITC-labeled MSLNpep together with either soluble or mature MSLN. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (D) HEK 293 and AsPC-1 cells were pretreated with an anti-MSLN antibody at room temperature for 1 h and incubated with FITC–MSLNpep (green) at 4 °C for 1 h. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (E) ELISA quantifying MSLNpep binding to soluble and mature MSLN. Data are mean ± standard error from 3 independent experiments. ** P < 0.01 by unpaired t test. (F) In silico modeling of MSLNpep binding to MSLN using the PEPFOLD program. MSLN, green; MSLNpep, blue.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Incubation, Labeling, Control, Magnetic Beads, Western Blot, Software, Staining, Enzyme-linked Immunosorbent Assay, In Silico

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Cytotoxicity of an MSLN-targeted mitochondrial-membrane-damaging peptide. (A) AsPC-1 cells were incubated at 37 °C for 2 h for 1 h with 25 μM FITC-labeled control peptide (Ctrlpep) or MSLNpep (green). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (B) Diagram of MSLNpep-KLA (L-form) and MSLNpep-kla (D-form). (C to E) AsPC-1 (C), MIA PaCa-2 (D), and HEK 293T (E) cells were incubated with phosphate-buffered saline (PBS), Ctrlpep-KLA, Ctrlpep-kla, MSLNpep-KLA, or MSLNpep-kla for 24 h. After incubation, cell viability was measured using Cell Counting Kit reagents. The half-maximal inhibitory concentration (IC50) values are presented as mean ± standard error from 3 independent experiments. Created with BioRender.com .

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Membrane, Incubation, Labeling, Control, Staining, Saline, Cell Counting, Concentration Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Inhibition of orthotopic pancreatic tumor growth by an MSLN-targeted mitochondrial-membrane-damaging peptide. (A) Treatment schema for mice bearing orthotopic AsPC-1-luc pancreatic tumors. Mice received PBS, Ctrlpep-kla, or MSLNpep-kla at 10 mg/kg body weight. (B) Whole-body bioluminescence imaging on days 0 and 16 after treatment. (C) Total bioluminescent flux after treatment. Data are mean ± standard error ( n = 5). *** P < 0.001; ns, not significant by 2-way ANOVA. (D to F) Total bioluminescent flux for each mouse after treatment with PBS (D), Ctrlpep-kla (E), or MSLNpep-kla (F). (G) Survival rates. (H) Body weights. (I) Serum aspartate aminotransferase (AST) levels. (J) Serum alanine aminotransferase (ALT) levels. (K) Serum alkaline phosphatase (ALP) levels. (L) Serum blood urea nitrogen (BUN) levels. (M) Serum creatinine (CRE) levels. Created with BioRender.com .

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Inhibition, Membrane, Imaging

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Cytotoxicity of an MSLN-targeted mitochondrial-membrane-damaging peptide in human pancreatic cancer patient-derived organoids (PDOs). (A) Immunoblot analysis of MSLN levels in human pancreatic cancer PDOs. Red, MSLN-high organoid; blue, MSLN-low organoid. AsPC-1 and MIA PaCa-2 cells served as controls. (B) Cytotoxicity of MSLNpep-kla in human pancreatic cancer PDOs. Red, MSLN-high organoid; blue, MSLN-low organoid. (C) Correlation between MSLN levels and IC50 values in PDOs ( R 2 = 0.7952). (D) Microscopic morphology of SPT#145 and SPT#144 organoids after treatment with Ctrlpep-kla (100 μM) or MSLNpep-kla (25, 50, and 100 μM). Scale bars, 200 μm.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Membrane, Derivative Assay, Western Blot

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Single-cell expression analysis of MSLN in human pancreatic cancer tissues. Violin plots showing the expression distribution of MSLN across various cell types identified within the pancreatic cancer tumor microenvironment (dataset from Ellerby et al. ). The y -axis represents log-normalized expression levels.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Single Cell, Expressing

Journal: Molecular Therapy Oncology

Article Title: Development of potent Affitin-based bispecific NK cell engagers for the therapy of MSLN-expressing cancers

doi: 10.1016/j.omton.2025.201095

Figure Lengend Snippet: Selection of Affitins specific for human mesothelin (A) Schematic representation of the sequential technical steps that led to the selection of antihuman mesothelin (hMSLN) Affitins. (B) Logo representation of Affitin sequences obtained by NGS. The size of each amino acid letter is proportional to its prevalence at a given position. (C) Heatmap representation of the entire dataset. Each line represents a unique sequence (redundant sequences were removed), and each column corresponds to an amino acid position. Colors represent the distance between an amino acid and the consensus sequence according to the Grantham matrix (see ). White lines delimit sequences from different selection rounds (see ). (D) Sequences were clustered into 82 clusters, and a consensus sequence was computed for each cluster. Hierarchical clustering was then applied to represent the clusters on a dendrogram. 25 sequences (yellow dots), representing the global variability, were arbitrarily chosen for further characterization.

Article Snippet: Maxisorp plates (Thermo Fisher Scientific) were coated with either 100 μL of 2 μg/mL recombinant hMSLN (R&D Systems, 3265-MS) or 66 μM NeutrAvidin (Thermo Fisher Scientific) and blocked with 300 μL of PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Selection, Sequencing

Journal: Molecular Therapy Oncology

Article Title: Development of potent Affitin-based bispecific NK cell engagers for the therapy of MSLN-expressing cancers

doi: 10.1016/j.omton.2025.201095

Figure Lengend Snippet: Characterization of the specificity of the most promising antihMSLN Affitins N13 and N18 Affitins were tetramerized on fluorescent streptavidin to facilitate detection. The Affitin tetramers were added to cocultures of fluorescent Meso34-hMSLN (hMSLN+) and unstained Meso34 cells (hMSLN−). Tetramers were either added to the culture medium and incubated statically for 30 min at 37°C or diluted in culture medium and applied to the cocultures under flow (10 μL/min) for 10 min at room temperature. Cells were then washed, stained with phalloidin, and analyzed by confocal microscopy (×60 obj). Scale bars, 20 μm.

Article Snippet: Maxisorp plates (Thermo Fisher Scientific) were coated with either 100 μL of 2 μg/mL recombinant hMSLN (R&D Systems, 3265-MS) or 66 μM NeutrAvidin (Thermo Fisher Scientific) and blocked with 300 μL of PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Incubation, Staining, Confocal Microscopy

Journal: Molecular Therapy Oncology

Article Title: Development of potent Affitin-based bispecific NK cell engagers for the therapy of MSLN-expressing cancers

doi: 10.1016/j.omton.2025.201095

Figure Lengend Snippet: Dimerization of N13 Affitin drastically improves its specific binding to cellular hMSLN (A) A homodimer of the N13 Affitin was produced, and its capacity to bind to recombinant hMSLN fragment 302–359 was assessed by surface plasmon resonance. The affinity constants obtained, calculated with the bivalent model, are displayed in the table in (B). (C–D) Meso34 (MSLN−) and Meso34-MSLN (MSLN+) cells were stained with a concentration range of N13 monomer or dimer, and N13 binding was quantified by flow cytometry ( n = 3 independent experiments). Results are displayed as the means of ratios of median fluorescence intensities (RMFI) +/− SEM, normalized to the median fluorescence intensity of the secondary antibody alone. (E) N13 dimer was diluted to 1 μM in culture medium, and a flow of this solution was applied multiple times on cocultures of fluorescent Meso34-hMSLN cancer cells (MSLN+), nonfluorescent human fibroblasts, and nonfluorescent human endothelial cells. Nuclei were stained with Draq5, endothelial cells were revealed by von Willebrand Factor (vWF) staining after fixation, and the specificity of dimer binding was assessed by confocal microscopy (×60 obj). Scale bars, 20 μm.

Article Snippet: Maxisorp plates (Thermo Fisher Scientific) were coated with either 100 μL of 2 μg/mL recombinant hMSLN (R&D Systems, 3265-MS) or 66 μM NeutrAvidin (Thermo Fisher Scientific) and blocked with 300 μL of PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Binding Assay, Produced, Recombinant, SPR Assay, Staining, Concentration Assay, Flow Cytometry, Fluorescence, Confocal Microscopy

Journal: Molecular Therapy Oncology

Article Title: Development of potent Affitin-based bispecific NK cell engagers for the therapy of MSLN-expressing cancers

doi: 10.1016/j.omton.2025.201095

Figure Lengend Snippet: Affitin-based bispecific NK cell engagers efficiently induce NK-mediated cytotoxicity on Meso34-hMSLN cells (A) Bispecific NK engagers (BiKEs), composed of either a monomer (C21(N13) 1 ) or a dimer (C21(N13) 2 ) of the N13 Affitin fused to the C21 anti-CD16 nanobody, were generated, and their capacity to bind to recombinant hMSLN fragment 302–359 was assessed by surface plasmon resonance. The affinity constants obtained, calculated using the Langmuir model (for C21(N13) 1 ) or the bivalent model (for C21(N13) 2 ), are displayed in the table in (B). (C) Meso34-hMSLN cells were stained with a concentration range of C21(N13) 1 and C21(N13) 2 BiKEs to validate their capacity to bind hMSLN. Binding was quantified by flow cytometry. (D–F) A Cr-51 cytotoxicity assay was performed using the Meso34-hMSLN cell line (hMSLN+) as targets and the NK92CD16h NK cell line as effectors, in the presence of several concentrations of C21(C5) 2 irrelevant BiKE (D), C21(N13) 1 (E), and C21(N13) 2 BiKEs (F). The assay was conducted with effector/target ratios of 20:1 (circles), 10:1 (squares), and 1:1 (triangles). (C–F) Results are expressed as the mean ± SEM of three independent experiments.

Article Snippet: Maxisorp plates (Thermo Fisher Scientific) were coated with either 100 μL of 2 μg/mL recombinant hMSLN (R&D Systems, 3265-MS) or 66 μM NeutrAvidin (Thermo Fisher Scientific) and blocked with 300 μL of PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Generated, Recombinant, SPR Assay, Staining, Concentration Assay, Binding Assay, Flow Cytometry, Cytotoxicity Assay