Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cellular expression of cathepsin V with PE results in (Met)enkephalin production in PC12 cells. a, expression of cathepsin V with PE results in ME production. The human cathepsin V cDNA in expression plasmid vector was transfected into PC12 neuroendocrine cells, and measurement of ME by RIA was conducted 3 days after transfection of PE. The coexpression of cathepsin V with PE resulted in increased cellular levels of (Met)enkephalin, expressed as x ± S.E. *, statistically significant (n = 3 for each experiment, p < 0.05 by Student's t test, repeated twice). b, shown is control expression of PE alone. Transfection of the PE cDNA in PC12 cells results in expression of PE shown as a band of ∼31 kDa (lane 2). Control experiments show that PE is absent in cells when transfected with vector alone (pcDNA3.1 without PE cDNA) (lane 1). c, shown is control expression of cathepsin V alone. Transfection of the cathepsin V cDNA results in expression of this protease, shown as a main band of ∼24-kDa cathepsin V (lane 2), likely corresponding to the mature form of the enzyme of 23,999 daltons (∼24 kDa) calculated molecular mass (31). Cell extract (15 μg protein) contained ∼10–25 ng of cathepsin V, based on the high sensitivity of the Western blot with standard cathepsin V (shown in supplemental Fig. S1). The Western blot detects low levels of cathepsin V, illustrated by detection of 25 ng of purified cathepsin V. Furthermore, the anti-cathepsin V Western blot shows specificity for detection of cathepsin V but does not detect the related cysteine cathepsins L, B, and H (supplemental Fig. S1).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Purification

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Expression of cathepsin V in human neuroblastoma SK-N-MC cells results in (Met)enkephalin production. a, cathepsin V expression mediates enkephalin production. Human cathepsin V was transfected in human SK-N-MC neuroblastoma cells, and cells were harvested 2 days later. (Met)enkephalin levels in cell extracts were measured by RIA, expressed as x ± S.E. *. statistically significant (n = 6 for each experiment, p < 0.01, Student's t test, repeated twice). b, shown is control expression of cathepsin V. Transfection of the cathepsin V cDNA results in expression of the protease, shown as an ∼24-kDa band, likely corresponding to the mature form of the enzyme. The two band areas of ∼38–40 and ∼40–45 kDa in b are consistent with preprocathepsin V and procathepsin V, respectively (8, 10, 31).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Gene silencing of cathepsin V reduces (Met)enkephalin in human neuroblastoma SK-N-MC cells. a, gene silencing of cathepsin V by siRNA reduces (Met)enkephalin production. Human neuroblastoma SK-N-MC cells were transfected with siRNA to cathepsin V as described under “Experimental Procedures.” As controls, transfection of scrambled sequences of siRNA was conducted as well as no siRNA. One day after transfection cells were harvested, and cell levels of (Met)enkephalin were measured by radioimmunoassay. A significant decrease in (Met)enkephalin occurred after transfection of cells with siRNA to cathepsin V (p < 0.05 by Student's t test) compared with the controls of scrambled siRNA or no siRNA. b, cathepsin V protein expression is substantially reduced by siRNA gene silencing. Silencing of cathepsin V expression was assessed by Western blots after transfection of cathepsin V siRNA into SK-N-MC cells. Cathepsin V was substantially reduced with 50 nm siRNA (lane 2) and partially reduced with 25 nm siRNA (lane 3) compared with control transfection with scrambled siRNA (lane 1). These data show decreased cathepsin V expression when cathepsin V siRNA is transfected in SK-N-MC cells.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Transfection, RIA Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: In vitro processing of PE by cathepsin V generates an intermediate present in human brain. a, in vitro processing of PE by cathepsin V is shown. PE was incubated with or without human cathepsin V for 30 min and subjected to SDS-PAGE and Western blot using anti-ME (panel i) or LE (panel ii). Results show the presence of an ∼24-kDa PE-derived band recognized by both anti-ME and anti-LE antisera. b, shown is the PE-derived ∼24-kDa intermediate in human brain cortex and hippocampus. Western blots of human brain cortex (panel i) and hippocampus (panel ii) with anti-ME and anti-LE are shown. Results show the presence of an ∼24-kDa band that is recognized by both antisera, indicating the presence of a PE-derived ∼24-kDa intermediate.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: In Vitro, Incubation, SDS Page, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: In vitro processing of PE by cathepsin V via cleavage at dibasic residues generates enkephalin peptides. a, shown is the PE precursor. The PE precursor contains several copies of enkephalin-related peptides consisting of ME, LE, ME-Arg-Gly-Leu (O), and ME-Arg-Phe (H), flanked by dibasic residues (KR, KK) that are known to be processed to generate mature enkephalin. b, shown are PE-derived products. Results of recombinant human cathepsin V cleavage of recombinant PE resulted in production of a ∼24-kDa (from Fig. 4). Peptide products identified by mass spectrometry are indicated by Lys-Arg-ME consisting of the KRYGGFM sequence (with the ME sequence underlined) and C-terminal peptides consisting of the sequence RFAEALPSDEEGESYSKEVPEMEKRYGGFMRF and KRFAEALPSDEEGESYSKEVPEMEKRYGGFMRF that contain the heptapeptide ME-Arg-Phe (underlined). Peptide products result from cleavage at the N-terminal side and between dibasic residues. Mass spectrometry data of peptide products are provided in supplemental Table S1, and MS/MS spectra of identified peptide products are shown in supplemental Fig. S2.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: In Vitro, Derivative Assay, Recombinant, Mass Spectrometry, Sequencing, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cathepsin V proteolytic activity with dibasic and monobasic peptide-MCA substrates Cathepsin V was incubated with peptide-MCA, and proteolytic activity of cathepsin V was measured as μmol of fluorescent AMC/h/mg of cathepsin V. All assays were performed in quadruplicate with each substrate; replicate values varied by less than 10%. These results indicate that cathepsin V cleaves at dibasic and monobasic residues. Boc, t -butoxycarbonyl.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Activity Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cathepsin V in human secretory vesicles and neuronal tissues. a, cathepsin V in isolated human secretory vesicles from sympathoadrenal pheochromocytoma. Western blot with anti-cathepsin V was assessed in soluble and membrane fractions (lanes 1 and 2, respectively) of secretory vesicles isolated from human pheochromocytoma of sympathoadrenal tissue. Blots illustrate the presence of mature cathepsin V of an ∼24-kDa band (31). b–d, shown is cathepsin V in human brain cortex and hippocampus. Cathepsin V in human neuronal tissues was analyzed by Western blots after immunoprecipitation of cathepsin V from human brain cortex (C) and hippocampus (H) (panel b, lanes 1 and 2, respectively), human SH-SY-5Y neuroblastoma cells (panel c), and human SK-N-MC neuroblastoma cells (panel d). Cathepsin V was observed as its mature form of ∼24 kDa (31). The band in panels b–d of ∼40–45 kDa are consistent to that of preprocathepsin V (8, 10, 31). The specificity of the anti-cathepsin V Western blot shows its sensitive detection of 25 ng or lower levels of standard cathepsin V, with no detection of the cysteine cathepsins L, B, or H (supplemental Fig. 1). In addition, control Western blot without primary antibody resulted in a lack of immunopositive bands.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Isolation, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Neuronal localization of cathepsin V with enkephalin in secretory vesicles assessed by immunofluorescent confocal microscopy in human SK-N-MC neuroblastoma cells. a, localization of cathepsin V with enkephalin in secretory vesicles is shown. Cathepsin V was observed (red fluorescence) as a punctate, discrete pattern of localization like that of (Met)enkephalin (green fluorescence). The colocalization of the protease with enkephalin was illustrated by the merged yellow fluorescence. Quantitation of the relative amount of cellular cathepsin V that is colocalized with enkephalin was assessed with the Pearson correlation coefficient (Rr value) (Table 2). Measurement of the Rr value as 0.45 indicates partial colocalization of cathepsin V with enkephalin. It is noted that cathepsin V is also observed in nuclei; this is consistent with other reports demonstrating nuclear functions of cathepsin V (52, 53). b, shown is an enlarged view of secretory vesicles containing cathepsin V and enkephalin. An enlarged image more clearly shows the secretory vesicle colocalization of cathepsin V (red immunofluorescence) with enkephalin (green immunofluorescence) as indicated by the merged yellow immunofluorescence; arrows indicate the secretory vesicles containing both cathepsin V and enkephalin. Controls using normal mouse IgG instead of mouse anti-cathepsin V resulted in markedly reduced immunofluorescence (supplemental Fig. 3), illustrating the specificity of the antibody to cathepsin V.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Confocal Microscopy, Fluorescence, Quantitation Assay, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Quantitation of cathepsin V localization with enkephalin and NPY in human neuroblastoma cells The colocalization of cathepsin V immunofluorescence with (Met)enkephalin in human neuroblastoma SK-N-MC cells and with NPY in human neuroblastoma SH-SY-5Y cells was quantitated by measuring the Pearson correlation coefficient (Rr) with the Velocity software as described under “Experimental Procedures.” A Pearson correlation of 1 indicates complete colocalization, and a value of 0 indicates no specific colocalization. Measurements of the Pearson correlation coefficient of 0.448 ± 0.018 for cathepsin V and (Met)enkephalin indicate their partial colocalization. The Pearson correlation coefficient of 0.736 ± 0.030 for cathepsin V and NPY in SH-SY-5Y cells indicates a reasonable degree of partial colocalization. Additional evaluation of cathepsin V localization with Lamp-1, a marker for lysosomes (54), indicated the Rr value of 0.535 ± 0.036, indicating partial colocalization. The Pearson correlation coefficient was measured with n = 5 cells. The mean ± S.E. is shown, and statistical significance of colocalization ( p < 0.0001 by Student's t test) was compared to the null hypothesis of no specific colocalization (Pearson correlation coefficient value of 0).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Quantitation Assay, Immunofluorescence, Software, Marker

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Localization of cathepsin V with Lamp-1 marker of lysosomes in human SK-N-MC cells. a, shown is cathepsin V localization assessed with Lamp-1. Cathepsin V subcellular distribution was evaluated with Lamp-1, a marker for lysosomes (32). Cathepsin V (red) and Lamp-1 (green) display partial colocalization (merged yellow immunofluorescence), shown by arrows. Quantitative analyses of their partial colocalization was conducted with the Pearson correlation coefficient, indicated as 0.535 ± 0.036 (see Table 2, legend). b, shown is an enlarged view of cathepsin V localization with Lamp-1. An enlarged image shows the distinct colocalization of cathepsin V (red) with Lamp-1 (green) shown by the merged yellow immunofluorescence.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Marker, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cellular production of NPY by cathepsin V. a, localization of cathepsin V and NPY in human SH-SY-5Y neuroblastoma cells is shown. Cathepsin V localization in human SH-SY-5Y neuroblastoma cells was observed by immunofluorescent confocal microscopy. Cathepsin V (red fluorescence) and NPY (green fluorescence) were discretely localized, with colocalization among many secretory vesicles (yellow immunofluorescence, indicated by arrows). Quantitation of the relative cathepsin V colocalization with NPY was assessed by the Pearson correlation coefficient (Rr value) (Table 2). The measured Rr value of 0.74 indicates that cathepsin V is partially colocalized with NPY. Controls using normal mouse IgG instead of mouse anti-cathepsin V resulted in markedly reduced immunofluorescence (supplemental Fig. S3), illustrating the specificity of the antibody to cathepsin V. b, NPY production by cathepsin V in PC12 cells is shown. PC12 cells were cotransfected with proNPY cDNA (in pcDNA3.1 vector) and cathepsin V cDNA (in pCMV6-XL5 vector), and cells were harvested 3 days later for analyses of cellular levels of NPY (by RIA). Results are shown as x ± S.E. *, statistically significant (n = 6, p < 0.001, Student's t test).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Confocal Microscopy, Fluorescence, Immunofluorescence, Quantitation Assay, Plasmid Preparation

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: Equilibrium binding of CD40/PDL1-bispecific antibody variants and the corresponding parental antibodies to their cell-expressed target molecules. (A,B) Domain architecture of the various antibodies variant (A) and the corresponding GpL fusion proteins (B). N297A indicates a point mutation which prevents or strongly reduces binding to FcγRs. Heavy and light chains of all constructs contained an N-terminal Flag tag for estimation of concentration in cell culture supernatants and gentle purification. CH1/2/3, heavy chain constant region 1/2/3; CL, light chain constant region; Fab, antigen binding fragment; scFv, single chain variable fragment; VH, heavy chain variable region; VL light chain variable region. (C) 200 ng of the indicated antibody variants were separated by SDS-PAGE and visualized by western blotting with αFlag. (D) Specific binding of the GpL-tagged antibody variants to CD40- and PDL1-expressing cells. One representative experiment is shown. Mean and single K D -values of three to six independent experiments are summarized in Table of the manuscript.

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Binding Assay, Variant Assay, Mutagenesis, Construct, FLAG-tag, Concentration Assay, Cell Culture, Gentle, Purification, SDS Page, Western Blot, Expressing

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: Binding affinities of investigated aCD40/PDL1-antibody fusion proteins. For statistical analysis see supplemental data Figure S1 .

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Binding Assay

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: PDL1-dependent CD40 agonism of CD40/PDL1-bispecific antibody variants. (A, B, C) HT1080-CD40 cells were stimulated in the presence of HEK293 cells transfected with empty vector (EV) or an PDL1-encoding expression plasmid with the indicated concentrations of αCD40-Fab 2 -HC:scFvPDL1 and αCD40-Fab 2 ( A ), αCD40-IgG1(N297A)-HC:scFvPDL1 and αCD40-IgG1(N297A) ( B ) or αPDL1-IgG1(N297A)-HC:scFvCD40 and αPDL1-IgG1(N297A) ( C ). Next day, CD40 activation was quantified by measuring upregulation of IL8 production by ELISA. Shown are data obtained by three independent experiments.

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Transfection, Plasmid Preparation, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: CD40 activation by membrane CD40L-expressing cells and PDL1-anchored CD40/PDL1-bispecific antibody variants. (A) HT1080-CD40 cells were stimulated with empty vector- (open bars) and PDL1-transfected (grey bars) HEK293 cells along with the indicated concentrations of the various antibody fusion proteins or with HEK293 cells transfected with a membrane CD40L expression vector (black bars). After overnight incubation IL8 was measured by ELISA. (B) Empty vector- (EV) and PDL1-transfected HEK293 cells were pre-incubated with 40 µg/mL of the parental anti-PDL1 antibody (αPDL1) and were then added together with the indicated CD40-targeting antibody fusion protein (10 ng/mL) to HT1080-CD40 cells. Next day, IL8 production was again determined as a readout of CD40 activity. (C) U2OS cells expressing endogenous CD40 were stimulated with empty vector- (open bars) and PDL1-transfected (black bars) HEK293 cells along with 200 ng/mL of the different antibody fusion proteins and next day IL8 production was evaluated. Experiments shown in A to C are representative for two to three independent experiments.

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Activation Assay, Membrane, Expressing, Plasmid Preparation, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: Binding affinities of different α41BB/PDL1-antibody fusion proteins. For statistical analysis see supplemental data Figure S3 .

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Binding Assay

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: PDL1-dependent 41BB agonism of 41BB/PDL1-bispecific antibody variants. (A) HT1080-41BB cells were stimulated in the presence of EV- and PDL1-transfected HEK293 cells with increasing concentrations of the indicated antibody variants. Next day, 41BB activation was evaluated by determination of IL8 production. ( B ) HT1080-41BB cells were stimulated with empty vector- (open bars) and PDL1-transfected (grey bars) HEK293 cells along with the indicated concentrations of α41BB-Fab 2 -HC:scFvPDL1, α41BB-IgG1(N297A)-HC:scFvPDL1 and αPDL1-IgG1(N297A)-HC:scFv41BB or with HEK293 cells which have been transfected with a membrane 41BBL expression vector (black bars). Next day, IL8 was measured by ELISA. (C) EV- and PDL1 transfected HEK293 cells were pre-incubated with 40 µg/mL of the parental PDL1-specific antibody (αPDL1) and were then transferred along with the antibody and 10 ng/mL of the 41BB/PDL1-bispecific constructs to HT1080-41BB cells. Next day, IL8 production was measured . For (A) three independent experiments have been averaged, for (B) and (C) one representative experiment of two with technical triplicates is shown.

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Transfection, Activation Assay, Plasmid Preparation, Membrane, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Construct

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: CD40/PDL1- and 41BB/PDL1-bispecific antibody fusion proteins block PDL1-PD interaction. (A) Affinity purified proteins (200 ng) were separated by SDS-PAGE and visualized by silver staining: (B) Gel filtration analysis of the indicated purified antibody fusion proteins. Arrows indicate peaks of non-aggregated protein species. Also remaining Flag peptide from the affinity purification is marked. (C) PDL1-dependent TNFR agonism of the purified proteins were evaluated by coculture assays of CD40- or 41BB-expressing HT1080 variants with empty vector (EV)- and PDL1-transfected HEK293 cells. After overnight stimulation TNFR activation was measured by IL8 ELISA. ( D ) Binding of PD1-GpL (300 ng/mL) to PDL1-expressing HEK293 transfectants was determined in the presence and absence of the indicated antibody variants. (E) TCR + PD1 + Jurkat cells expressing luciferase under the control of a NFAT response element were co-cultivated with CHO-K1 cells expressing a TCR agonist and PDL1. Cocultures were treated with the indicated concentrations of different PDL1-targeting antibody fusion proteins for 6 hours and NFAT-driven luciferase expression was measured. For ( C ) three independent experiments have been averaged, for ( D, E ) one representative experiment of three is shown.

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Blocking Assay, Affinity Purification, SDS Page, Silver Staining, Filtration, Purification, Expressing, Plasmid Preparation, Transfection, Activation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Control

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: T-cell costimulation by αCD3 expressing tumor cells and α41BB-IgG1(N297A)-HC:scFvPDL1. (A) Scheme of experimental co-culture design. (B) FACS analysis of PDL1-expression on ES2.scFvCD3 ovarian cancer cells with and without IFNγ pretreatment. (C) Left panels: representative flow cytometry of 41BB cell surface expression on resting CD3 + T-cells, T-cells activated with ES2.scFvCD3 cells or activated with a PDL1-deficient variant of the latter. Right panel: Averaged results of experiments with 5 independent donors. D-G IFNγ-pretreated ES2-scFvCD3 cells were co-cultured with CD3 + T-cells in the indicated target to effector ratios with or without 10 µg/mL α41BB-IgG1(N297A)-HC:scFvPDL1. Microscopy was performed after 3 days (D) . Viability ( E ) and CD25 expression of CD4+ ( F ) and CD8+ cells ( G ) were analyzed on day 4. (H) ES2-scFvCD3 and the corresponding PDL1-KO variant were pretreated with IFNγ and co-cultured with CD3 + T-cells (E:T = 10:1) with or without 10 µg/mL α41BB-IgG1(N297A)-HC:scFvPDL1. After 4 days viability was analyzed. Shown are the results obtained with T-cells of 8 independent donors.

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Expressing, Co-Culture Assay, Flow Cytometry, Variant Assay, Cell Culture, Microscopy

Journal: Theranostics

Article Title: CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism

doi: 10.7150/thno.66119

Figure Lengend Snippet: T-cell costimulation by a CD19-BiTe and α41BB-IgG1(N297A)-HC:scFvPDL1. K562 cells and K562-CD19 transfectants were seeded in 96-well plates (35 x 10 3 cells/well). Next day, cells were challenged as indicated with PBMCs, Empty vector or PDL1 transfected HEK293 cells and α41BB-IgG1(N297A)-HC:scFvPDL1. After an additional day IL2 production was analyzed by ELISA. Shown are the averaged results of 6 independent experiments.

Article Snippet: The PDL1 encoding expression plasmid (pCMV6-XL4) was purchased from Origene Technologies, Inc.

Techniques: Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay