Journal: Cancer immunology, immunotherapy : CII

Article Title: Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

doi: 10.1007/s00262-012-1264-y

Figure Lengend Snippet: CpG ODN-conjugated HMFG-2 mAb treatment in vivo induced a significant tumor growth delay in immunodeficient nu/nu mice. a Schematic representation of the treatment regimen. Nude mice (nu/nu) were implanted s.c. with 3 × 106 KCM cells on day 0. On day 4, groups of mice (n = 4 mice/group) were treated i.t. with CpG ODN (50 µg), HMFG-2 mAb (50 µg), CpG ODN-conjugated HMFG-2 mAb, and PBS (50 µl). Each group of mice was treated every 48 h. Mice were randomized at day 4. b Tumor volume was measured with digital caliper every 2 days for 20 days and tumor growth is shown. Data shown are the mean ± SE of two experiments (***p < 0.001 compared to CpG ODN HMFG-2 mAb-treated mice vs. all other groups). c Tumor burden measured after treatment was suspended (n = 4 mice/group). Tumor burden increased in both the CpG ODN and CpG ODN-HMFG-2 mAb groups (*p = 0.038 and p = 0.027, respectively)

Article Snippet: CpG ODN type B 1826 and FITC–CpG ODN were obtained from Trilink (San Diego, CA, USA).

Techniques: In Vivo

Journal: Cancer immunology, immunotherapy : CII

Article Title: Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

doi: 10.1007/s00262-012-1264-y

Figure Lengend Snippet: Enhanced ADCC triggered in vivo by CpG ODN-conjugated HMFG-2 mAb is mutually dependent on NK cells and macrophages. a Schematic representation of the treatment regimen (n = 4). Depletion of NK cells (anti-asialo GM1) or macrophages (carrageenan) was initiated 3 days prior to KCM tumor challenge. Depletion was carried out for 3 consecutive days prior to tumor challenge and then every 7 days to maintain depletion. Ctrl mice received i.p. injection of IgG isotype Ctrl antibody. At day 9 post tumor challenge, mice were treated with CpG ODN conjugated with HMFG-2 mAbs every 48 h. All mice were killed at day 24. b Tumor volume was measured with digital caliper every 2 days and resulting tumor burden is shown. Data shown are the mean ± SE of two experiments. Significantly lower tumor burden (***p < 0.001) in the non-depleted macrophages/NK cells group compared to macrophages and/or NK cells depleted groups

Article Snippet: CpG ODN type B 1826 and FITC–CpG ODN were obtained from Trilink (San Diego, CA, USA).

Techniques: In Vivo, Injection

Journal: Cancer immunology, immunotherapy : CII

Article Title: Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

doi: 10.1007/s00262-012-1264-y

Figure Lengend Snippet: CpG ODN-conjugated HMFG-2 mAb retains specificity to endogenous human MUC1 expressed on KCM cells. a Schematic representation of CpG ODN-conjugated HMFG-2 mAb. b Schematic representation of PDA × MUC1.Tg mice from which KCM cells were derived. c Staining of MUC1+ KCM tumor cells with HMFG-2 antibody before and after conjugation with CpG ODN. Antibody-binding capability was revealed using a PE-conjugated anti-IgG mouse antibody. Flow cytometry analysis of unconjugated mAb (top) and CpG ODN-conjugated mAb (bottom) is shown (15 µg/mL). Similar results were obtained with 1, 5, 7, and 11 µg/mL. Isotype Ctrl is shown as a closed histogram

Article Snippet: CpG ODN type B 1826 and FITC–CpG ODN were obtained from Trilink (San Diego, CA, USA).

Techniques: Derivative Assay, Staining, Conjugation Assay, Binding Assay, Flow Cytometry

