Journal: bioRxiv

Article Title: SUMO Inhibition Plus CD40 Agonism Increases Anti-Tumor Immunogenicity Through Interferon Mediated Macrophage Activation

doi: 10.1101/2024.12.03.626688

Figure Lengend Snippet: A) Experimental timeline. Mice were injected with 2,000 KPC46 cells and enrolled into one of four groups: untreated, TAK-981, anti-CD40, and combination. Mice were enrolled and randomized when tumors reached 3-5mm in diameter as measured by ultrasonography. Mice were dosed with TAK-981 at 15mg/kg every 48 hours and αCD40 at 100µg every 7 days. B) Survival curves for untreated (median survival: 19 days), TAK-981(median survival: 15.5 days, p= 0.879), αCD40(median survival: 20 days, p=0.417), and combination (median survival: 24.5 days, p=0.0005). Survival curves were analyzed with the log rank test. C) Tumors were generated as in panel A and treated for 12 days with either vehicle + IgG control, TAK-981, αCD40, or TAK-981+αCD40. Tumor weights for vehicle + IgG (1.10 g), TAK-981(0.85 g, p=0.31), αCD40 (0.76 g, p=0.11), combination (0.57 g, p= 0.01). Weight comparisons to the vehicle + IgG group were conducted using a two tailed t-test. D) Mean tumor diameters for vehicle + IgG (12.3 mm), TAK-981 (11.13 mm, p=0.67), αCD40 (11.69, p=0.93), and combination (9.05mm, p=0.05). Comparisons to vehicle + IgG group were conducted using a two tailed t-test. E) Representative hematoxylin and eosin staining for tumors in the various treatment groups.

Article Snippet: Anti-CD40 antibody was obtained from BioXCell (BioXCell clone FGK4.5/FGK45, Cat. No.: BP0016-2) and diluted in a pH 7.0 dilution buffer supplied by BioXCell (Cat. No. IP0070) according to manufacturer instructions.

Techniques: Injection, Generated, Control, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: SUMO Inhibition Plus CD40 Agonism Increases Anti-Tumor Immunogenicity Through Interferon Mediated Macrophage Activation

doi: 10.1101/2024.12.03.626688

Figure Lengend Snippet: A) Umap of tumor cell populations sorted by cell type (left) and treatment condition (right). B) Stacked bar graph of tumor cellular composition for each treatment condition. T Cell Quantification (Vehicle + IgG: 3.05%, TAK-981: 7.4%, αCD40: 1.4%, Combination: 10.8%, Effect Size for Combination vs Vehicle + IgG: 1.54 . Macrophage quantification (Vehicle + IgG: 7.1%, TAK-981: 8.9%, αCD40: 6.7%, combination: 13.0%, Effect Size for anti-CD40 vs Combination: 0.79). C) Umap of lymphoid immune cell populations per treatment condition. D) Umap of myeloid immune cell populations per treatment condition. E) GSEA Analysis Pairwise Comparisons for IFNα-Macrophages (Left): Combination vs αCD40 (NES: 2.33, p<0.0001), Combination vs TAK-981 (NES:2.24, p<0.0001), and Combination vs Vehicle + IgG (IFNα-NES: 2.70, p<0.0001). T Cells (Right): Combination vs αCD40 (NES: 1.75, p= 0.005), Combination vs TAK-981 (NES: 1.16, p=0.42), and Combination vs Vehicle + IgG (NES: 2.17, p<0.0001. F) GSEA Analysis Pairwise Comparisons for IFNγ-Macrophages (Left): Combination vs αCD40 (NES: 1.82, p=0.0002), Combination vs TAK-981 (NES: 1.75, p<0.0001), and Combination vs Vehicle + IgG (NES: 2.18, p<0.0001). T Cells (Right): Combination vs anti-CD40 (NES: 1.69, p=0.0009), Combination vs TAK-981 (NES: 0.97, p=0.99), and Combination vs Vehicle + IgG (-NES: 1.94, p <0.0001).G) Violin plots for MHC-I expression (H2-K1, H2-D1) for TAK+ αCD40, monotherapy, and vehicle + IgG conditions. Pairwise comparison differential expression coefficients for H2-K1 (Combination vs anti-CD40: 0.76, Combination vs TAK: 0.06, Combination vs Vehicle + Control IgG: 0.60; p<0.002 for all pairwise comparisons) and H2-D1 (Combination vs anti-CD40: 0.68, Combination vs TAK-981: 0.13, Combination vs Vehicle + IgG: 0.63; p<0.001 for all comparisons)

