Journal: Nature Communications

Article Title: Synergistic cancer immunotherapy combines MVA-CD40L induced innate and adaptive immunity with tumor targeting antibodies

doi: 10.1038/s41467-019-12998-6

Figure Lengend Snippet: Therapeutic efficacy of rMVA-CD40L in unrelated, large, established tumor models. a Experimental layout: briefly, C57BL/6 ( b – e ) or Balb/c mice ( f – i ) received either B16.OVA ( b , c ), MC38.WT ( d , e ), CT26.WT ( f , g ) or CT26.HER2 ( h , i ) cells subcutaneously in the flank. Seven to 14 days later, when tumors were above 60 mm 3 , mice were immunized intravenously either with PBS or with 5 × 10 7 TCID 50 of the mentioned rMVA viruses. b , c B16.OVA; b tumor size follow-up ( n = 5 mice/group) and c overall survival ( n = 10 mice/group) of mice injected either with PBS, MVA-OVA, or MVA-OVA-CD40L; d , e MC38.WT tumor-bearing mice were grouped 18 days after tumor cell inoculation, when tumors were above 90 mm 3 ; d tumor size follow-up ( n = 10 mice/group) and e overall survival ( n = 10 mice/group) of mice injected either with PBS, MVA-TAA, or MVA-TAA-CD40L until day 60 after treatment; f , g CT26.WT; f tumor size follow-up ( n = 5 mice/group), and g overall survival ( n = 5 mice/group) of mice injected either with PBS, MVA-TAA, or MVA-TAA-CD40L; h , i CT26.HER2; h tumor size follow-up ( n = 6 mice/group), and i overall survival ( n = 6 mice/group) of mice injected either with PBS or MVA-HER2-CD40L. b , d , f , and h Data are expressed as mean ± SEM. Panel b is representative of at least two independent experiments. Panel c represents overall survival of two merged independent experiments. The antitumor efficacy of MVA-HER2-CD40L in h and i has been tested in the CT26.HER2 tumor model in at least two independent experiments. One-way ANOVA at day 20 after tumor inoculation was performed on b , d , f , and h . Log-rank test on mouse survival was performed for c , e , g , and i . * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For anti-HER2 experiments, 5 µg anti-HER2-Trastuzumab-hIgG1 or hIgG1 (Invivogen) were injected i.p. at days −2, 1, and 4 after immunization

Techniques: Injection

Journal: Nature Communications

Article Title: Synergistic cancer immunotherapy combines MVA-CD40L induced innate and adaptive immunity with tumor targeting antibodies

doi: 10.1038/s41467-019-12998-6

Figure Lengend Snippet: Combination of MVA-CD40L and tumor-targeting antibodies enhances tumor growth control. a – d Enhanced tumor growth control when rMVA-CD40L immunization is combined with tumor-targeting antibodies. a , b B16.OVA tumor-bearing mice received PBS or were immunized with 5 × 10 7 TCID 50 of rMVA-CD40L (Day 0). Mice received 200 µg of either IgG2a or anti-TRP-1 antibody i.p. at days −2, 2, 6, and 10. a Tumor size follow-up ( n = 5 mice/group) and b overall survival. c , d Balb/c mice bearing 85–100 mm 3 CT26.HER2 received PBS or were immunized with 5 × 10 7 TCID 50 of MVA-HER2-CD40L (Day 0). Mice received 5 µg of either IgG1 or anti-HER2 antibody i.p. at days –2, 1, and 4. c Tumor size follow-up ( n = 5 mice/group) and d overall survival. Data in a and c are expressed as mean ± SEM, representative of at least two independent experiments. One-way ANOVA was performed on a . Log-rank test on mouse survival was performed for b . * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For anti-HER2 experiments, 5 µg anti-HER2-Trastuzumab-hIgG1 or hIgG1 (Invivogen) were injected i.p. at days −2, 1, and 4 after immunization

Techniques:

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A Analysis of the immunohistochemical pathological reports for HER2 of 13 CRC patients in Jiangsu Provincial Hospital. B Western blot analysis of HER2 expression in murine and human CRC cell lines. C Expression and localization of HER2 detected by immunofluorescence on MC38 HER2 , MC38 Ctrl , CT26 HER2 , and CT26 Ctrl cells. D The mRNA expression of HER2 was measured by qPCR. ** P < 0.01. E The expressions of HER2 on the cell membrane were detected using flow cytometry. F The expression and localization of HER2 in HCT116 and HT29 cells were determined by immunofluorescence. Scale bars, 10 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Immunohistochemical staining, Western Blot, Expressing, Immunofluorescence, Flow Cytometry