Journal: Cancer immunology, immunotherapy : CII

Article Title: Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

doi: 10.1007/s00262-012-1264-y

Figure Lengend Snippet: NK cell-mediated ADCC against KCM cells is enhanced when HMFG-2 mAb is conjugated to CpG ODN. a Cell proliferation assays were performed with KCM cells co-cultured with CpG ODN-conjugated or unconjugated HMFG-2 mAb (15 µg/ml). Proliferation was determined by bioreduction of a tetrazolium salt to a formazan at 490 nm. b Antibody-induced apoptosis analysis was determined in the presence of 15 µg/ml HMFG-2 mAb by flow cytometry. Annexin-V/7ADD staining was used to detect apoptotic cells. c Tumor cells (target cells) were mixed with CpG ODN-conjugated or unconjugated HMFG-2 (15 µg/ml) and 30 % cold-murine serum and cultured for 2 h at 37 °C. CDC was quantified by propidium iodide exclusion assay (PI−) on target cells. Positive Ctrl (TNP derivatization of KCM cells plus anti-DNP antibodies) showed significantly lower cell viability (***p = 0.0002 as compared to all other groups). d ADCC activity in KCM tumor cells coated with unconjugated HMFG-2 mAb as targets. Freshly isolated NK/LAK cells were used as effectors cells at E:T ratios of 50:1, 25:1, 12.5:1, 6.25:1. Significantly higher lysis (**p = 0.019 and ***p = 0.0001) in the HMFG-2 mAb-treated cells compared to IgG Ctrl-treated cells. E) ADCC activity using CpG ODN-conjugated HMFG-2 mAb (15 µg/ml) coated KCM tumor target cells versus unconjugated HMFG-2-treated cells. Isolated NK/LAK cells were used as effectors cells at E:T ratios of 50:1. Significantly higher lysis (***p = 0.0005) observed in CpG ODN-conjugated HMFG-2 treatment versus unconjugated HMFG-2 treatment. Each assay was repeated at least three times. Data shown are the mean ± SE of triplicate determinations

Article Snippet: CpG ODN type B 1826 and FITC–CpG ODN were obtained from Trilink (San Diego, CA, USA).

Techniques: Cell Culture, Flow Cytometry, Staining, Propidium Iodide Exclusion Assay, Activity Assay, Isolation, Lysis

Journal: Cancer immunology, immunotherapy : CII

Article Title: Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

doi: 10.1007/s00262-012-1264-y

Figure Lengend Snippet: a Immobilized CpG ODN-conjugated HMFG-2 mAb induced perforin up-regulation in NK cells. b Inhibition of Src family kinases or TLR9 signaling does not impair enhanced ADCC activity. a Immobilization of CpG ODN, HMFG-2 mAbs, or CpG ODN-conjugated HMFG-2 mAbs all induced upregulation of perforin on isolated NK cells when cultured for 18 h. Cells were incubated with PE-conjugated anti-NK1.1, and an intracellular staining with FITC-conjugated anti-perforin performed. Data shown are the mean ± SE of triplicate determinations (**p < 0.01 when compared to all treatment groups). b ADCC using isolated NK cells pre-incubated with pp2 (a Src inhibitor) (10 µM) or chloroquine (TLR9 inhibitor) (10 µM), and Ctrls (pp3, vehicle, or non-pretreatment) before the assay was performed. Data shown are the mean ± SE of triplicate determinations (***p < 0.001 for all groups compared to the vehicle Ctrl). c,d Fluorescent microscopy (×40) and flow cytometry analysis of immobilized FITC–CpG ODN incubated with activated NK cells. Immobilized FITC–CpG ODN is not internalized after overnight incubation with activated NK cells

Article Snippet: CpG ODN type B 1826 and FITC–CpG ODN were obtained from Trilink (San Diego, CA, USA).

Techniques: Inhibition, Activity Assay, Isolation, Cell Culture, Incubation, Staining, Microscopy, Flow Cytometry

Journal: Cancer immunology, immunotherapy : CII

Article Title: Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

doi: 10.1007/s00262-012-1264-y

Figure Lengend Snippet: CpG ODN-conjugated HMFG-2 mAb does not require MyD88 to trigger enhanced NK cell-mediated ADCC. ADCC activity was analyzed on opzonized KCM tumor cells using CpG ODN-conjugated mAb (15 µg/ml). Isolated NK cells from wild type, MyD88−/− mice were used as effectors cells at E:T ratios of 100:1, 50:1, 10:1. Compared to the MyD88−/− NK cells, NK cells from WT mice were significantly lower (*p = 0.048 and **p = 0.008). Each assay was repeated at least three times

Article Snippet: CpG ODN type B 1826 and FITC–CpG ODN were obtained from Trilink (San Diego, CA, USA).

Techniques: Activity Assay, Isolation