agonistic anti cd27 antibody  (Celldex Inc)

Journal: Nature Communications

Article Title: Harnessing multivalency and FcγRIIB engagement to augment anti-CD27 immunotherapy

doi: 10.1038/s41467-025-67882-3

Figure Lengend Snippet: A hCD27 + Jurkat NF-κB GFP reporter cells were co-cultured with either WT CHO or hFcγRIIB + CHO cells in the presence of the indicated antibodies. NF-κB activation was quantitated by GFP expression using flow cytometry. GFP production from Jurkat cells after co-culture with WT CHO cells (above) or hFcγRIIB + CHO cells (below) and the specified antibodies; hIgG1 (left), hIgG1 NA (center), or hIgG1 V11 (right). Data points are the mean ± SEM (n = 3) from 3 independent experiments. Statistical significance was determined by one-way ANOVA (Tetra anti-hCD27 versus anti-CD27 IgG), with significance values indicated in the figure. B Human CFSE-labeled PBMCs were stimulated with sub-optimal anti-CD3 and the indicated antibodies, hIgG1 (left), hIgG1 NA (center), or hIgG1 V11 (right). CD8 + (above) and CD4 + (below) T cell proliferation was assessed by CFSE dilution, with CFSE low cells identified by comparison to cells that were cultured in the absence of any stimulation. Each data point is the mean ± SEM (n = 5) from 5 independent experiments across 5 different donors. Statistical significance between bivalent anti-CD27 and bivalent isotype control (colored numbers) and tetravalent anti-CD27 and tetravalent isotype control (black numbers) was determined by one-way ANOVA, with significance values indicated in the figure. Source data are provided as a file.

Article Snippet: A.Al-S. and S.H.L. are inventors on patents pertaining to the generation and therapeutic use of agonist anti-CD27 antibodies and have received research funding from Celldex Therapeutics in relation to anti-CD27 antibodies.

Techniques: Cell Culture, Activation Assay, Expressing, Flow Cytometry, Co-Culture Assay, Labeling, Comparison, Control

Journal: Nature Communications

Article Title: Harnessing multivalency and FcγRIIB engagement to augment anti-CD27 immunotherapy

doi: 10.1038/s41467-025-67882-3

Figure Lengend Snippet: A , B Jurkat cells expressing hCD27-GFP + fusion protein were stimulated with the specified antibodies at the indicated concentrations, before PFA fixation and confocal imaging. A Representative images at 100 nM (white corresponds to hCD27-GFP fluorescence; clustered areas of the signal are indicated by green arrows) and B quantification of the number of clusters per cell section, performed using ImageJ. Data shown are mean ± SEM of CD27 clusters per cell section from 2 independent experiments (n = 54 cells). Statistical significance was determined by one-way ANOVA, with significance values indicated in the figure. C , D hCD27-GFP + Jurkat cells were stimulated with 1 nM tetra anti-hCD27 hIgG1 V11 and either WT CHO cells or hFcγRIIB + CHO cells for the specified time points before PFA fixation and confocal imaging using Oxford Nanoimager. C Representative images of hCD27-GFP + Jurkat cells with WT CHO (upper panels) or hFcγRIIB + CHO (lower panels) for the specified time points. Dashed blue and green lines (determined by brightfield and confocal images) indicate the plasma membrane of CHO and Jurkat cells, respectively. D Quantification of intracellular GFP MFI of individual cell sections of hCD27-GFP + Jurkat cells after stimulation. Data are the mean ± SEM from 2 independent experiments (n = 24 cells). Statistical significance was determined by one-way ANOVA, with significance values indicated in the figure. E , F hCD27 + Jurkat NF-κB GFP reporter cells were stimulated with the indicated concentrations of either bivalent anti-hCD27 hIgG1 V11 (left panel) or Tetra anti-hCD27 hIgG1 V11 (right panel) in the presence of WT CHO cells (WT CHO), WT CHO cells and a goat anti-hFc f(ab) 2 (WT CHO + Xlink) or hFcγRIIB + CHO cells (hFcγRIIB + CHO). GFP reporter production was determined by flow cytometry and presented as E mean fluorescence intensity (MFI) of GFP + cells and F percentage of GFP + cells . Data points are the mean ± SEM (n = 3) from 3 independent experiments. Statistical significance between hFcγRIIB + CHO and WT CHO + Xlink was determined by one-way ANOVA on AUC values, with significance values indicated in the figure. Source data are provided as a file.

Article Snippet: A.Al-S. and S.H.L. are inventors on patents pertaining to the generation and therapeutic use of agonist anti-CD27 antibodies and have received research funding from Celldex Therapeutics in relation to anti-CD27 antibodies.

Techniques: Expressing, Imaging, Fluorescence, Clinical Proteomics, Membrane, Flow Cytometry

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor site influences efficacy of MEKi and immunotherapy combinations in pre-clinical model of cholangiocarcinoma

doi: 10.1097/HEP.0000000000001439

Figure Lengend Snippet: (A) Splenocytes from pmel-1 mice were isolated and grown in culture in the presence of IL-2, gp100 and either monotherapy or dual therapy with MEKi and CD27 agonist. After 4 days, cells were characterized by flow cytometry. The percentage of cells expressing (B) CD69 or (C) CD44 was quantified (n=26). P-values were calculated by a one-way ANOVA, followed by multiple comparisons testing. Error bars represent mean ± SD. *p<0.05, ****p<0.0001. (D) Representative histograms show CD69 or CD44 expression gated on lymphocytes/single cells/live cells/CD3 + CD8 + .

Article Snippet: Rat IgG2a CD27 agonist Ab (RRID: AB_3665171) was from Celldex, Inc. (Phillipsburg, NJ) under material transfer agreement.

Techniques: Activation Assay, Isolation, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs

doi: 10.3389/fimmu.2023.1023064

Figure Lengend Snippet: Activation of Tregs and Tconvs in CD70 transgenic mice. Spleen cells from CD27 +/- mice expressing or not a CD70 tg were harvested at 6-8 wk of age and stained ex vivo for indicated proteins. (A) Spleen cells were stained for CD4, Foxp3 and the indicated markers. Singlets were selected by gating events in the diagonal of FSC-H vs FSC-A plots. Representative flow cytometry plots of indicated marker on gated viable CD4 + cells. (B) Proportion of positive cells among viable CD4 + Tregs and Tconvs (left panels; proportion of Tregs and intensity of Foxp3 expression (right panels). Data are from 3-4 independent experiments with 5-8 mice per group. Bars represent median ± SD. Unpaired t-test was used to determine statistical differences followed by FDR correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. (C) Representative merged (n =8) t-distributed stochastic neighbor embedding (t-SNE) plot after dimensionality reduction and unsupervised clustering of flow cytometry data from CD4 + Tregs and Tconvs.

Article Snippet: Agonistic anti-CD27 mAb treatment: mice were injected i.p. with 100 or 200μg of agonistic anti-CD27 mAb (BioXCell BE0348), at days 0 and 3, or isotype control (BioXCell BE0089).

Techniques: Activation Assay, Transgenic Assay, Expressing, Staining, Ex Vivo, Flow Cytometry, Marker

Journal: Frontiers in Immunology

Article Title: Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs

doi: 10.3389/fimmu.2023.1023064

Figure Lengend Snippet: Injection of agonistic anti-CD27 mAb induces some features of Th1-type Tregs. WT mice were injected i.p. with agonistic anti-CD27 mAb (at days 0 and 3) and spleen cells were analyzed ex vivo by flow cytometry at day 6. (A) Spleen cells were stained for CD4, Foxp3 and the indicated markers? Singlets were selected by gating events in the diagonal of FSC-H vs FSC-A plots. Representative flow cytometry plots of indicated marker on gated viable CD4 + cells. (B) Proportion of Tregs and intensity of Foxp3 expression. (C) Proportion of CD4 + Tregs and Tconvs expressing the proliferation marker Ki67, transcription factors (Eomes, T-bet), cytokines (IFN-γ and IL-10) and inhibitory receptors (TIGIT, ICOS, CTLA-4 and PD-1). Data are from 3 independent experiments with 4 mice per group. Bars represent median ± SD. Unpaired t-test was used to determine statistical differences followed by FDR correction for multiple comparisons (*p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant).

Article Snippet: Agonistic anti-CD27 mAb treatment: mice were injected i.p. with 100 or 200μg of agonistic anti-CD27 mAb (BioXCell BE0348), at days 0 and 3, or isotype control (BioXCell BE0089).

Techniques: Injection, Ex Vivo, Flow Cytometry, Staining, Marker, Expressing

Journal: Frontiers in Immunology

Article Title: Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs

doi: 10.3389/fimmu.2023.1023064

Figure Lengend Snippet: Unchanged suppressive capacity of Tregs in CD11c- Cd70 tg;CD27 +/- mice. CD4 + Foxp3 + cells were sorted from CD27 +/- or CD11c- Cd70 tg;CD27 +/- mice and co-cultured with CFSE-labeled, naive conventional CD4 + T cells from CD45.1 mice in the presence of soluble anti-CD3 mAb and irradiated splenocytes. After 3 days, flow cytometry profiles of CFSE were analyzed. Percent of suppression of proliferation as compared to cultures in which Treg cells were omitted. Data are representative of 4 independent experiments with n = 5 per group. Values are presented as the median ± SD and were compared by two-tailed unpaired student’s t-test. ns, not significant.

Article Snippet: Agonistic anti-CD27 mAb treatment: mice were injected i.p. with 100 or 200μg of agonistic anti-CD27 mAb (BioXCell BE0348), at days 0 and 3, or isotype control (BioXCell BE0089).

Techniques: Cell Culture, Labeling, Irradiation, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs

doi: 10.3389/fimmu.2023.1023064

Figure Lengend Snippet: Cell autonomous activation/differentiation of Tregs upon CD27 engagement. (A, B) 5 x 10 5 Tregs purified from Foxp3eGFP CD90.1 mice were injected i.v. into CD11c- Cd70 tg;CD27 -/- recipients and either WT (A) or CD27 -/- (B) as control recipients. (C) Tregs were purified from Foxp3eGFP CD90.1 mice either CD27 competent or deficient (CD27 -/- ) and 5 x 10 5 cells were injected i.v. into CD11c- Cd70 tg;CD27 -/- recipients. Spleen cells were analyzed ex vivo by flow cytometry 7 days after injection. Data show the proportion of transferred Tregs (Foxp3 + CD90.1 + ) expressing the proliferation marker Ki67, transcription factors (eomes, T-bet), chemokine receptor CXCR3 and inhibitory receptors (ICOS, CTLA-4 and PD-1) as well as the absolute number of CD90.1 + Tregs recovered. Controls include CD27 and CD70 staining. Data are representative of 3 independent experiments with 4 mice per group. Bars represent median ± SD. Unpaired t-test was used to determine statistical differences followed by FDR correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).

Article Snippet: Agonistic anti-CD27 mAb treatment: mice were injected i.p. with 100 or 200μg of agonistic anti-CD27 mAb (BioXCell BE0348), at days 0 and 3, or isotype control (BioXCell BE0089).

Techniques: Activation Assay, Purification, Injection, Control, Ex Vivo, Flow Cytometry, Expressing, Marker, Staining

Journal: Frontiers in Immunology

Article Title: Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs

doi: 10.3389/fimmu.2023.1023064

Figure Lengend Snippet: Common transcriptional signature of CD27 engagement in Tregs versus Tconvs. (A) Left panel: principal component analysis (PCA), a dimensionality reduction method, show the separation among Tregs and Tconvs due to CD70 Tg . Variance in PC1 and PC2 is shown. (B) Left panel: differentially expressed genes of Tconvs and Tregs are clustered based on occurrence. Clusters 1 and 3 are upregulated specifically in Treg F1 CD70 Tg and Tconv F1 CD70 Tg , respectively, whereas clusters 4 and 6 consist of genes downregulated specifically in Treg F1 CD70 Tg and Tconv F1 CD70 Tg , respectively. Shared up/down regulated genes are found in cluster 2 and 5. Values are represented as Log2 fold-change obtained from median CPM of each gene. Selected genes for each cluster are displayed in the right margin. The number of genes in each cluster is shown in the left margin. Right panel, selected pathways enriched in clusters 1, 2, and 3 using clusterProfiler R package with default parameters and presented as −Log 10 of p-value. (C) GSEA performed on F1 CD70 Tg data set and Tconv F1 CD70 Tg differentially expressed genes as gene sets. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated. F1 mice: CD27 +/- ; F1 CD70 Tg mice: CD11c- Cd70 tg;CD27 +/- .

Article Snippet: Agonistic anti-CD27 mAb treatment: mice were injected i.p. with 100 or 200μg of agonistic anti-CD27 mAb (BioXCell BE0348), at days 0 and 3, or isotype control (BioXCell BE0089).

Techniques:

Journal: Frontiers in Immunology

Article Title: Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs

doi: 10.3389/fimmu.2023.1023064

Figure Lengend Snippet: Common enriched pathways in Tregs from CD11c- Cd70 tg;CD27 +/- , Eomes Tg and CXCR3 + Tregs. (A, B) GSEA performed on F1 CD70 Tg data set and Treg Eomes Tg (A) or Treg Cxcr3 + (B) differentially expressed genes as gene sets. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated. (C) Bubble plot for selected pathways (database MsigDB; https://www.gsea-msigdb.org/gsea/msigdb/ ) enriched from genes upregulated in Treg F1 CD70 Tg , Treg Cxcr3 + and Treg Eomes Tg using clusterProfiler R package with default parameters and presented as −Log 10 of p-value. Color intensity indicates p-value and bubble size indicates number of enriched genes.

Article Snippet: Agonistic anti-CD27 mAb treatment: mice were injected i.p. with 100 or 200μg of agonistic anti-CD27 mAb (BioXCell BE0348), at days 0 and 3, or isotype control (BioXCell BE0089).

Techniques:

Journal: Frontiers in Immunology

Article Title: Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs

doi: 10.3389/fimmu.2023.1023064

Figure Lengend Snippet: List of reagents and resources.

Article Snippet: Agonistic anti-CD27 mAb treatment: mice were injected i.p. with 100 or 200μg of agonistic anti-CD27 mAb (BioXCell BE0348), at days 0 and 3, or isotype control (BioXCell BE0089).

Techniques: Purification, Staining, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy

doi: 10.1186/2051-1426-2-7

Figure Lengend Snippet: TNFR-targeted agents in development

Article Snippet: CD27 , CDX-1127 , Fully human anti-CD27 agonist monoclonal antibody , Celldex Therapeutics , I.

Techniques: Transduction, Recombinant