Journal: Journal of Biological Chemistry

Article Title: Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

doi: 10.1074/jbc.m705725200

Figure Lengend Snippet: FIGURE 2. IL-15 stabilizes IL-15R. Human 293 cells were transfected with plasmids expressing 0.05 g IL-15R (A) or IL-15sR (B) alone or in combina- tion with 0.05 g of IL-15t, as indicated. After 72 h, IL-15R production in the culture supernatants and cell extracts was analyzed by Western immunoblot using a goat anti-IL-15R antibody. Sample dilutions of 1:2 and 1:4 were loaded as indicated to quantify the produced IL-15R. Mock indicates trans- fectionwithacontrolplasmidonly(GFP).Theamountofundilutedcellextract and culture supernatants loaded on the gel were 1:200 and 1:300, respec- tively. Arrows indicate the position of IL-15R bands, and arrowheads indicate the position of molecular weight markers. Similar transfection efficiencies were verified by co-transfection of GFP expression vectors. C, mRNA expres- sion levels of IL-15 and IL-15R plasmids transfected individually or together in human 293 cells. The expression of cellular gene GAPDH was used as con- trol (lower panel). The location of mRNAs was identified by Northern blot analysis and is indicated by arrows.

Article Snippet: Human IL-15 levels were measured by ELISA (Quantikine human IL-15 immunoassay; R & D Systems) or by Western immunoblot (using the polyclonal goat anti-human IL-15 antibody AF315, R & D Systems).

Techniques: Transfection, Expressing, Western Blot, Produced, Molecular Weight, Cotransfection, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

doi: 10.1074/jbc.m705725200

Figure Lengend Snippet: FIGURE4.IntracellularassociationofIL-15withtheIL-15R.A,human293 cells were transfected with IL-15-expressing plasmid (IL-15t) alone or in com- bination with IL-15R-expressing plasmid producing either full-length (IL- 15R) or soluble form (IL-15sR). Complexes were immunoprecipitated from cell extracts or culture medium with an anti-IL-15R antibody and were sub- sequently examined by Western immunoblot analysis using an anti-IL-15 antibody. The three bands corresponding to the unglycosylated, partially, and fully N-glycosylated forms of IL-15 are indicated. B, human 293 cells were transfected with 0.05 g of IL-15FLAG DNA alone, IL-15R DNA alone, or IL-15FLAGIL-15RDNAsincombination.Cellstransfectedwiththeindivid- ual plasmids were harvested and washed separately at 4 °C and divided into two tubes. Recombinant human IL-15 protein was added to the cells as indi- cated using 50 excess of the expected IL-15 production. The cells were lysed, and IL-15 was immunoprecipitated using anti-FLAG antibody. The complexes were separated on SDS-PAGE, and the IL-15R was visualized by Western immunoblot. The positions of the glycosylated and unglycosylated IL-15R are indicated.

Article Snippet: Human IL-15 levels were measured by ELISA (Quantikine human IL-15 immunoassay; R & D Systems) or by Western immunoblot (using the polyclonal goat anti-human IL-15 antibody AF315, R & D Systems).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Recombinant, SDS Page