Article Snippet: Anti-CD40 antibody was obtained from BioXCell (BioXCell clone FGK4.5/FGK45, Cat. No.: BP0016-2) and diluted in a pH 7.0 dilution buffer supplied by BioXCell (Cat. No. IP0070) according to manufacturer instructions.

Techniques: Expressing, Comparison, Control

Journal: bioRxiv

Article Title: SUMO Inhibition Plus CD40 Agonism Increases Anti-Tumor Immunogenicity Through Interferon Mediated Macrophage Activation

doi: 10.1101/2024.12.03.626688

Figure Lengend Snippet: A) Violin plots for TGBβ1 (combination vs αCD40: −0.35, combination vs TAK-981: −0.72, combination vs vehicle + IgG: −0.93, p<0.001 for all pairwise comparisons) and TGFβ2(combination vs anti-CD40: −1.35, combination vs TAK-981: −0.76, combination vs vehicle + IgG: −1.22, p<0.001 for all pairwise comparisons) differential expression coefficient levels in the combination treatment compared to monotherapy and untreated groups. B) Cell chat graphs showing decreased macrophage/epithelial and macrophage fibroblast interactions for combination vs αCD40, combination vs TAK-981, and combination vs vehicle + IgG conditions. C) Relative TGFβ information flow between epithelial cells/macrophages, fibroblasts/macrophages, and macrophages/macrophages for each of the following pairwise comparisons: combination vs αCD40, combination vs TAK-981, combination vs vehicle + IgG. For the following conditions, there were significant decreases in TGFβ signaling for all pairwise comparisons: macrophages vs. macrophages, macrophages vs. fibroblasts, fibroblasts vs. macrophages, and epithelial cells vs. macrophages.

Article Snippet: Anti-CD40 antibody was obtained from BioXCell (BioXCell clone FGK4.5/FGK45, Cat. No.: BP0016-2) and diluted in a pH 7.0 dilution buffer supplied by BioXCell (Cat. No. IP0070) according to manufacturer instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: SUMO Inhibition Plus CD40 Agonism Increases Anti-Tumor Immunogenicity Through Interferon Mediated Macrophage Activation

doi: 10.1101/2024.12.03.626688

Figure Lengend Snippet: A) Representative immunofluorescence images of CD8 (orange) and CD4 (green) T cell infiltration in an untreated orthotopically generated tumor taken at the time of the animal’s euthanasia. B) Representative immunofluorescence images of CD8 (orange) and CD4 (green) T cell infiltration in an orthotopically generated tumor exposed to combination treatment and taken at the time of the animal’s euthanasia. C) T cell flow cytometry of tumors treated with vehicle + IgG, TAK-981, αCD40, or TAK-981 + αCD40 after 12 days of treatment. (Left) Flow cytometry quantification for CD3+ T Cells (vehicle + IgG:6.77%, TAK-981: 5.05%, αCD40: 11.28%, combination: 23.13%, p<0.02 for all pairwise comparisons to combination group), CD8+ T Cells (vehicle + IgG:1.08%, TAK-981: 0.48%, αCD40: 1.50%, combination: 9.04%, p<0.01 for all pairwise comparisons to combination group). (Right) Representative flow cytometry plots for each condition. D) (Left) Flow cytometry quantification for CD4+ T Cells (vehicle + IgG: 3.87%, TAK-981: 2.43%, αCD40: 6.61%, combination: 7.22%, ANOVA p= 0.14), TReg Cells (vehicle + IgG: 45.05%, TAK-981:52.33%, αCD40: 43.70%, combination: 18.40%, p<0.001 for combination vs vehicle + IgG and TAK-981. (Right) Representative flow cytometry plots for each condition. E) Spleen flow cytometry after 12 days of CD8 depleting antibody with sample flow cytometry plots for CD8 and CD4 T Cells (CD8 T Cells: KLH: 12.54%, αCD8: 0.05%, p<0.001). F) Survival curves for Selective CD8 T Cell Depletion. Tumors were generated via orthotopic injection of 2,000 KPC46 cells into mouse pancreases, and treatments were started after tumors reached 3-5 mm in diameter as measured via ultrasound imaging. Vehicle + IgG (median survival: 16 days), Combination + KLH isotype control (median survival: 21 days), Combination + αCD8 depletion antibody (median survival: 20 days). Survival comparisons were done with Log Rank test. Vehicle + IgG vs TAK-981 + αCD40 + CD8 depletion p = 0.04. Combination + KLH vs TAK+ αCD40 +CD8 depletion p = 0.67. G) Survival curves for B6 Nude Mice bearing orthotopically injected KPC46 pancreatic tumors either treated with vehicle + IgG controls (median survival 14 days) or TAK-981 + αCD40 combination (median survival 17 days), p= 0.032 as computed by the log rank test.

Article Snippet: Anti-CD40 antibody was obtained from BioXCell (BioXCell clone FGK4.5/FGK45, Cat. No.: BP0016-2) and diluted in a pH 7.0 dilution buffer supplied by BioXCell (Cat. No. IP0070) according to manufacturer instructions.

Techniques: Immunofluorescence, Generated, Flow Cytometry, Injection, Imaging, Control

Journal: bioRxiv

Article Title: SUMO Inhibition Plus CD40 Agonism Increases Anti-Tumor Immunogenicity Through Interferon Mediated Macrophage Activation

doi: 10.1101/2024.12.03.626688

Figure Lengend Snippet: A) Immunohistochemistry staining for F4/80 and corresponding quantification in B6 nude mouse tumors treated with vehicle + IgG (39.5%) or TAK-981 +αCD40 (65.95%), p=0.008. B) Survival curves for clodronate-mediated macrophage Depletion. Vehicle + IgG (median survival: 13.5 days), TAK-981 + αCD40 +PBS liposome (median survival: 23 days), TAK-981 + αCD40 + clodronate liposome (median survival: 14 days). Survival comparisons with Log Rank test: Vehicle + IgG vs TAK-981 + αCD40 + PBS liposome, p = 0.01; Vehicle + IgG vs. TAK-981 + αCD40 + clodonrate liposome p = 0.39. C) (Left) Flow cytometry for spleen macrophages (vehicle + IgG: 8.57 %, TAK-981 + αCD40 + PBS liposome: 7.8 %, TAK-981 + αCD40 + clodronate liposome: 4.24%, p=0.01) with p-values comparing PBS liposome to clodronate liposome group. (Right) Representative flow cytometry plots for PBS liposome and clodronate liposome groups. D) Flow cytometry for tumor-associated macrophages with representative flow cytometry plots (vehicle + IgG: 11.9%, TAK-981 + αCD40 + PBS: 22.5%, TAK-981 + αCD40 +Clodronate: 12.6%, p=0.014), tumor resident macrophages (vehicle + IgG: 0.78%, TAK-981 + αCD40 +PBS: 1.10%, TAK-981 + αCD40 + clodronate: 0.67%, p=0.11), tumor dendritic cells (vehicle + IgG: 63.2%, TAK-981 + αCD40 + PBS.: 55.6%, TAK-981 + αCD40 + clodronate: 14.9%, p<0.01), and splenic dendritic cells (vehicle + IgG: 13.4%, TAK-981 + αCD40 +PBS: 6.2%, TAK-981 + αCD40 + clodronate: 6.2%, p=0.2) with p-values comparing PBS liposome to clodronate liposome groups. E) Flow cytometry for tumor specific CD8+ T cells (vehicle + Control IgG: 1.07%, TAK-981 + αCD40 + PBS: 4.95%, TAK-981 + αCD40 + clodronate: 3.85%, p=0.25), CD69+ CD8+ T Cells (TAK-981 + αCD40 + PBS: 39.5%, TAK-981 + αCD40 + clodronate: 15.6%, p=0.01), PD-1+ CD8+ T cells (TAK-981 + αCD40 + PBS: 78.7%, TAK-981 + αCD40 + clodronate: 65.5%, p=0.052) with p-values comparing PBS liposome to clodronate liposome groups.

