Journal: Clinical Cancer Research

Article Title: A Novel Chemoimmunomodulating Property of Docetaxel: Suppression of Myeloid-Derived Suppressor Cells in Tumor Bearers

doi: 10.1158/1078-0432.ccr-10-0733

Figure Lengend Snippet: Fig. 4. A, MDSCs from docetaxel-treated mice express M1 markers. MDSCs were affinity column purified from three mice per group representing naive mice, docetaxel-treated naive mice, tumor bearers, and docetaxel-treated tumor bearers. They were immunostained for CCR7 and iNOS to represent M1 markers or MR to represent a specific M2 marker before flow cytometric analysis. A, histogram representation of MR, CCR7, and iNOS levels in naive mice, docetaxel-treated naive mice, tumor bearers, and docetaxel-treated tumor bearers. B, columns, mean fluorescence intensity units of CCR7, MR, and iNOS from three independent experiments; bars, SE. C, in vivo docetaxel-treated MDSCs have increased differentiation markers. MACS column– purified MDSCs from the above treatment groups were analyzed by flow cytometry for MHC class II, CD11c, CD40, CD80, and CD85 expression. Gray filled histograms are isotype controls, dotted lines represent the docetaxel-treated MDSCs, and black solid lines represent untreated MDSCs.

Article Snippet: Anti-Ly6G and anti-Ly6C ylated antibodies, FITC-conjugated anti-LY6C, APCgated anti-LY6C, PE-conjugated anti-Ly6G, FITCgated anti-mouse CD40, PE-conjugated anti-mouse , FITC-conjugated anti-mouse CD80, APC-conjugated ouse CD11c, and PE-conjugated anti–mouse I Ad purchased from BD Biosciences; rat anti-mouse MR (CD206) was from AbD Serotec; and streptavidin beads were purchased from Miltenyi Biotec.

Techniques: Affinity Column, Purification, Marker, Fluorescence, In Vivo, Flow Cytometry, Expressing

Journal: Clinical and Vaccine Immunology

Article Title: Evidence of a Functional B-Cell Immunodeficiency in Adults Who Experience Serogroup C Meningococcal Disease

doi: 10.1128/cvi.00485-08

Figure Lengend Snippet: FIG. 3. T-cell help to B cells. B-cell activation in response to T-cell help was assessed by CD86 expression on cells stimulated with --dex along with T cells activated through CD3 and CD28. PBLs were incubated in the presence of various stimuli for 4 days, with some having previously been labeled with CFSE. Cells were then stained for CD19 and CD86. Mean increases in CD86 expression are shown for control or patient B cells after incubation of lymphocytes with either anti-CD3/CD28 (a) or both anti-CD3/CD28 and --dex (b). There was no significant difference in the ability of patient or control B cells to be activated in response to either CD3/CD28 stimulation alone or in response to the combination of --dex with the two T-cell stimuli. (c) PBLs from two donors were stimulated as described above, but with the addition of a blocking anti-CD154 monoclonal antibody or an isotype control antibody (mouse immunoglobulin) in some of the wells. (d and e) CD19 B-cell proliferation of patient or control B cells after incubation of PBLs with CD3/CD28-activated T cells (d) or with CD3/CD28 and --dex (b). There was no significant difference in the ability of patient or control B cells to proliferate in response to the T-cell activation signals alone, but additional proliferation in response to --dex was reduced in the patient cells in comparison with that in controls (P 0.015; Wilcoxon signed rank test).

Article Snippet: Anti-human CD154 antibody was purchased from R&D Systems Inc. - -Dex was prepared by us as described previously (20, 25).

Techniques: Activation Assay, Expressing, Incubation, Labeling, Staining, Control, Blocking Assay, Comparison

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Dendritic cells in the bladder microenvironment of mice responding to CD40 agonism have higher expression of IL-15Rα. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody. ( B ) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 24 post-tumor implantation (n = 3-6 mice per group; bars represent SD). Responders (gray circles) and non-responders (blue circles) to anti-CD40 antibody therapy are indicated. ( C ) IL-15Rα mean fluorescence intensity across mice on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 24 post-tumor implantation (n = 3-6 mice per group). *p < 0.05, **p < 0.01.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Expressing, Luciferase, Imaging, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Endogenous IL-15 participates in the therapeutic activity of CD40 agonism. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody and/or anti-IL-15 blocking antibody or isotype-matched control antibody. ( B ) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 12 post-tumor implantation (n = 5 mice per group; bars represent SD). ( C ) Representative flow cytometry plots and quantification across mice (n = 5 mice per group) of CD44 hi CD122 + CD8 T cells (top) and CXCR6 + CD44 hi CD122 + CD8 T cells (bottom) in the bladders of mice treated as outlined in A. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Blocking Assay, Control, Luciferase, Imaging, Tumor Implantation, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: DCs in human non-muscle invasive bladder cancer express high levels of IL-15. Eight primary tumor samples from either untreated NMIBC patients (n=3) or from NMIBC patients that had progressed from NMIBC to MIBC following BCG therapy (n=5) were included in this single-cell RNA sequencing analysis. A) Phenograph clustering was performed and 29 clusters were identified. Canonical lineage markers for cell populations were utilized to assign cell identities to each cluster. B) tSNE plots were created for four representative markers: CD40, CD40L, IL15, and IL15RA. C) A matrix plot with column scaled expression of the same four markers of interest is shown to highlight the distribution and intensity of expression for each gene across each subset.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: CD40 agonism induces IL-15/IL-15Rα upregulation on dendritic cells in the bladder tumor microenvironment. Mice bearing orthotopic MB49 bladder tumors were treated intravesically with anti-CD40 antibody, BCG, or isotype-matched control antibody on days 6 and 9 post-tumor implantation. ( A ) Representative histogram and ( B ) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). ( C ) Representative flow cytometry plots and ( D ) quantification across mice of IL-15 and IL-15Rα surface expression on cDC1s in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). Left graph is gated on cDC1s and depicts proportions of cDC1s that are double-positive for surface IL-15 and IL-15Rα. Right graph is gated on IL-15Rα-expressing cDC1s and depicts proportions of cDC1s expressing IL-15Rα that is occupied by IL-15. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Control, Tumor Implantation, Fluorescence, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: CD40 agonism induces IL-15Rα upregulation on dendritic cells in the UPPL1541 bladder tumor microenvironment. Mice bearing orthotopic UPPL1541 bladder tumors were treated intravesically with anti-CD40 antibody or isotype-matched control antibody on days 6 and 9 post-tumor implantation. ( A ) Representative histogram and ( B ) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). **p < 0.01, ***p < 0.001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Control, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances primary anti-tumor activity. ( A ) Schematic of the treatment of humanized hCD40/hFcyR mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody 2141-V11 and/or IL-15 or control (isotype-matched control antibody and/or vehicle). ( B ) Representative intravital luciferase imaging (left) and luminescence quantification (right) across mice at day 14 post-tumor implantation (n = 5 mice per group; bars represent SD). ( C ) Survival (top) and representative intravital luciferase imaging (bottom) of surviving mice at day 85 post-tumor implantation treated as outlined in A. ( D ) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across mice treated with anti-CD40 antibody 2141-V11 and IL-15 or control in the absence or presence of additional anti-CD8 T cell depleting antibody (n = 5 mice per group; bars represent SD). ( E ) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across WT or Batf3 -/- mice treated with anti-CD40 antibody 1C10 and IL-15 or control (n = 5 mice per group; bars represent SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Control, Luciferase, Imaging, Tumor Implantation

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination of Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances anti-tumor activity against UPPL1541 tumors. ( A ) Schematic of the treatment of humanized hCD40/hFcyR mice bearing orthotopic UPPL1541 bladder tumors with anti-CD40 antibody 2141-V11 and/or IL-15 or control (isotype-matched control antibody and/or vehicle). ( B ) Representative bladder ultrasound imaging (left) at day 45 post-tumor implantation and disease-free survival (right) of mice treated as outlined in A. *p < 0.05, **p < 0.01.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Control, Imaging, Tumor Implantation

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: IL-15 therapy does not modulate myeloid CD40 expression. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with IL-15 or vehicle control. ( B ) Bladder weights across mice at day 12 post-tumor implantation (n = 4-5 mice per group; bars represent SD). ( C ) CD40 mean fluorescence intensity across mice on total dendritic cells (DC; defined as F4/80 − Ly-6G − CD11c + MHCII + ), type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), monocytes (defined as CD11b hi Ly6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 4-5 mice per group). ns = not significant.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Expressing, Control, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances anti-tumor memory responses. ( A ) Schematic of subcutaneous rechallenge (ten-fold tumor cell dose) of humanized hCD40/hFcyR mice surviving long-term (>90 days after initial orthotopic MB49 bladder tumor implantation) following initial therapy with anti-CD40 antibody 2141-V11 with or without IL-15. ( B ) Tumor growth (in the absence of any additional therapy) of long-term survivors compared with naïve mice receiving an equivalent MB49 tumor cell implant (n = 2-5 mice per group; bars represent SD). ( C ) Representative flow cytometry plots and quantification across mice (n = 2-5 mice per group) of CD44 hi CD122 + CD8 T cells (top), CD44 hi CD122 + CD62L + CD8 T cells (middle), and CD11b + KLRG1 + CD27 − NK cells (bottom) in the peripheral blood of mice surviving long-term (day 110) after primary tumor implantation and tumor rechallenge. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Tumor Implantation, Flow Cytometry