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A In vitro growth inhibition assay for HCT116 cells treated with MMAE and Paclitaxel for 24 h. B In vitro growth inhibition assay for HCT116 and HT29 cells. C In vitro growth inhibition assay for MC38 HER2 and MC38 Ctrl cells treated with RC48 for 48 h. D In vitro growth inhibition assay for MC38 Ctrl and MC38 HER2 cells treated with MMAE for 24 h. E Flow cytometry analysis of cell cycle after treatment with different concentrations of RC48 for 48 h. F Clonogenic assay of HCT116, CT26 HER2 and MC38 HER2 treated with RC48. Data represent the mean ± SEM of three replicates. * P < 0.05, ** P < 0.01, versus as indicated. n.s. not significant.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: In Vitro, Growth Inhibition Assay, Flow Cytometry, Clonogenic Assay

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A MC38 Ctrl cells were treated with different concentrations of Disitamab (0, 10, 30, 100 μg/ml) for 48 h, and the expression of p-TBK1, p-IRF3, TBK1, IRF3 protein was detected by immunoblotting. B MC38 HER2 and MC38 Ctrl cells were treated with 100 μg/ml RC48 for 48 h and 30 μM CPT11 for 12 h and the expression of p-TBK1, p-IRF3, TBK1, IRF3 protein was detected by immunoblotting. C HCT116 was treated with 100 μg/ml RC48 for 48 h and 30 μM CPT11 for 12 h. D The expressions of HER2 on the cell membrane were detected using flow cytometry. MFI Mean fluorescence intensity. E Co-immunoprecipitation of endogenous HER2 and STING in HCT116. F The expression and localization of HER2 in MC38 HER2 were detected by immunofluorescence. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 10 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Expressing, Western Blot, Flow Cytometry, Fluorescence, Immunoprecipitation, Immunofluorescence

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A – E MC38 HER2 (1 × 10 6 ) cells were inoculated in the C57BL/6 mice. Drugs were given as described in Materials and Methods. B , C Tumor volume and body weight were measured every two days. D , E Solid tumors were separated and weighed after the mice were sacrificed. The data represent the mean ± SEM of 10 mice per group. F Paraffin sections of tumor tissues were analyzed by H&E staining, PCNA immunohistochemistry staining, and TUNEL fluorescent staining. G The survival of tumor-bearing mice. H – M Same experiment with CT26 HER2 in BALB/c mice. The data represent the mean ± SEM of 7 mice per group. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 50 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Staining, Immunohistochemistry, TUNEL Assay

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A Immunofluorescence staining for p-TBK1 and p-IRF3 in tumor tissue in C57BL/6. B , C Immunohistochemical detection of IFN-β secretion in tumor tissue in BALB/c and C57BL/6. D LDH release assay was performed using Tils achieved from the syngeneic model of CT26 HER2 as described in material and methods. E , F Examination of CD3 + , CD8 + , IFN-γ secretion in the tumor by flow cytometry in BALB/c and C57BL/6. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 100 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Lactate Dehydrogenase Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: RC48 exhibits the capability to bind to HER2 receptors and undergo internalization, subsequently releasing the toxin MMAE. MMAE inhibits tubulin, leading to the disruption of the cell cycle and resulting in the efficient elimination of tumor cells. Moreover, through its interaction with HER2, RC48 can alleviate the inhibition induced by internalized HER2 on the STING pathway and its downstream signaling. These combined mechanisms enable RC48 to effectively impede the proliferation of HER2-positive tumors while also enhancing their sensitivity to immunotherapy.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Inhibition

Journal: Nature Communications

Article Title: Unlocking the potential of allogeneic Vδ2 T cells for ovarian cancer therapy through CD16 biomarker selection and CAR/IL-15 engineering

doi: 10.1038/s41467-023-42619-2

Figure Lengend Snippet: a – g In vitro ADCC assay. Three human ovarian cancer cell lines (OVCAR3-FG, OVCAR8-FG, and SKOV3-FG) and four effector cells (Vδ2T, MCAR-Vδ2T, MCAR15-Vδ2T, and MCAR-T as a benchmark control) were included in the study. The same CD16 Hi donor PBMCs were used to generate all 4 types of effector cells. a Experimental design. Data were collected at 24 h after co-culture. Anti-HER2 Ab: monoclonal antibody trastuzumab. b FACS detection of HER2 expression on the indicated tumor cell lines. c Killing of OVCAR3-FG tumor cells by Vδ2T cells in the presence of titrated amounts of either isotype control or anti-HER2 Ab ( n = 3). Tumor cell killing data of ( d ) OVCAR3-FG, ( e ) OVCAR8-FG, and ( f ) SKOV3-FG in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 0.5:1; n = 3). g ELISA measurements of IFN-γ production at 24 h in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 1:1; n = 3). h , i In vitro repeated tumor cell challenge assay studying ADCC. h Experimental design. Effector cells were mixed with tumor cells and rechallenged every 3 days. Tumor cell killing data was measured at 24 h post co-culture with or without the addition of anti-HER2 antibody (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 2:1; n = 3). i KO OVCAR3-FG tumor cell killing data collected over time. Representative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by Student’s t test ( c ) or by one-way ANOVA ( d – g ). Sour c e data and exact p values are provided as a Source Data file.

Article Snippet: InVivo SIM anti-human HER2 (Trastuzumab Biosimilar, Cat. SIM0005) was purchased from BioXCell.

Techniques: In Vitro, ADCC Assay, Control, Co-Culture Assay, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Integrated Analytical System for Clinical Single‐Cell Analysis

doi: 10.1002/advs.202200415

Figure Lengend Snippet: Antibodies for flow and direct single cell staining

Article Snippet: 3 , HER2 , Tumor cells , BioRad , MCA1788 , ICR55 , Rat , BT474, HCC‐1954.

Techniques: Control

Journal: iScience

Article Title: From byproduct to biotherapeutic: Comparative study of buffy coats and leukoreduction system chambers for NK cell-based immunotherapies

doi: 10.1016/j.isci.2026.114907

Figure Lengend Snippet: Functional characterization of NK cells (A) Degranulation capacity of NK cells following a 2 h co-culture with K562 leukemia cells, measured by surface CD107a expression ( n = 5). (B and C) Europium-based cytotoxicity assay assessing the specific lysis of K562 target cells at E:T ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 on day 7 (B) and day 14 (C) of culture to monitor functional decline over time ( n = 6 for both time points). Here, all comparisons were not significant and are summarized as such (n.s.: p > 0.05). (D) IFN-γ release following 16 h of co-culture of NK cells with K562 cells, quantified via Luminex assay ( n = 5). (E) Kinetic live-cell killing assay using the Incucyte monitoring of Nuclight Red signal loss in K562 cells over 72 h at an E:T ratio of 1:1 ( n = 6). (F) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis thresholds during Incucyte co-culture with K562 cells. (G) CD107a degranulation assay of NK cells with or without pre-incubation with trastuzumab (anti-HER2 antibody) during a 2 h co-culture with MDA-MB-453 cells ( n = 5). (H) Incucyte-based cytotoxicity assay shows the real-time lysis of MDA-MB-453 cells by trastuzumab-loaded NK cells at an E:T ratio of 1:1 over 72 h ( n = 6). (I) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the ADCC assay. (n varies as not all samples reached the benchmark values within the observation period). All ADCC experiments have been performed after 14 days of in vitro NK cell expansion. N/A indicates that statistical analysis was not possible for this group. 9J) CD107a degranulation of unmodified NK cells and ErbB2-CAR-NK cells after 2 h co-culture with MDA-MB-453 cells ( n = 5). (K) Incucyte-based cytotoxicity assay showing killing dynamics of ErbB2-CAR-NK cells against MDA-MB-453 cells at an E:T ratio of 1:1 over 72 h ( n = 6). (L) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the CAR-mediated cytotoxicity assay. (n varies as not all samples reached the benchmark values within the observation period). All CAR-killing experiments have been performed after 14 days of in vitro NK cell expansion. Unless otherwise stated, n refers to biologically independent donor samples and is indicated per group. Data are shown as mean ± SEM. p-values were determined by Student’s t test with Welsh correction (A, D, F, G, I, J, and L) or by one-way ANOVA (B and C) with Tukey post-test (K and L).

Article Snippet: Mouse Monoclonal anti-CD340 (Her2) , Miltenyi Biotec , Cat# 130-124-474.

Techniques: Functional Assay, Co-Culture Assay, Expressing, Cytotoxicity Assay, Lysis, Luminex, Degranulation Assay, Incubation, ADCC Assay, In Vitro