Article Snippet: Anti-CD40 antibody was obtained from BioXCell (BioXCell clone FGK4.5/FGK45, Cat. No.: BP0016-2) and diluted in a pH 7.0 dilution buffer supplied by BioXCell (Cat. No. IP0070) according to manufacturer instructions.

Techniques: Immunohistochemistry, Staining, Flow Cytometry, Control

Journal: Nature communications

Article Title: Anti-CTLA4 treatment reduces lymphedema risk potentially through a systemic expansion of the FOXP3 + T reg population.

doi: 10.1038/s41467-024-55002-6

Figure Lengend Snippet: Fig. 2 | Increased infiltration with CD4+, CD25+ and FOXP3+ cells, as well as increased CD4, CD25 and CTLA4 expression characterizes lymphedema tissue. A–C Representative immunohistological images and quantification of histological analysis in paraffin skin tissue sections. An increased infiltration of CD4+ (A), CD25+ (B) and FOXP3+ (C) cells was detected in skin sections from lymphedema patients. Arrows indicate positive cells. D The evaluation of the CD4, CTLA4, CD25 and

Article Snippet: Treatment administration: 100 μg InVivoPlus anti-mouse CTLA4 (Cat. Nr. BP0131, clone 9H10, BioXCell) or the corresponding InVivoPlus polyclonal Syrian hamster IgG control antibody (Cat. Nr. BP0087, BioXCell) diluted in InVivoPure pH7.0DilutionBuffer (Cat. Nr. IP0070, BioXCell)were administeredonday3, 6, 9 and 12 after surgery through intraperitoneal injection.

Techniques: Expressing

Journal: Nature communications

Article Title: Anti-CTLA4 treatment reduces lymphedema risk potentially through a systemic expansion of the FOXP3 + T reg population.

doi: 10.1038/s41467-024-55002-6

Figure Lengend Snippet: Fig. 3 | Anti-CTLA4 treatment exhibits efficacy in the lymphedema mouse tail model. A Surgery was performed on day 0 (D0), treatment was given on day 3, 6, 9 and 12 (D3, D6, D9, D12) and evaluation/endpoint was in day 14 (D14). B Anti- CTLA4 administration (aCTLA4) following the surgical induction of lymphedema leads to significantly reduced edema 1 and 2 weeks (W) after surgery. (N(C) = 10 and N(aCTLA4)=10) C Representative photographs of tails of control and anti-CTLA4 (aCTLA4)–treated mice two weeks postoperatively. D Representative images of near-infrared intravital microscopy of the tail lymphatic network ~1.5 cm distally to the surgical site, visualized through the uptake and transport of a lymphatic- specific fluorescent tracer 10 and 20 min after infusion near the tip of the tail. Quantification of lymphatic vascular transport based on fluorescence intensity revealed significantly increased lymphatic vessel function in the anti-CTLA4

Article Snippet: Treatment administration: 100 μg InVivoPlus anti-mouse CTLA4 (Cat. Nr. BP0131, clone 9H10, BioXCell) or the corresponding InVivoPlus polyclonal Syrian hamster IgG control antibody (Cat. Nr. BP0087, BioXCell) diluted in InVivoPure pH7.0DilutionBuffer (Cat. Nr. IP0070, BioXCell)were administeredonday3, 6, 9 and 12 after surgery through intraperitoneal injection.

Techniques: Control, Intravital Microscopy

Journal: Nature communications

Article Title: Anti-CTLA4 treatment reduces lymphedema risk potentially through a systemic expansion of the FOXP3 + T reg population.

doi: 10.1038/s41467-024-55002-6

Figure Lengend Snippet: Fig. 4 | Anti-CTLA4 treatment does not influence the Th1/Th2 balance but results in a distinct serum cytokine milieu. A, B The evaluation of the Th1 (Il2, Infγ, Tnfα and Cxcr3) and Th2 (Il10, Il13, Ccr4 and Ccr8) marker mRNA expression in lymphedematous tail skin suggests that the Th1/Th2 immune response balance is not affected after 2 weeks of anti-CTLA4 treatment (aCTLA4) in lymphedema. C Quantification of altered tissue cytokines, indicating that only Il1-α increases locally in the lymphedematous tissue in response to anti-CTLA4 treatment. D Normalized heatmap of serum cytokines. Quantification of significantly altered serum cytokines, indicating the increased levels of Tnfα, Ccl3, Ccl4 and Il5 while the levels ofCcl5 decrease in response to the treatment. (N(C) = 10 mice and N(aCTLA4) =9 mice). E Representative flow cytometry analysis graphs of the phenotypic characterization performed to assess the circulating lymphoid compartment using PBMCs (gated on CD45+/CD11b−) in mice treated with anti-CTLA4 versus controls.

Article Snippet: Treatment administration: 100 μg InVivoPlus anti-mouse CTLA4 (Cat. Nr. BP0131, clone 9H10, BioXCell) or the corresponding InVivoPlus polyclonal Syrian hamster IgG control antibody (Cat. Nr. BP0087, BioXCell) diluted in InVivoPure pH7.0DilutionBuffer (Cat. Nr. IP0070, BioXCell)were administeredonday3, 6, 9 and 12 after surgery through intraperitoneal injection.

Techniques: Marker, Expressing, Cytometry

Journal: Cancer Science

Article Title: Intratumor Fusobacterium nucleatum promotes the progression of pancreatic cancer via the CXCL1‐CXCR2 axis

doi: 10.1111/cas.15901

Figure Lengend Snippet: Effects of intratumor Fusobacterium nucleatum on myeloid‐derived suppressor cell (MDSC) recruitment to the tumor immune microenvironment. (A) Experimental schema. KPC cells were subcutaneously implanted into C57/B6 mice. From 1 week after implantation, the mice were intratumorally injected with F. nucleatum or BHI and treated with an anti‐Ly6G Ab or isotype control Ab every 3 days for 2 weeks. (B) Representative quantified graph of tumor weights. (C) Representative in situ images of tumors. Tumor growth promoted by F. nucleatum was significantly suppressed by the anti‐Ly6G Ab. (D) Representative flow cytometric analysis of CD11b + Gr‐1 + MDSCs, CD8 + T cells, CD11c + MHCII + F4/80 − dendritic cells, and F4/80 + macrophages gated on CD45 + cells. (E) Representative immunohistochemical staining of CD8a + T cells. Original magnification, ×100. Scale bar, 50 μm. * p < 0.05, ** p < 0.01.

Article Snippet: To deplete myeloid‐derived suppressor cells (MDSCs), six doses of 100 μg/dose of InVivoMAb anti‐mouse Ly6G (clone 1A8; BioXCell, Cat # BP0075‐1) or isotype control antibody (clone 2A3, BioXcell, Cat # BP0089) diluted in InVivoPure pH 7.0 Dilution Buffer (BioXcell, Cat # IP0070) was intraperitoneally injected at 72‐h intervals unless specified otherwise.

Techniques: Derivative Assay, Injection, Control, In Situ, Immunohistochemical staining, Staining