Journal: Cancer Biotherapy & Radiopharmaceuticals

Article Title: In Vitro Performance of Published Glypican 3-Targeting Peptides TJ12P1 and L5 Indicates Lack of Specificity and Potency

doi: 10.1089/cbr.2019.2888

Figure Lengend Snippet: Commercial anti-GPC3 antibody confirms expression of GPC3. Flow cytometry histograms confirming that (A) A431 cell lines do not express GPC3 and that (B) HepG2 and (C) G1 do.

Article Snippet: Experimental samples were stained with PE-conjugated anti-GPC3 antibody (R&D Systems, Minneapolis, MN), PE-conjugated IgG2 isotype control (Biolegend, San Diego, CA), conjugated specific or nonspecific peptide and incubated for 1–2 h on ice in the dark.

Techniques: Expressing, Flow Cytometry

Journal: Cancer Biotherapy & Radiopharmaceuticals

Article Title: In Vitro Performance of Published Glypican 3-Targeting Peptides TJ12P1 and L5 Indicates Lack of Specificity and Potency

doi: 10.1089/cbr.2019.2888

Figure Lengend Snippet: TJ12P1 binds nonspecifically. (A) Flow cytometry histograms of GPC3 + (G1 and HepG2) and GPC3 − (A431) cells incubated with 325 nM of sulfo-Cy5-TJ12P1 for 1 h demonstrating nonspecific association of TJ12P1 to all cell lines. (B) Bar graphs of MFI values for all cell lines ether unstained or incubated with 325 nM of the specific (sulfo-Cy5-TJ12P1) or nonspecific (TJ12P1 scramble) for 1 h indicating that nonspecific peptide had more binding to all cell lines tested ( p < 0.05). (C) Biolayer interferometry response curve shows the association and dissociation of TJ12P1 peptide to recombinant GPC3 at various concentrations (125–1000 nM) compared to a known GPC3-specific molecule ( K D of 4–6 nM) at 150 nM. TJ12P1 failed to demonstrate concentration-dependent binding behavior consistent with normal, specific equilibrium binding, and no K D could be calculated. MFI, mean fluorescence intensity. * indicates p < 0.05.

Article Snippet: Experimental samples were stained with PE-conjugated anti-GPC3 antibody (R&D Systems, Minneapolis, MN), PE-conjugated IgG2 isotype control (Biolegend, San Diego, CA), conjugated specific or nonspecific peptide and incubated for 1–2 h on ice in the dark.

Techniques: Flow Cytometry, Incubation, Binding Assay, Recombinant, Concentration Assay, Fluorescence

Journal: Cancer Biotherapy & Radiopharmaceuticals

Article Title: In Vitro Performance of Published Glypican 3-Targeting Peptides TJ12P1 and L5 Indicates Lack of Specificity and Potency

doi: 10.1089/cbr.2019.2888

Figure Lengend Snippet: L5 variant binds nonspecifically. (A) Flow cytometry histograms show the Cy5 emission shift in GPC3 + HepG2 (Lo and Hi were used to differentiate the two HepG2 cell populations with different extent of peptide association) and G1 cells compared to GPC3 − A431 cells. (B) Bar graph shows MFI values for all cell lines ether unstained or incubated with 300 nM of the specific (sulfo-Cy5-KKK-L5) or nonspecific (sulfo-Cy5-KKK-L5 scramble) for 1 h. MFI values suggest that the nonspecific peptide binds better to all cell lines than the specific peptide. * p < 0.05, ** p < 0.005, *** p < 0.0001. (C) Biolayer interferometry response curve shows the association and dissociation of KKK-L5 and KKK-L5 scramble to recombinant human GPC3 at various concentrations (62.5–500 nM) compared to a known GPC3-specific molecule ( K D of 4–6 nM) at 150 nM. KKK-L5 failed to demonstrate concentration-dependent binding behavior consistent with normal, specific equilibrium binding, and no K D could be calculated.

Article Snippet: Experimental samples were stained with PE-conjugated anti-GPC3 antibody (R&D Systems, Minneapolis, MN), PE-conjugated IgG2 isotype control (Biolegend, San Diego, CA), conjugated specific or nonspecific peptide and incubated for 1–2 h on ice in the dark.

Techniques: Variant Assay, Flow Cytometry, Incubation, Recombinant, Concentration Assay, Binding Assay

Journal: Scientific Reports

Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

doi: 10.1038/s41598-017-14793-z

Figure Lengend Snippet: SDC2 and GPC3, representative HSPGs, act as endocytic receptors for 3D8 scFv on HeLa and HEK293 cells. ( a ) Schematic diagrams of SD2 and GPC3, which are expressed on the cell surface, and their recombinants, which are expressed intracellularly (controls). The parts of the core protein are shown without attached of GAGs. SP, signal peptide; Δ, deletion; TM, transmembrane domain; CD, cytoplasmic domain; GPI, GPI anchor; GFP, green fluorescent protein; HA, HA tag. ( b – e ) Confocal microscopy. HeLa cells were transfected with plasmids encoding SDC2-GFP ( b ) and HA-GPC3 ( d ) which are expressed on the cell surface, or with plasmids encoding SDC2ΔTM-GFP ( c ) and GPC3- nf GPI-GFP ( nf indicates non-functional) ( e ), which are expressed intracellularly. At 18 h post-transfection, cells were incubated with 10 μM 3D8 scFv for 1 h at 4 °C or 6 h at 37 °C. Cells transfected with plasmids containing the GFP gene were incubated with a rabbit anti-3D8 scFv antibody, followed by a TRITC-conjugated goat anti-rabbit IgG antibody. Cells transfected with the plasmid encoding the HA-GPC3 gene were incubated with a rabbit anti-3D8 scFv antibody and a mouse anti-HA antibody, followed by TRITC-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG, respectively. Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm. ( f , g ) Cell surface expression of endogenous SDC2 and GPC3 was examined by flow cytometry using core protein-specific antibodies. ( h , i ) Co-immunoprecipitation. HeLa ( h ) and HEK293 cells ( i ) were transfected with plasmids encoding SDC2-GFP and HA-GPC3 and treated for 6 h at 37 °C with 5 μM 3D8 scFv 24 h later. Cells were collected and lysed for co-immunoprecipitation assays. Co-immunoprecipitation was performed using an anti-GFP antibody. Samples were analyzed by immunoblotting with antibodies specific for SDC2 and the His tag (left panels of h, i). Samples from the co-immunoprecipitation performed using the anti-GPC3 antibody were analyzed by immunoblotting with antibodies specific for GPC3 and the His tag (right panels of h, i). Cells not treated with the 3D8 scFv protein were used as a negative control. Proteins in the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting. Asterisks denote endogenous SDC2.

Article Snippet: To express GPC3 in the cytoplasm as an N-terminal GPC3- nfGPI-G FP fusion protein, pCMV6-AC-GPC3-turboGFP, in which the GPI anchor is non-functional, was purchased from Origene (cat# RG205911).

Techniques: Confocal Microscopy, Transfection, Functional Assay, Incubation, Plasmid Preparation, Staining, Expressing, Flow Cytometry, Immunoprecipitation, Western Blot, Negative Control

Journal: Scientific Reports

Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

doi: 10.1038/s41598-017-14793-z

Figure Lengend Snippet: CD44 and BCAN, representative CSPGs, act as endocytic receptors for 3D8 scFv on HeLa and HEK293 cells. ( a ) Schematic diagrams of CD44 and BCAN, which are expressed on the cell surface, and their recombinants, which are expressed intracellularly (controls). SP, signal peptide; Δ, deletion; TM, transmembrane domain; CD, cytoplasmic domain; GPI, GPI anchor; GFP, green fluorescent protein; HA, HA tag. ( b – e ) Confocal microscopy. HeLa cells were transfected with plasmids encoding CD44-GFP ( b ) and HA-BCAN ( d ), which are expressed on the cell surface, or with plasmids encoding CD44ΔTM-GFP ( c ) and BCAN-ΔGPI-GFP ( e ), which are expressed intracellularly. At 18 h post-transfection, cells were incubated with 10 μM 3D8 scFv under the specified conditions. After fixation and permeabilization, cells transfected with plasmids containing the GFP gene were incubated with a rabbit anti-3D8 scFv antibody, followed by a TRITC-conjugated goat anti-rabbit IgG antibody. Cells transfected with the plasmid encoding the HA-BCAN gene were incubated with a rabbit anti-3D8 scFv antibody and a mouse anti-HA antibody, which were detected by TRITC-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG, respectively. Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm. ( f , g ) Cell surface expression of endogenous CD44 and BCAN was examined by flow cytometry. ( h,i ) Co-immunoprecipitation. HeLa ( h ) and HEK293 cells ( i ) were transfected with plasmids encoding CD44-GFP and HA-BCAN and 24 h later treated with 5 μM 3D8 scFv for 6 h at 37 °C. Cells were collected and lysed for co-immunoprecipitation assays. Co-immunoprecipitation was performed using an anti-GFP antibody, Samples were analyzed by immunoblotting with antibodies specific for CD44 and the His tag (left panels of h, i). Samples from the co-immunoprecipitation performed with the anti-HA antibody, were analyzed by immunoblotting with antibodies specific for the HA tag and the His tag (right panels of h, i). Cells not treated with 3D8 scFv protein were used as a negative control. Proteins from the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting. Asterisks denote endogenous CD44.

Article Snippet: To express GPC3 in the cytoplasm as an N-terminal GPC3- nfGPI-G FP fusion protein, pCMV6-AC-GPC3-turboGFP, in which the GPI anchor is non-functional, was purchased from Origene (cat# RG205911).

Techniques: Confocal Microscopy, Transfection, Incubation, Plasmid Preparation, Staining, Expressing, Flow Cytometry, Immunoprecipitation, Western Blot, Negative Control