cd40 agonist  (Celldex Inc)

Journal: bioRxiv

Article Title: Antigen presentation requirements for effective cDC1-based cancer immunotherapy

doi: 10.64898/2026.01.24.701412

Figure Lengend Snippet: ( A ) Schematic of the workflow used to generate cDC1s from WT, MHC-I KO , or MHC-II KO mice in vitro . BM cells were cultured in medium containing Flt3L and GM-CSF, followed by FACS purification, poly dI:dC stimulation, and incubation with OVA. ( B ) Flow cytometry histogram plots showing cell surface expression of MHC-I (H2-K b ) and MHC-II (I-A/I-E) on cDC1s after 16 hours under the indicated conditions. n = 6 (WT), n = 2 (MHC-I KO ), n = 4 (MHC-II KO ); representative data from at least two independent experiments. ( C ) Bulk RNA-seq analysis of cDC1s in steady-state conditions (no stim) or following stimulation with poly dI:dC with or without αCD40, as indicated. The heatmaps show log 2 (TPM + 1) gene expression, grouped according to cDC1 functions. ( D ) Mapping of bulk RNA-seq profiles from in vitro -derived cDC1s onto mouse or human tumor-infiltrating DC scRNA-seq reference datasets using ProjectLSI. Reference scRNA-seq cells are displayed as dots, and bulk RNA-seq samples are overlaid as shapes, corresponding to individual genotypes, as indicated.

Article Snippet: For experiments assessing CD40 stimulation, cDC1s were stimulated with 20 ng/mL mGM-CSF, 20 μg/mL poly dI:dC, and 200 μg/mL OVA, in the presence or absence of 20 μg/mL agonistic anti-CD40 antibody (αCD40; Leinco Technologies, Fenton, MO, USA).

Techniques: In Vitro, Cell Culture, Purification, Incubation, Flow Cytometry, Expressing, RNA Sequencing, Gene Expression, Derivative Assay

Journal: bioRxiv

Article Title: Antigen presentation requirements for effective cDC1-based cancer immunotherapy

doi: 10.64898/2026.01.24.701412

Figure Lengend Snippet: ( A ) Representative flow cytometry histograms (left) showing CD40 surface expression on WT and MHC-II KO cDC1s after 4-hour stimulation in vitro , with quantification of CD40 + frequency and CD40 MFI (right). n = 4 per group for all conditions; cumulative data from two independent experiments. ( B ) Schematic of the in vivo experimental design. cDC1s were stimulated in vitro with either poly dI:dC or poly dI:dC and αCD40 and pulsed with OVA for 4 hours, then prepared for vaccination as described in the Materials and Methods. WT mice were implanted with B16-OVA tumors on day 0 and treated intratumorally on days 4 and 7 with either PBS or cDC1 vaccines. ( C ) Representative mean tumor area (mm 2 ) over time. ( D ) Individual tumor growth curves corresponding to mice in ( C ). ( C, D ) n = 5 mice per group; representative of one of two independent experiments. ( E ) Cumulative mouse survival representing time to humane endpoint, defined as tumors reaching 15 mm in any direction. n = 10 mice per group, cumulative data from two independent experiments. ( A, C ) Data shown are the mean ± S.E.M. and were analyzed by two-way ANOVA, followed by Tukey’s multiple-comparisons test applied to ( A) . ( E ) Survival was analyzed by log-rank (Mantel-Cox) test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For experiments assessing CD40 stimulation, cDC1s were stimulated with 20 ng/mL mGM-CSF, 20 μg/mL poly dI:dC, and 200 μg/mL OVA, in the presence or absence of 20 μg/mL agonistic anti-CD40 antibody (αCD40; Leinco Technologies, Fenton, MO, USA).