Journal: Oncoimmunology

Article Title: A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy

doi: 10.1080/2162402X.2021.1893500

Figure Lengend Snippet: In vitro characterization of IL15/IL15Rα-Fc complexes . (a) Schematic diagrams of all four IL15/IL15Rα-Fc complexes in theoretical dimeric Fc fusion formats. (b) All four complexes were shown on reduced SDS-PAGE, before and after deglycosylation treatment, using Invitrogen SeeBlue Plus2 Pre-Stained Standard as the protein molecular weight (MW) marker. (c) WT-FL complex on SDS-PAGE under reduced and non-reduced conditions, using Life Technologies BenchMark Unstained Protein Ladder as the protein MW marker (the size of selected bands was shown), with IL15Rα-Fc and IL15 bands were marked with arrows. (d) SE-HPLC chromatography using G4000SW column. Major peak of WT-FL (19.0 minutes) corresponding to MW around 600 kDa, and SU complexes (22.3 minutes) corresponding to around 120 kDa, and salt buffer peak (26.5 minutes). (e) SE-HPLC results of the 6 standard MW marker (Sigma Gel Filtration Markers Kit) mixed together. Each MW marker was also run separately to confirm the position, except for the 150 and 66 kDa markers which did not separate in the mixture. (f) The fitted curve for WT-FL complex MW calculation. The size of 600 kDa of the WT-FL complex was calculated based on the fitted curve derived from the top three high MW standard markers that was run separately. (g) Schematic diagram of WT-FL complex in hexameric format, and accelerated stability test of WT-FL at 37°C over 4 weeks; monomer % represents the percentage of monomers in HPLC profile for each time point, based on AUC analysis excluding buffer peak. (h) In vitro cell proliferation assays using mouse lymphoblast cell line CTLL-2. Mean + SD (n = 3). * p < .0001 when FL-complexes treatment groups were compared with SU-complexes treatment groups at indicated concentration, respectively

Article Snippet: IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech).

Techniques: In Vitro, SDS Page, Staining, Molecular Weight, Marker, Chromatography, Filtration, Derivative Assay, Concentration Assay

Journal: Oncoimmunology

Article Title: A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy

doi: 10.1080/2162402X.2021.1893500

Figure Lengend Snippet: In vitro binding of hu3F8 and hu3F8-BsAb to IL15-stimulated lymphocyte subsets . (a) Representative immunophenotypic appearance of normal PBMC-derived CD3 + CD56 − T cells and CD3 − CD56 + NK cells (left panel). Representative flow cytometric appearance of PBMC and M14 tumor cells mixed together at 1:1 ratio (middle panel). Binding of hu3F8 and hu3F8-BsAb to M14 tumor cells (right two panels). (b) Expression of CD16 (FcγRIII) on T cells and NK cells. (c) Binding of hu3F8 IgG1 via FcγRIII to CD16-expressing NK cells but not T cells. (d) Binding of hu3F8-BsAb via CD3 to CD3-expressing T cells but not NK cells. PBMCs were cultured in complete RPMI medium either without (Medium) or with 1 nM WT-FL complex for 72 hrs before FACS analysis. Numbers in right upper corner of each histogram box represent MFI ratios calculated as geo-MFI of antigen-specific staining (red-line histogram) divided by geo-MFI of isotype-control IgG staining (black filled peak)

Article Snippet: IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech).

Techniques: In Vitro, Binding Assay, Derivative Assay, Expressing, Cell Culture, Staining, Control

Journal: Oncoimmunology

Article Title: A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy

doi: 10.1080/2162402X.2021.1893500

Figure Lengend Snippet: In vitro cytotoxicity by IL15-stimulated PBMCs . PBMCs from one healthy donor was cultured in vitro in medium either without (Medium) or with soluble IL15 or different IL15/IL15Rα complexes. After 72 hrs of culture, the PBMCs were harvested, re-adjusted in numbers and tested in an in vitro cytotoxicity ( 51 Cr-release) assay against M14 human melanoma cells either in the absence or presence of different antibodies. Results are presented as percentage of tumor cell lysis (Mean + SEM, n = 3). * p < .01 when WT-complexes treatment groups were compared with MUT-complexes treatment groups at indicated concentration or E:T ratio, respectively. (a) Antibody-independent cytotoxicity titrated by IL15 concentrations. E:T ratio at 10:1. (b) Antibody-dependent cytotoxicity titrated by hu3F8 (middle panel) or hu3F8-BsAb (right panel). IL15 complexes concentration at 0.001 nM, and E:T ratio at 10:1. (c) Cytotoxicity titrated by E:T ratios. IL15 complexes concentration at 1 nM, and antibodies concentration at 0.01 ug/mL

Article Snippet: IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech).

