mouse anti wt1 mab  (Novus Biologicals)

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) Binding of WT1-specific mAbs to immobilized WT1 peptide or DP4/WT1 monomers was assessed by ELISA. Left: mAbs were added to the ELISA plate coated with WT1 330-348 or CLIP (control). Right: mAbs were added to the ELISA plate coated with DP4/WT1330-348 monomers or DP4/CLIP monomers (control). The data shown represent the mean ± SD of experiments performed in triplicate. Results are representative of two independent experiments.ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Bonferroni tests. (B) Quantification of binding between WT1-specific mAb, 5H2, and WT1 328-348 . Interaction strength was measured by a biolayer interferometry (BLI) binding assay. (C) Quantification of binding between WT1-specific mAb, 5H2, and DP4/WT1 328-348 monomers. Interaction strength was measured by a BLI binding assay. (D) Flow cytometry plot of surface CAR expressions (stained by protein L) versus tag (ΔNGFR) expressions. (E) IFN-γ secretion by WT1-TCR T cells against K562 cells expressing the indicated HLA-II molecules pulsed with WT1 328-348 at an E:T ratio of 5:1 for 20-24 h measured by ELISpot. n = 3 donors. (F) Western blot analysis of WT1 expressions in K562/WT1 KO , K562/WT1 OE , and wild-type K562 (wt).

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Flow Cytometry, Staining, Expressing, Enzyme-linked Immunospot, Western Blot

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) Schematic representation of the WT1-TCR and WT1-CAR constructs. (B) Cytotoxicity of WT1-TCR or WT1-CAR T cells against the indicated K562 expressing DP4 with WT1 gene knockout (KO) or overexpression (OE) at an E:T ratio of 5:1 for 18 h, measured by flow cytometry-based killing assay. n = 3 donors. (C) Cytotoxicity of WT1-TCR or -CAR T cells against WT1 OE K562 expressing the indicated HLA-II molecules at an E:T ratio of 5:1 for 18 h was measured by flow cytometry-based killing assays. HLA-II-null, WT1 OE K562 (WT) was used as a negative control. n = 3 donors. (B-C) Data shown represents the mean ± SD of all donors. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA multiple comparison test with Bonferroni tests.

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Construct, Expressing, Gene Knockout, Over Expression, Flow Cytometry, Negative Control, Comparison

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) WT1-TCR or CAR T cells were stimulated with T2/DP4 pulsed with different deletion peptides for 20-24 h. MAGE-A3 243-258 was used as a negative control. IFN-γ secretion was measured by ELISpot. n = 6 donors. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA multiple comparison test with Bonferroni tests. (B) WT1-TCR or CAR T cells were stimulated with T2/DP4 pulsed with WT1 328-348 (WT) or WT1 328-348 peptide with alanine substitution at indicated amino acid positions for 20-24 h. IFN-γ secretion was measured by ELISpot analysis. n = 3 donors. (A-B) Data showing the mean ± SD of all donors. (C) Competitive binding assays with T2 cells expressing HLA-DP4, pulsed with graded concentrations of WT1 328-348 alanine mutant at specified positions in the presence of 1 µM biotinylated-CLIP peptide. Poor binders (Methods) are marked in red. Data showing the mean ± SD from three independent studies. (D) Structural modelling of WT1 330-348 presented in HLA-DP4 by AlphaFold3 showing HLA surface electrostatic charge (red: negative, blue: positive), peptide-HLA H-bonds (light blue dash), intra-peptide H-bonds (yellow dash), anchors identified in 2B (magenta), residues forming H-bonds (yellow).

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Negative Control, Enzyme-linked Immunospot, Comparison, Binding Assay, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: WT1-TCR or CAR T cells were stimulated with (A) T2/DP2 or (B) T2/DP5 pulsed with different deletion peptides, for 20-24 h. MAGE-A3 243-258 was used as a negative control. IFN-γ secretion was measured by ELISpot analysis. n = 6 donors. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA multiple comparison test with Bonferroni tests.

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Negative Control, Enzyme-linked Immunospot, Comparison

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) WT1-CAR T cells were stimulated with T2/DR1501 or T2/ DQ9.2 pulsed with WT1 328-348 (WT) or WT1 328-348 substituted with alanine at indicated positions for 20-24 h. IFN-γ secretion was measured by ELISpot. n = 3 donors. (B) Competitive binding assay with T2 cells expressing HLA-DR1501, pulsed with graded concentration of WT1 328-348 alanine mutant at specified positions in the presence of 1 µM biotinylated-CLIP peptide. Poor binders (Methods) are marked in red. Data showing the mean ± SD from 3 independent studies. (C) Structural modelling of WT1 330-348 presented in HLA-DR1501 by AlphaFold3 showing HLA surface electrostatic charge (red: negative, blue: positive), peptide-HLA H-bonds (light blue dash), intra-peptide H-bonds (yellow dash), anchors identified in 2B (magenta), residues forming H-bonds (yellow).

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Enzyme-linked Immunospot, Competitive Binding Assay, Expressing, Concentration Assay, Mutagenesis

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) Long-term cytotoxicity of WT1-CAR T cells against WT1 OE K562 expressing the indicated HLA-II molecules at an E:T ratio of 1:5 was measured by impedance-based xCELLigence killing assays. Target cell cytotoxicity was calculated based on cell index (Methods). n = 5 donors. Data showing the mean ± SD of all donors. P values calculated at endpoint (85 h) with two-way ANOVA multiple comparison test with Tukey tests. (B) Cell cluster populations (>3%) that were identified to be significantly influenced by time in all T cells as determined by two-way paired ANOVA. (C) Long-term cytotoxicity of WT1-TCR or CAR T cells against target cells K562/DP4+DQ1502+DR9.1/WT1 at an E:T ratio of 20:1 measured by fluorescence signal-based xCELLigence killing assay. Target cell cytotoxicity was calculated based on total fluorescence intensity (Methods). n = 5 donors. Data showing the mean ± SD of all donors. P value calculated with cytotoxicity value at 85 h with paired t-test. (D) Cell cluster populations (>2%) that were identified to be significantly influenced by time as determined using two-way paired ANOVA. (E) Cell cluster populations (>5%) that were identified to be significantly influenced by effector cell type as determined using two-way paired ANOVA followed by t-test comparison of each day.

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Expressing, Comparison, Fluorescence

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) Flow cytometric gating for CD45 + CD3 + T cell population before CyTOF analysis for (D) and (E). (B) (Left) UMAP plots for all profiles from CyTOF, colored according to the identified cell types. (Right) UMAP plots for all profiles showing origin of cells (WT1-TCR or WT1-CAR). (C) Heatmap of the median marker intensities of all the lineage and phenotype markers across the 21 cell populations identified in (B) (excluding CD8 c9, DN 3, identified to be T cell-K562 cell doublets populations).

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Marker

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) WT1-CAR T cells were stimulated with T2 cells expressing the indicated HLA-II molecules pulsed with WT1 330-348 or WT1 235-243 (control) or cross-reactive peptide candidates for 20-24 h (Supplementary Table S3); IFN-γ secretion was measured by ELISpot analysis. The data shown represent the mean ± SD of experiments performed in triplicate. Results are representative of two independent experiments. (B) Surface HLA-II and intracellular WT1 expressions in primary human CD34 + hematopoietic samples from three different healthy donors were measured by flow cytometric analysis after staining with isotype control, anti-HLA-II mAb or anti-WT1 mAb.

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Expressing, Control, Enzyme-linked Immunospot, Staining

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: (A) Cytotoxicity of WT1-CAR or control-CAR (CD19-specific) T cells against indicated cell lines and human primary CD34 + hematopoietic cells at an E:T ratio of 5:1 for 6 h as measured by flow cytometry-based killing assays. n = 6 donors. (B) Cytotoxicity of WT1-TCR or CAR T cells against the indicated leukemia and lymphoma cell lines at an E:T ratio of 1:1 for 18 h was measured by flow cytometry-based killing assays. n = 3 donors. (C) Cytotoxicity of WT1-CAR or CD19-CAR (control) transduced T cells against primary ALL or AML samples at an E:T ratio of 5:1 for 18 h was measured by flow cytometry-based killing assays. n = 3 donors. (B-C) HLA-II and WT1 expression were measured by flow cytometric analysis. < 3% positive (-); 3-33% positive (+); 33-66% positive (++); > 66% positive (+++). (A-C) Data shown represents the mean ± SD of all donors. ****P<0.0001 by two-way ANOVA with Bonferroni tests.

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Control, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Peptide-Specific CARs Recognize WT1 Promiscuously Presented by Diverse HLA Class II Alleles

doi: 10.1101/2025.06.30.660492

Figure Lengend Snippet: Expressions of surface HLA-II and intracellular WT1 proteins of (A) cell lines and (B) primary ALL or AML samples were measured by flow cytometric analysis after staining with isotype control or anti-HLA-II mAb or anti-WT1 mAb.

Article Snippet: For intracellular WT1 expression detection, target cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with mouse anti-WT1 mAb (Novus Biologicals), followed by BV421-anti-mouse IgG (Poly4053, BioLegend).

Techniques: Staining, Control

Journal: Cell reports

Article Title: A molecular atlas of the human postmenopausal fallopian tube and ovary from single-cell RNA and ATAC sequencing

doi: 10.1016/j.celrep.2022.111838

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse WT1 Clone 6F-H2 , Thermo Fisher Scientific , Cat#MA1–46028; RRID:AB_962464.

Techniques: Recombinant, Saline, RNAscope, Multiplex Assay, Diagnostic Assay, Software, Gene Expression, Microscopy, Real-time Polymerase Chain Reaction