Techniques: Flow Cytometry, Expressing, In Vitro, In Vivo, Vaccines

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A Immunofluorescence staining in tumors of patients with metastatic melanoma resistant to αPD1 therapy. DNA (blue), CD8α (green), CD20 (red) and CD11b (yellow). Scale bars indicate 2 mm (left) and 50 μm (right). These results are representative of immunofluorescence staining performed on melanoma tumors from two patients. B – F Single-cell (sc) RNA-Seq analysis identifies CD20 + CD11b + PD-L1 + regulatory B cells in patient melanoma tumors. References: ( B ) Cell 2018, 175, 984–997. doi: j.cell.2018.09.006. C Cell 2018, 175, 998–1013.e1–e20. doi: j.cell.2018.12.034. Samples with total B-cell size <5 were excluded. D Frequency of CD20 + CD11b + PD-L1 + regulatory B cells in patient melanoma tumors at baseline and post αPD1 treatment. Data were derived from ( C ). E , F Patient melanoma scRNA-Seq analysis reveals a high CD40 mRNA expression in CD20 + CD11b + PD-L1 + Bregs. E n = 818 B cells from 26 patients, two-sided t -test; and ( F ) n = 1379 B cells from 30 patients. One-way analysis of variance (ANOVA) with post hoc test.

Article Snippet: Mouse splenocytes were isolated from C57BL/6 mice and were cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, CAT#: C2825) for five days.

Techniques: Immunofluorescence, Staining, RNA Sequencing, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A 1014 cells were injected in C57BL/6 female mice. Treatment of C57BL/6 mice with aCD40 (30 μg/mouse every 3 days, intraperitoneal) started at Day 10 post tumor cell inoculation. Analysis of CD40 levels by flow cytometry of CD45 + CD19 + B cells prepared from tumor samples at day 22 post-treatment ( n = 5 per group) using a one-sided Mann–Whitney U test. B Splenocytes were isolated from C57BL/6 mice by magnetic separation, serum-free starved for 4hrs, and stimulated with or without 12.5 μg/ml aCD40. After 4 days of culture, splenic B cells were isolated by magnetic separation and stained for flow cytometric analysis ( n = 6 per group). C , D Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (every 3 days, intraperitoneal), 100 μg/mouse αCTLA4 (every 3 days, intraperitoneal), 30 μg/mouse agonist CD40 (aCD40, every 3 days, intraperitoneal), or IgG control antibody, starts at day 10 post tumor cell inoculation. E Frequency of CD19 + CD11b + regulatory B cells in 1014 melanoma tumors or tumor draining lymph node (TDLN) after 22 days of aCD40 treatment (every 3 days, intraperitoneal) TDLN IgG, n = 5; TDLN aCD40, n = 5; Tumor IgG, n = 4; Tumor aCD40, n = 5. F Flow cytometric analysis of CD45 + CD19 + B cells in the tumor microenvironment of 1014 melanoma after 3 weeks treatment of aCD40. Data were replicates from one experiment ( n = 5 mice per group). G C57BL/6 mice were intravenously injected with formalin-fixed 2 × 10 6 metastatic B16F10-Luc2 cells via the tail vein. Splenic CD8 + T and total B cells were isolated by magnetic separation after 2 days of tumor antigen priming. Total B cells were stimulated with 12.5 μg/ml aCD40 for 2 days and CD11b + B cells were isolated by magnetic separation. CD8 + cytotoxic T cell killing assay were performed via co-culturing CD11b + B cells, CD8 + T cells and B16F10-Luc2 cells in the conditioned RPMI complete medium containing 0.5 μg/ml of IL-7, 30U/ml of IL-2, and CD3/CD28 Dynabeads at a bead-to-cell ratio 2:1. Dead cells were removed and the luciferase activity of remaining live tumor cells was quantified to calculate % inhibition of CTL killing capacity. ( n = 3 independent experiments). Created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 . Exact p values are provided as a Source Data file. H Splenic B cells were stimulated with 12.5 μg/ml aCD40 for 4 days. The CD11b - and CD11b + B cells were isolated by magnetic separation and restimulated with 12.5 μg/ml aCD40 for 30 min. Whole-cell extracts were harvested and immunoblotted. β-actin was used as a loading control for densitometry quantification. n = 3 independent experiments. One-way analysis of variance (ANOVA) with post hoc test. Source data are provided as a Source Data file.

Article Snippet: Mouse splenocytes were isolated from C57BL/6 mice and were cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, CAT#: C2825) for five days.

Techniques: Injection, Flow Cytometry, MANN-WHITNEY, Isolation, Staining, Control, Luciferase, Activity Assay, Inhibition

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 300 mg/kg rigosertib (RGS, 5 days a week, oral gavage) or vehicle control (H 2 O) starts at day 10 post tumor cell inoculation. B Tumor samples were collected from each treatment group at 4 weeks of treatment and immune profiled by FACS analysis. All graphs show Mean ± SEM, n = 5 samples per group. C 1014 tumor cells were treated with 1 μM trametinib (T) and/or 1 μM RGS for 30–60 min. Whole-cell extracts were harvested and immunoblotted. β-actin was used as a loading control for densitometry quantification. n = 3 independent experiments. Source data are provided as a Source Data file. D RGS and T induce CD40 expression in 1014 cells. Cells were treated with RGS + /- T for 24 h. CD40 protein expression on the cell surface was detected by anti-CD40-FITC staining and flow cytometric analysis. n = 3 independent experiments. E Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (every 3 days, intraperitoneal), 300 mg/kg rigosertib (RGS, 5 days a week, oral gavage), and/or 1 mg/kg trametinib (T, 5 days a week, oral gavage), starts at day 8 post tumor cell inoculation, n = 7 ~ 10 mice per group. Created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 . Exact p values are provided as a Source Data file. F Definition of time to resistance to drug: (1) the tumor is > 100 mm 3 and (2) has a > 30% increase of tumor volume compared to the previous measurement. Survival curves (resistance-free probabilities or survival probabilities) of treatment groups are estimated using the Kaplan–Meier Plotter and compared using the log-rank test, n = 7 ~ 9 mice per group. Exact p values are provided as a Source Data file. G Mouse body weight recorded for 20 days of treatment. Veh, n = 10; T, n = 8; RGS, n = 7, RGS + T, n = 9 mice. H Tumor and ( I ) Tumor-draining lymph node (TDLN) samples were collected from each treatment group at day 20 of treatment and immune profiled by FACS analysis, Veh, n = 5; T, n = 4; RGS, n = 4, RGS + T, n = 4 tumors. For tumor samples, we collected 200,000 live singlet cells from each tumor and recovered ~6000 CD45 + cells per tumor, regardless of treatment groups. Tc, CD8 + CD3 + cytotoxic T cells. Th, CD4 + CD3 + T helper cells. Exact p values are provided as a Source Data file.

Article Snippet: Mouse splenocytes were isolated from C57BL/6 mice and were cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, CAT#: C2825) for five days.

Techniques: Control, Expressing, Staining

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A , B Splenocytes were isolated from C57BL/6 mice, serum-free starved for 4 h, and stimulated with 12 μl/ml anti-CD3/CD28 Dynabeads plus 12.5 μg/ml agonist CD40 (aCD40). After 5 days of culture, cells were stained for FACS analysis. Exact p values are provided as a Source Data file. C Tumor volume of 1014 melanoma, created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 , exact p values are provided as a Source Data file; and D Mouse body weight of tumor-bearing C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (2 lead-in doses, every 3 days, intraperitoneal), 30 μg/mouse aCD40 (every 3 days, intraperitoneal), plus 300 mg/kg rigosertib (RGS, 5 days a week, oral gavage) and/or 1 mg/kg trametinib (T, 5 days a week, oral gavage), starts at day 8 post tumor cell inoculation ( n = 10 per group). E Serum level of liver/kidney enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) at day 27 post-treatment ( n = 5 per group). Live CD45 + leukocytes in ( F ) Tumor and ( G ) Tumor-draining lymph node (TDLN) samples were concatenated after downsampling to, 25,000 and 10,000 events respectively, for t-SNE analysis through flow cytometry. All graphs show Mean ± SEM, n = 5 per group. H – K Frequency of B cells and effector cells in 1014 tumors and TDLN samples at day 27 post-treatment ( n = 5 per group). Exact p values are provided as a Source Data file. L L308 mouse protein array of 1014 tumor lysate samples at day 27 post-treatment. Data were pooled from replicates of one experiment ( n = 5 mice per group). Exact p values are provided as a Source Data file.

Article Snippet: Mouse splenocytes were isolated from C57BL/6 mice and were cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, CAT#: C2825) for five days.

Techniques: Isolation, Staining, Flow Cytometry, Protein Array

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A Correlation heatmap of top 25% differentially altered genes summarized across all treatment groups (15,903 genes plotted). Heatmap3 was used for cluster analysis and visualization. Significantly differential expressed genes with absolute fold change ≥ 2 and FDR adjusted p value ≤ 0.05 were detected by DESeq2. B CIBERSORT analysis of a leukocyte signature matrix (LM22) to deconvolution of 1014 tumors. Exact p values are provided as a Source Data file. C , D Hallmark Gene Set Enrichment Analysis (GSEA) of the transcriptomic profile of 1014 tumors at baseline or with agonist CD40 (aCD40) plus αPD1 treatment at 22 days using GSEA package. E – K TCR-β chain sequencing results were evaluated using the Archer Immunoverse analyzer. CDR3 sequences and frequency tables were extracted from the manufacturers’ analysis platform and imported for analysis using the Immunarch package ( https://immunarch.com ) in R. Comparisons were made within specific tumor types and across different tumor types (CD40-Ctrl vs CD40-OE) to evaluate drug effects, with the analysis conducted on a natural logarithmic scale. Number of ( E ) total and ( F ) unique TCR clonotypes. Exact p values are provided as a Source Data file. G Treatment effect (ratio of TCR clonotypes among treatment to clonotypes among Veh + IgG control) comparisons between CD40-Ctrl and CD40-OE tumors were performed using multiple comparisons test for generalized linear hypothesis test based on the negative Binomial generalized linear model. All graphs show Mean ± SD. H Estimated distribution of clonotype abundances. I Table summary of FDR (False Discovery Rate) adjustment. J Estimated relative abundance (also known as clonal space homeostasis), which characterizes the proportion of repertoire occupied by clonal groups with specific abundances indicated. K The top 20 most abundant TCRs of small- and medium-expanded clonotypes in TRBV26 are shown. Flow between samples indicates shared TCRs. Boxed CDR3s indicate most clonal TCRs in TRBV26. The Holm correction was used to adjust the p value for multiple comparisons. A – K Pooled values of n = 3 tumors per group.

Article Snippet: Mouse splenocytes were isolated from C57BL/6 mice and were cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, CAT#: C2825) for five days.

Techniques: Sequencing, Control

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A PDO cultures were developed using previously frozen patient melanoma tissue that was thawed and immediately subjected to fine needle aspiration. IL-2 was added to all cultures to ensure the survival and proliferation of T cells. PDOs were grown in organoid media containing 5% Matrigel. After allowing PDOs to establish for 3–4 days, treatments were initiated as follows: RGS, CD40 agonist (aCD40), αPD1, RGS + aCD40, RGS + αPD1, or RGS + aCD40 + αPD1. Cultures were allowed to grow for 10 days, and the organoid area was assessed and quantified at day 10, PDO-2624, n = 18 per group; PDO-3453A, n = 18 per group; PDO-3529 primary, n = 12 per group; PDO-3529LN, n = 12 per group; PDO-3101, n = 12 per group. All graphs show Min to Max with all points +/− SD. Exact p values are provided as a Source Data file. B , C , F Melanoma scRNA-Seq dataset . Analysis of malignant and immune cell gene expression of 6173 scRNA-seq profiles from 32 human melanoma tumors. D , E , G Melanoma scRNA-Seq dataset . Transcriptome analysis of 16,291 individual immune cells from 49 tumor samples of melanoma patients treated with checkpoint inhibitors. C , E Functional enrichment of Geneset and KEGG pathway over-representation analysis were performed on differentially expressed genes of CD20 + CD11b + PD-L1 + Bregs in patient melanoma tumors using WebGestaltR package (NULL). F , G Patient melanoma scRNA-Seq analysis reveals a distinct transcriptome profile of CD20 + CD11b + PD-L1 + Bregs. All graphs show Median ± SD. H The Breg gene signature (Sig. Bregs) is associated with poorer patient OS in multiple cancer types. Kaplan-Meier plots show OS for patients in the TCGA pan-cancer datasets . Patients were split into high and low expression of the Breg gene curated signature. Median OS survival is represented on the graph when available. P-values were calculated by log-rank test. GBM, glioblastoma; LGG, low-grade glioma; LUSC, lung squamous cell carcinoma; STAD, stomach adenocarcinoma; MESO, mesothelioma; OV, ovarian carcinoma.

Article Snippet: Mouse splenocytes were isolated from C57BL/6 mice and were cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, CAT#: C2825) for five days.

Techniques: Gene Expression, Functional Assay, Expressing