Journal: Clinical & Translational Immunology

Article Title: Novel combination immunotherapy for pancreatic cancer: potent anti‐tumor effects with CD40 agonist and interleukin‐15 treatment

doi: 10.1002/cti2.1165

Figure Lengend Snippet: Combining IL‐15 with a CD40 agonist results in decreased tumor volume and increased survival. C57BL/6j mice were injected with either 0.5 × 10 6 Panc02 or KPC cells subcutaneously. When tumors reached a size of 25–35 mm 2 , mice were randomised and treated with isotype control, IL‐15, CD40 agonist or IL‐15 + CD40. (a) Treatment scheme showing timing of dosing is indicated for IL‐15 (2.5 µg) with black arrows and for CD40 agonist or the corresponding isotype with red arrows (five doses of 12.5 µg for Panc02 or first dose 200 µg and consecutive four doses 100 µg for KPC). (b, c) Tumor growth kinetics are depicted [ n = 5 or 6 mice per group, representative data of 3 (Panc02) or 2 (KPC) independent experiments]. One‐way ANOVA with Bonferroni post hoc . (d, e) Survival of Panc02 ( n = 17) and KPC ( n = 11) mice treated as indicated. Pooled data of 3 (Panc02) or 2 (KPC) independent experiments. Survival was determined by tumor size reaching 150 mm 2 . Log‐rank test. (f, g) Waterfall plots showing the % change in tumor area relative to baseline after 34 days (Panc02, n = 17) or 35 days (KPC, n = 11). Pooled data of 3 (Panc02) or 2 (KPC) independent experiments. All data represent mean ± SEM. * P ˂ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: A mouse agonistic CD40 monoclonal antibody (Clone FGK‐45, BioXCell, obtained via Bio‐connect, Huissen, The Netherlands) or corresponding isotype control (Clone 2A3, BioXCell) was administered i.p. at days 0, 3, 7, 10 and 14 at a dosage of 12.5 μg per mouse for Panc02 or 200 μg (day 0) and 100 μg (days 3, 7, 10 and 14) for KPC tumors.

Techniques: Injection, Control

Journal: Clinical & Translational Immunology

Article Title: Novel combination immunotherapy for pancreatic cancer: potent anti‐tumor effects with CD40 agonist and interleukin‐15 treatment

doi: 10.1002/cti2.1165

Figure Lengend Snippet: Immune cell depletion. C57BL/6j mice bearing Panc02 or KPC tumors were treated with isotype control or the IL‐15 + CD40 agonist combination regimen alone or with depleting antibodies against CD4, CD8, asialo‐GM1 (NK cell depletion). (a, b) Tumor growth kinetics of Panc02 or KPC tumors either non‐treated (isotype), treated with the combination regimen only (no depletion) or combination and depletion antibodies. One‐way ANOVA with Bonferroni post hoc . Data points represent mean ± SEM. n = 5–7 mice/group, representative data of two independent experiments. (c, d) Waterfall plots showing the % change in tumor area relative to baseline after 17 days. (e–k) Survival of Panc02 or KPC‐bearing mice either non‐treated (isotype), treated with the combination regiment (no depletion) or the combination and depletion antibodies against CD4 (e, f) , CD8 (g, h) , asialo‐GM1 (i, j) or CD8 + asialo‐GM1 (k, l) . Data pooled from two independent experiments with n = 10 or 11 (Panc02) or n = 11–13 (KPC). Log‐Rank test. ns P ≥ 0.05; * P < 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: A mouse agonistic CD40 monoclonal antibody (Clone FGK‐45, BioXCell, obtained via Bio‐connect, Huissen, The Netherlands) or corresponding isotype control (Clone 2A3, BioXCell) was administered i.p. at days 0, 3, 7, 10 and 14 at a dosage of 12.5 μg per mouse for Panc02 or 200 μg (day 0) and 100 μg (days 3, 7, 10 and 14) for KPC tumors.

Techniques: Control

Journal: Clinical & Translational Immunology

Article Title: Novel combination immunotherapy for pancreatic cancer: potent anti‐tumor effects with CD40 agonist and interleukin‐15 treatment

doi: 10.1002/cti2.1165

Figure Lengend Snippet: Characterisation of tumor‐infiltrating lymphocytes. C57BL/6j mice bearing KPC tumors were treated with isotype control, IL‐15, CD40 agonist or the combination of the latter. (a) Tumors were harvested at day 8 post‐treatment initiation. Single‐cell suspensions were acquired after enzymatic digestion for flow cytometry analysis. Immune cell populations indicated as fold change of absolute number of cells and CD69 expression (MFI) on NK, NKT and CD8 + T cells. Data pooled from three independent experiments, n = 13–16/group. One‐way ANOVA with Bonferroni. * P < 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. (b) Heatmap of gene expression of relevant genes for the quantified immune subsets. n = 5 tumors/group.

Article Snippet: A mouse agonistic CD40 monoclonal antibody (Clone FGK‐45, BioXCell, obtained via Bio‐connect, Huissen, The Netherlands) or corresponding isotype control (Clone 2A3, BioXCell) was administered i.p. at days 0, 3, 7, 10 and 14 at a dosage of 12.5 μg per mouse for Panc02 or 200 μg (day 0) and 100 μg (days 3, 7, 10 and 14) for KPC tumors.

Techniques: Control, Flow Cytometry, Expressing, Gene Expression

Journal: Clinical & Translational Immunology

Article Title: Novel combination immunotherapy for pancreatic cancer: potent anti‐tumor effects with CD40 agonist and interleukin‐15 treatment

doi: 10.1002/cti2.1165

Figure Lengend Snippet: Characterisation of DCs in tumor and TDLN. C57BL/6j mice bearing KPC tumors were treated with isotype control, IL‐15, CD40 agonist or the combination of the latter. Tumors or TDLN were harvested at day 8 post‐treatment initiation. Single‐cell suspensions were acquired after enzymatic digestion for flow cytometry analysis. (a, b) DCs or CD103 + DCs in tumors. (c, d) DCs or CD103 + DCs in TDLN. Data pooled from three independent experiments, n = 10–16/group. One‐way ANOVA with Bonferroni. * P < 0.05; ** P ≤ 0.01; **** P ≤ 0.0001. (e) Heatmap of gene expression of relevant genes for the quantified immune subsets. n = 5 tumors/group.

Article Snippet: A mouse agonistic CD40 monoclonal antibody (Clone FGK‐45, BioXCell, obtained via Bio‐connect, Huissen, The Netherlands) or corresponding isotype control (Clone 2A3, BioXCell) was administered i.p. at days 0, 3, 7, 10 and 14 at a dosage of 12.5 μg per mouse for Panc02 or 200 μg (day 0) and 100 μg (days 3, 7, 10 and 14) for KPC tumors.

Techniques: Control, Flow Cytometry, Gene Expression

Journal: Clinical & Translational Immunology

Article Title: Novel combination immunotherapy for pancreatic cancer: potent anti‐tumor effects with CD40 agonist and interleukin‐15 treatment

doi: 10.1002/cti2.1165

Figure Lengend Snippet: Rechallenge experiments. C57BL/6j mice cured from Panc02 or KPC tumors after treatment with IL‐15 + CD40 agonist were re‐injected with the same tumor type at the contralateral side of the abdomen. (a, b) Tumor kinetics and survival (log‐rank test) of mice rechallenged with Panc02 tumor cells, n = 16. (c, d) Tumor kinetics and survival of mice rechallenged with KPC tumor cells, n = 9. (e, f) Flow cytometry quantification of intra‐tumoral CD8 + Effector or Memory T cells of KPC tumor‐bearing mice after 8 days following treatment ( n = 9). One‐way ANOVA with Bonferroni. ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: A mouse agonistic CD40 monoclonal antibody (Clone FGK‐45, BioXCell, obtained via Bio‐connect, Huissen, The Netherlands) or corresponding isotype control (Clone 2A3, BioXCell) was administered i.p. at days 0, 3, 7, 10 and 14 at a dosage of 12.5 μg per mouse for Panc02 or 200 μg (day 0) and 100 μg (days 3, 7, 10 and 14) for KPC tumors.

Techniques: Injection, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity

doi: 10.1084/jem.20171440

Figure Lengend Snippet: Combination of CSF-1R inhibition and αCD40 treatment leads to rejection of MC38 tumors. (A) αCSF-1R treatment indicates up-regulation of genes belonging to activation signatures. Single-cell suspension of whole tumors was obtained 16 h after in vivo treatment with either mIgG1 or αCSF-1R (30 mg/kg; n = 4 each; tumors were treated on day 10 and 11, respectively, after inoculation at a mean tumor volume of ∼200 mm 3 ), and RNA sequencing was performed. Data were compared with activation signatures (Table S1), and genes regulated by αCSF-1R compared with control belong to at least one of the general activation signatures (genes marked in orange are associated with general activation, and genes marked in blue are associated with suppression). (B) MC38 tumor–bearing mice were treated using 30 mg/kg mIgG1, 30 mg/kg αCSF-1R weekly, 4 mg/kg αCD40 once, or a combination of both targeting antibodies, starting treatment at either 80 mm 3 or 200 mm 3 tumor volume. (C) MC38 tumor–bearing mice were either treated with 30 mg/kg mIgG1 ( n = 10) or four times with 4 mg/kg of an anti-CD8α antibody ( n = 9) to deplete CD8 + T cells. Isotype control treatment started on day 10, and the anti-CD8α treatment was given on days 7, 9, and 11. Graphs show mean ± SEM of tumor volumes and statistical analyses by two-tailed Student’s t test for each time point depicted. (D) MC38 tumor–bearing mice were treated using 30 mg/kg mIgG1 or 10 mg/kg αPD-1 (three times a week for 2 wk), starting treatment at either 120 mm 3 or 225 mm 3 tumor volume. (E) MC38 tumor–bearing mice were treated using 30 mg/kg mIgG1, 4 mg/kg αCTLA-4 weekly, 10 mg/kg αPD-1 once, or a combination of both targeting antibodies as well as 4 mg/kg αCD40 once plus 30 mg/kg αCSF-1R weekly, starting treatment at 285 mm 3 tumor volume. (F) C57BL/6 mice were inoculated with the indicated syngeneic tumors E0771 and T241 ( n = 10 per group and model) and were treated using mIgG1, αCSF-1R, αCD40, or a combination of both targeting antibodies. (B and D–F) Animals were graphically censored in Kaplan-Meier curves once the tumor volume reached ≥700 mm 3 , and the numbers in graphs indicate the amount of tumor-free mice within the specific group ( n = 10 for all groups depicted). Asterisks indicate log-rank tests between αCD40- and αCSF-1R+αCD40–treated animals, and detailed log-rank test results for the other groups are available in Table S2; all animal survival experiments (B–F) were performed at least twice. *, P < 0.05; **, P < 0.01.

Article Snippet: 10 mice per group were treated with 30 mg/kg murine IgG1 isotype control (clone MOPC-21; BioXCell) and 4 mg/kg rat IgG2a isotype control (clone 2A3; BioXCell), 30 mg/kg anti–CSF-1R antibody (clone 2G2; ), 4 mg/kg αCD40 antibody (rat IgG2a clone FGK.45; BioXCell), or the combination of both targeting antibodies. αCD40 or the matching isotype control was only administered once, whereas αCSF-1R antibody was administered weekly unless otherwise stated (maximum four times total).

Techniques: Inhibition, Activation Assay, In Vivo, RNA Sequencing Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity

doi: 10.1084/jem.20171440

Figure Lengend Snippet: TAMs are required for αCSF-1R+αCD40-mediated tumor rejection. (A and B) Immune infiltrate of MC38 tumors on day 10 upon start of treatment. MC38 tumor–bearing mice were treated with either 30 mg/kg mIgG1, 30 mg/kg αCSF-1R weekly, 4 mg/kg αCD40 once, or a combination of both targeting antibodies. Data shown in A and B are pooled from two independent experiments, and T reg cells were assessed in the third experiment. Graphs show means ± SEM; statistical analysis by one-way ANOVA and Tukey correction (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (C) A single combined dose of αCSF-1R and αCD40 antibodies is sufficient for tumor rejection. MC38 tumor–bearing mice were treated using 30 mg/kg mIgG1, 4 mg/kg αCD40 once, or in combination with 30 mg/kg αCSF-1R given weekly (four times total), once (first treatment day), or twice on first and second treatment day. This experiment is exemplary out of two independent experiments. (D) Predepletion of macrophages completely abolishes tumor rejection in MC38 tumor–bearing mice. Mice were inoculated with MC38 tumors and received on days 3 and 9 upon inoculation of either 30 mg/kg mIgG1, mIgG1 incompetent in binding to FcγR (mIgG1-FcØ), or αCSF-1R clone 2G2 incompetent in binding to FcγR (αCSF-1R–FcØ). On day 11, at a mean tumor volume of 120 mm 3 , mice were treated with a single dose of either 130 mg/kg mIgG, 4 mg/kg αCD40, or a combination of 30 mg/kg αCSF-1R+αCD40 (FcγR competent or incompetent versions). Depletion of TAMs was confirmed by flow cytometry on day 11 from scout animals (Fig. S3 D). (C and D) Mice were graphically censored once the tumor size reached ≥700 mm 3 , and numbers in graphs indicate the amount of tumor-free mice within the specific group from n = 10 for all groups depicted.

Article Snippet: 10 mice per group were treated with 30 mg/kg murine IgG1 isotype control (clone MOPC-21; BioXCell) and 4 mg/kg rat IgG2a isotype control (clone 2A3; BioXCell), 30 mg/kg anti–CSF-1R antibody (clone 2G2; ), 4 mg/kg αCD40 antibody (rat IgG2a clone FGK.45; BioXCell), or the combination of both targeting antibodies. αCD40 or the matching isotype control was only administered once, whereas αCSF-1R antibody was administered weekly unless otherwise stated (maximum four times total).

Techniques: Binding Assay, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity

doi: 10.1084/jem.20171440

Figure Lengend Snippet: CSF-1R inhibition accelerates αCD40-mediated TAM reprogramming toward a strong proinflammatory phenotype. (A and B) Monocytic MDSCs and TAMs were sorted from tumors 16 h after treatment using 30 mg/kg mouse IgG1 + 4 mg/kg rat IgG2a, 30 mg/kg αCSF-1R rat + 4 mg/kg IgG2a, 4 mg/kg αCD40 + 30 mg/kg IgG1, or a combination of both targeting antibodies, and RNA sequencing was performed ( n = 4 each). Heat maps show genes up- or down-regulated, driven by either αCD40 alone (compared to IgG and αCSF-1R) or only occurring in the combination of αCSF-1R+αCD40 (compared with IgG, αCD40, and αCSF-1R). Genes depicted were also found by either being a member of M1- and/or M2-related signatures described in Table S1, or by being differentially regulated in M1 versus M2 based on a gene expression dataset comprising activated M1 and M2a macrophages GSE5099 and GSE58318 . Criteria for differential regulation: absolute log2 ratio >1 and an adjusted p-value <0.05. P-values were derived using R function aov() . Genes with contradicting information on direction of regulation (e.g., up-regulated in M2 according to one source but down-regulated in M2 in another source) were eliminated from the visualization. To determine genes regulated by combination (vs. IgG, αCSF-1R, and αCD40), p-values were computed using R function aov() with Tukey correction for multiple comparison of treatment groups. Genes were deemed as specifically regulated by combination if there was differential expression between combination and all monotherapies (P < 0.05 and absolute log2 ratio >1). (C) Selected genes from NanoString analysis of whole tumor tissue from treatment groups at the same time point as A and B, expressed as counts normalized to housekeeping genes Ppia , Polr2a , Eef1g , Sdha , and Rpl19 . (D) Protein data of selected cytokines and chemokines from treatment groups at the same time point as A–C by ELISA or by multiplex bead assay from whole tumor tissue. (C and D) Data are depicted as means ± SEM and analyzed using one-way ANOVA and Tukey correction. P-values are shown only for the comparison of αCD40 with αCSF-1R+αCD40, unless αCSF-1R treatment alone had significant impact on mRNA levels (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: 10 mice per group were treated with 30 mg/kg murine IgG1 isotype control (clone MOPC-21; BioXCell) and 4 mg/kg rat IgG2a isotype control (clone 2A3; BioXCell), 30 mg/kg anti–CSF-1R antibody (clone 2G2; ), 4 mg/kg αCD40 antibody (rat IgG2a clone FGK.45; BioXCell), or the combination of both targeting antibodies. αCD40 or the matching isotype control was only administered once, whereas αCSF-1R antibody was administered weekly unless otherwise stated (maximum four times total).

Techniques: Inhibition, RNA Sequencing Assay, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay

Journal: The Journal of Experimental Medicine

Article Title: Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity

doi: 10.1084/jem.20171440

Figure Lengend Snippet: Tumor rejection by αCSF-1R+αCD40 combination depends on CD8 + T cells. (A) Rechallenge of tumor-free mice upon αCD40 or αCSF-1R+αCD40 leads to rapid rejection of MC38 tumors. Mice were rechallenged with 5 × 10 6 MC38 tumor cells into the contralateral flank from the first tumor, and tumor volume was monitored. Data are pooled from four rechallenge experiments (total n = 5 naive mice, n = 7 αCD40, and n = 47 αCSF-1R+αCD40). (B) MC38, E0771, and T241 tumor–bearing mice were treated with mIgG1 weekly, αCSF-1R weekly, αCD40 once, or αCSF-1R weekly + αCD40 once, and splenocytes were analyzed on day 10. CD8 + T cells were analyzed for effector memory status (CTL EM ) or activated/central memory (activated CTL/CTL CM ), and the number of FoxP3 + T reg cells was assessed by flow cytometry. Graphs show means ± SEM, statistical analysis by one-way ANOVA, and Tukey correction with n = 3 to 5 per group as depicted from one out of a minimum of two independent experiments. (C) Rejection of MC38 tumors is mediated by CD8 + T cells. MC38 tumor–bearing animals were treated with depletion antibodies against CD4 + , CD8 + T cells, or NK cells starting on days −3 and −1 before therapy with mIgG1, αCD40, or αCSF-1R+αCD40 on day 0. An additional three doses of depleting antibodies were given at days 1, 4, and 7. Mice were graphically censored once the tumor sized reached ≥700 mm 3 , and numbers in graphs indicate amount of tumor-free mice within the specific group ( n = 10 for all groups depicted). Data depicted are exemplary from two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: 10 mice per group were treated with 30 mg/kg murine IgG1 isotype control (clone MOPC-21; BioXCell) and 4 mg/kg rat IgG2a isotype control (clone 2A3; BioXCell), 30 mg/kg anti–CSF-1R antibody (clone 2G2; ), 4 mg/kg αCD40 antibody (rat IgG2a clone FGK.45; BioXCell), or the combination of both targeting antibodies. αCD40 or the matching isotype control was only administered once, whereas αCSF-1R antibody was administered weekly unless otherwise stated (maximum four times total).

Techniques: Flow Cytometry