Journal: bioRxiv

Article Title: Targeting Mesothelin via the native T cell receptor

doi: 10.64898/2025.12.02.689353

Figure Lengend Snippet: (A) Specificity of generated cell lines (n = 29) evaluated by IFNγ ELISpot. 21 of the 29 lines exhibited anti-MSLN activity (defined as detection of >50 SFC/2×10 5 input cells). (B) Mean (± SEM) fold expansion of MSLN-specific T cells in the responder cohort (n=21). (C) Phenotype, memory/activation profile and exhaustion markers of MSLN-specific T cells in responders, reported as % expression (mean±SEM, n=8-21). MSLN, mesothelin; ELISpot, enzyme-linked immunospot; IFNγ, interferon gamma; SFC, spot-forming cells.

Article Snippet: To detect MSLN expression in cancer cell lines, 2 × 105 cells were washed twice and stained with 2 μl of a monoclonal, anti-human, recombinant MSLN antibody (RRID:AB_2733680) (Miltenyi Biotec), diluted 1:50 in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Generated, Enzyme-linked Immunospot, Activity Assay, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Targeting Mesothelin via the native T cell receptor

doi: 10.64898/2025.12.02.689353

Figure Lengend Snippet: (A) Representative donor data (left panel) and summary results (mean±SEM, n=21; right panel) showing CD8⁺/IFNγ⁺ and CD4⁺/IFNγ⁺ T cells, as assessed by ICS. (B) Representative donor data (left panel) and summary results (mean±SEM; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4⁺ and CD8⁺ MSLN-specific T cells, as assessed by ICS. (C) Polyfunctionality of MSLN-specific T cells as measured by FluoroSpot (IFNγ/Granzyme B/TNFα) (n=6). Top panel shows the total frequency (SFC/2×10 5 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.

Article Snippet: To detect MSLN expression in cancer cell lines, 2 × 105 cells were washed twice and stained with 2 μl of a monoclonal, anti-human, recombinant MSLN antibody (RRID:AB_2733680) (Miltenyi Biotec), diluted 1:50 in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Staining

Journal: bioRxiv

Article Title: Targeting Mesothelin via the native T cell receptor

doi: 10.64898/2025.12.02.689353

Figure Lengend Snippet: (A) Representative donor data showing lysis of MSLN-loaded autologous PHA blasts in the absence or presence of MHC class I or class II blocking antibodies (triplicates, mean ± SD). (B) Summary cytotoxicity data (n = 17). (C) Killing of cell lines endogenously expressing MSLN by partially HLA-matched MSLN-specific T cells from three independent donors (triplicates, mean ± SD). Cytotoxicity in all panels was assessed by ⁵¹Cr release assay following a 5–8 h co-culture at a 40:1 effector-to-target (E:T) ratio. PHA, phytohemagglutinin; MHC, major histocompatibility complex.

Article Snippet: To detect MSLN expression in cancer cell lines, 2 × 105 cells were washed twice and stained with 2 μl of a monoclonal, anti-human, recombinant MSLN antibody (RRID:AB_2733680) (Miltenyi Biotec), diluted 1:50 in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Lysis, Blocking Assay, Expressing, Release Assay, Co-Culture Assay, Immunopeptidomics

Journal: bioRxiv

Article Title: Targeting Mesothelin via the native T cell receptor

doi: 10.64898/2025.12.02.689353

Figure Lengend Snippet: (A) Illustration of the experimental procedure for the 3D co-culture assay using GFP/FFluc+ tumor spheroids and effector T cells. (B-C) Tumor spheroid bioluminescence in the absence of T cells (Tumor), and in the presence of MSLN or Control T cells at the indicated E:T ratios. Tumor spheroids were derived from the CFPAC-1 (B) and MS751 (C) cell lines.

Article Snippet: To detect MSLN expression in cancer cell lines, 2 × 105 cells were washed twice and stained with 2 μl of a monoclonal, anti-human, recombinant MSLN antibody (RRID:AB_2733680) (Miltenyi Biotec), diluted 1:50 in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Co-culture Assay, Control, Derivative Assay

Journal: bioRxiv

Article Title: Targeting Mesothelin via the native T cell receptor

doi: 10.64898/2025.12.02.689353

Figure Lengend Snippet: (A) To identify immunogenic peptide epitopes T cell lines were screened against individual MSLN minipools by IFNγ ELISpot. Panel A shows results for donor 15 (results reported as SFC/2×10 5 ), with the potential stimulatory peptides shown in panel B. (C) To identify the immunogenic epitopes the MSLN T cell line was exposed to each of the peptides identified in panel B – screening was performed by ICS for IFNγ and results reported as % of CD3+/IFNγ+ cells. (D) Summary of identified immunogenic MSLN peptides across all 21 cell lines and color coded to identify the number of responders, with immunodominant peptides (found to elicit responses in ≥3 donors) shown in panel E.

Article Snippet: To detect MSLN expression in cancer cell lines, 2 × 105 cells were washed twice and stained with 2 μl of a monoclonal, anti-human, recombinant MSLN antibody (RRID:AB_2733680) (Miltenyi Biotec), diluted 1:50 in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Targeting Mesothelin via the native T cell receptor

doi: 10.64898/2025.12.02.689353

Figure Lengend Snippet: Identification of HLA-restricting alleles for the immunodominant MSLN peptides. (A) For peptide 30: ICS (IFNγ production) analysis to determine whether reactive T cells were detected in the CD4+ or CD8+ T cell compartment (left panel). ⁵¹Cr release assay (5-8 hr co-culture) using peptide-pulsed autologous and partially HLA-matched PHA blasts as targets (right panel). Results show % specific lysis (mean±SD), E:T 40:1. Panels B-D show similar data for peptides 145, 91, and 128/129, respectively. HLA, Human Leukocyte Antigen.

Article Snippet: To detect MSLN expression in cancer cell lines, 2 × 105 cells were washed twice and stained with 2 μl of a monoclonal, anti-human, recombinant MSLN antibody (RRID:AB_2733680) (Miltenyi Biotec), diluted 1:50 in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich).

Techniques: Release Assay, Co-Culture Assay, Lysis

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: Clinical and Experimental Medicine

Article Title: A proteomics approach to identify predictive blood biomarkers for pleural mesothelioma in prospective cohorts

doi: 10.1007/s10238-026-02058-x

Figure Lengend Snippet: Proteomics Workflow in EPIC and MoMar Cohorts. Left (EPIC): SWATH LC-MS/MS profiled 323 plasma proteins from 21 asbestos-exposed cases (PM within 5 years) and 21 matched controls. A GLM identified 12 DEPs ( p ≤ 0.05, fold change ≥ 1.3 ≤ 0.75); four were ELISA-validated. Calretinin and mesothelin benchmark ELISA assays were performed. Combined markers underwent ROC analysis Right (MoMar): Three DEPs from EPIC were quantified by ELISA alongside calretinin and mesothelin. ROC analysis of the combined protein panel assessed reproducibility and predictive accuracy in the pre-diagnosis cohort

Article Snippet: The measurements of serum calretinin and mesothelin were determined by ELISA assays in the EPIC samples using the Calretinin ELISA kit (DLD Diagnostika, Hamburg, Germany) and Human Mesothelin ELISA kit (R&D systems Inc.) as previously described [ ].

Techniques: Data-independent acquisition, Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Clinical and Experimental Medicine

Article Title: A proteomics approach to identify predictive blood biomarkers for pleural mesothelioma in prospective cohorts

doi: 10.1007/s10238-026-02058-x

Figure Lengend Snippet: ELISA validation of candidate DEPs in pre-diagnostic EPIC sera. Box plots display serum concentrations of B2M (ng/mL), C4 (µg/mL), TF (µg/mL), and DCD (µg/mL) in cases sampled 0–2 years and 2–5 years before PM diagnosis versus matched controls. B2M showed a borderline significant increase in the 0–2 year group (t-test and GLM p < 0.05); TF, C4, and DCD trended similarly but were not significant. Asterisks indicate nominal p < 0.05

Article Snippet: The measurements of serum calretinin and mesothelin were determined by ELISA assays in the EPIC samples using the Calretinin ELISA kit (DLD Diagnostika, Hamburg, Germany) and Human Mesothelin ELISA kit (R&D systems Inc.) as previously described [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Diagnostic Assay

Journal: Clinical and Experimental Medicine

Article Title: A proteomics approach to identify predictive blood biomarkers for pleural mesothelioma in prospective cohorts

doi: 10.1007/s10238-026-02058-x

Figure Lengend Snippet: ELISA replication of candidate DEPs in MoMar plasma. Box plots show concentrations of B2M (ng/mL), TF (µg/mL), and C4 (µg/mL) in pre-diagnostic cases versus asbestos-exposed non-cancer controls. No significant differences were observed for any marker

Article Snippet: The measurements of serum calretinin and mesothelin were determined by ELISA assays in the EPIC samples using the Calretinin ELISA kit (DLD Diagnostika, Hamburg, Germany) and Human Mesothelin ELISA kit (R&D systems Inc.) as previously described [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Diagnostic Assay, Marker

Journal: Clinical and Experimental Medicine

Article Title: A proteomics approach to identify predictive blood biomarkers for pleural mesothelioma in prospective cohorts

doi: 10.1007/s10238-026-02058-x

Figure Lengend Snippet: ELISA quantification of calretinin and mesothelin in pre-diagnostic EPIC and MoMar samples. ( A ) EPIC serum: calretinin (ng/mL) and mesothelin (nM) levels in cases sampled 0–2 years and 2–5 years before diagnosis versus controls. ( B ) MoMar plasma: calretinin and mesothelin levels in 0–2 years and 2–5 years pre-diagnostic cases compared to asbestos-exposed non-cancer controls. Calretinin was significantly elevated in MoMar cases at 0–2 years ( p < 0.01), whereas EPIC increases were not significant. Mesothelin levels reached significance only in MoMar ( p < 0.0001). Asterisks indicate significance (** p < 0.01; *** p < 0.0001)

Article Snippet: The measurements of serum calretinin and mesothelin were determined by ELISA assays in the EPIC samples using the Calretinin ELISA kit (DLD Diagnostika, Hamburg, Germany) and Human Mesothelin ELISA kit (R&D systems Inc.) as previously described [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Biomarker Discovery, Clinical Proteomics

Journal: Clinical and Experimental Medicine

Article Title: A proteomics approach to identify predictive blood biomarkers for pleural mesothelioma in prospective cohorts

doi: 10.1007/s10238-026-02058-x

Figure Lengend Snippet: Receiver operating characteristics (ROC) analysis of combined biomarker panels in pre-diagnostic PM cases. ( A ) EPIC cohort: ROC curves for models using B2M alone (mod1, AUC = 0.71), B2M + C4 + TF (mod2, AUC = 0.81), Mesothelin + Calretinin (mod3, AUC = 0.65), and all five markers (mod4, AUC = 0.88; p-value = 0.17 vs. mod1 by DeLong’s test). ( B ) MoMar cohort: ROC curves for the same models (mod1 AUC = 0.55), mod2 (AUC = 0.75), mod3 (AUC = 0.84), and mod4 (AUC = 0.91; p = 0.001 vs. mod1)

Article Snippet: The measurements of serum calretinin and mesothelin were determined by ELISA assays in the EPIC samples using the Calretinin ELISA kit (DLD Diagnostika, Hamburg, Germany) and Human Mesothelin ELISA kit (R&D systems Inc.) as previously described [ ].

Techniques: Biomarker Discovery, Diagnostic Assay

Journal: Research

Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

doi: 10.34133/research.1105

Figure Lengend Snippet: Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

Article Snippet: The cells were again centrifuged and resuspended with 1 μl (10 μl/10 6 cells) Human Mesothelin Alexa Fluor 488-conjugated Antibody (R&D Systems, FAB32652G) and 0.5 μl of Invitrogen CellTrace Calcein Red-Orange, AM (Fisher Scientific, 50-113-7411) in 500 μl of flow cytometry buffer.

Techniques: Binding Assay, Electroporation, Membrane, Flow Cytometry, Control, Expressing, Imaging, Cell Characterization

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