Techniques: In Vitro, Cell Culture, Release Assay, Lysis, Concentration Assay

Journal: Oncoimmunology

Article Title: A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy

doi: 10.1080/2162402X.2021.1893500

Figure Lengend Snippet: In vitro immunophenotype of IL15-stimulated PBMCs . PBMCs from healthy donors (n = 5) were cultured in vitro in medium either without (Medium) or with 1 nM WT-FL or MUT-FL complex. At 24, 72 and 168 hr cells were tested by flow cytometry for expression of different surface markers. Analysis is based on gating on T cells (CD3 + CD56 − lymphocytes) or NK cells (CD3 − CD56 + lymphocytes). Results are presented as geo-MFI ratio of the marker of interest, individual for each donor. Lines represent Mean (n = 5) of those 5 individual values. * p < .01 when WT-FL treatment groups were compared with MUT-FL treatment groups at indicated time point, respectively. (a) Surface expression of FasL. (b) Surface expression of TRAIL. (c) Surface expression of CD16 and NKp46. (d) Surface expression of NKG2D. (e) Intracellular co-expression of Perforin and Granzyme-B. PBMCs from one healthy donor was used in this case. Results are presented as geo-MFI ratio of the marker of interest, at each time point of testing. To enable Perforin and Granzyme-B retention, Brefeldin-A was added to cultures for the last 6 hr of each time point. Representative FACS dot-plots at 72 hr time point were shown in (f)

Article Snippet: IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech).

Techniques: In Vitro, Cell Culture, Flow Cytometry, Expressing, Marker

Journal: Oncoimmunology

Article Title: A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy

doi: 10.1080/2162402X.2021.1893500

Figure Lengend Snippet: WT-FL IL15 complex promotes in vivo lymphocyte expansion and improves serum half-life in mice . (a) Lymphocyte expansion in healthy C57BL/6 mice. Absolute cell counts were calculated by multiplying the white blood cell count from CBC and percentage of positive cells from FACS. Mean ± SEM (n = 5). * p < .001 when WT-FL treatment group was compared with either no treatment (PBS) group or MUT-SU treatment group, respectively; ns, p > .05 when WT-FL treatment group was compared with MUT-SU treatment group. (b) Pharmacokinetics of IL15 complexes. Quantitation of serum IL15 complexes was carried out by ELISA, and the data were depicted using GraphPad Prism software. Mean + SD (n = 5). Pharmacokinetic analysis was carried out by non-compartmental analysis of the serum concentration-time data using WinNonlin software program, and presented in the

Article Snippet: IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech).

Techniques: In Vivo, Cell Counting, Drug discovery, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Software, Concentration Assay

Journal: Oncoimmunology

Article Title: A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy

doi: 10.1080/2162402X.2021.1893500

Figure Lengend Snippet: WT-FL IL15 complex improves serum half-life in mice

Article Snippet: IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech).

Techniques:

Journal: Oncoimmunology

Article Title: A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy

doi: 10.1080/2162402X.2021.1893500

Figure Lengend Snippet: WT-FL IL15 complex potentiates anti-tumor effects of antibody immunotherapy against tumor cell line xenografts . Treatment schedules were marked on the figures, and doses of IL15 complexes, antibodies and effector cells were detailed in the Results. Data shown as mean + SEM (n = 5); (a-b) iv tumor plus iv effector cells model : Bioluminescence changes of IMR32 neuroblastoma during treatment (a) and representative images at day 25 (b). * p < .05 when FL-complexes treatment groups were compared with all other four groups at indicated time point, respectively. (c) sc tumor plus sc effector cells (1:1 mixing) model : tumor volume changes of IMR32 neuroblastoma. * p < .05 when FL-complexes treatment groups were compared with No treatment group at indicated time point, respectively; ns, p > .05 when SU-complexes treatment groups were compared with No treatment group at indicated time point, respectively. (d) sc tumor plus iv effector cells model : % tumor growth (calculated as tumor volume at indicated time point divided by tumor volume at Day 8 before the treatment start) of M14 melanoma. * p < .0001 when FL-complexes treatment groups were compared with all other four groups at indicated time point, respectively. (e ) C57BL/6 mice grafted with sc GD2(+) murine melanoma cells : tumor volume changes of B78/D14 melanoma. * p < .0001 when FL-complexes treatment groups were compared with SU-complexes treatment groups at indicated time point, respectively

Article Snippet: IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech).

Techniques: