Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Inhibition of growth and HER2 signaling by HER2Ab-NSCs cells. (A) Inhibition in growth of BT474 cells using supernatant of HER2Ab-NSCs. (B) Co-Culture assay using BT474 cells with either vector control or HER2Ab-NSCs cells. (C-D) Inhibition of PI3K-AKT signaling using purified anti-HER2Ab released by NSC. Right panel in C and D demonstrates significant decrease in relative densitometric units. Trastuzumab was used as positive control in A, C and D. The experiments were repeated three times. * indicates p<0.05 and ** indicates p<0.01.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Co-culture Assay, Plasmid Preparation, Control, Purification, Positive Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Binding of anti-HER2Ab to HER2 overexpressing breast cancer cells. (A) BT474Br were stained with trastuzumab as a positive control (B-F) MCF 7, MDA-MB-361, BT474Br, ZR-75-30 and SKBR3 cells stained with anti-HER2Ab secreted by NSC. The experiments were repeated two times.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Staining, Positive Control

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Gating strategy for detection of hHER2-CAR-T cells. A median of > 90% of mouse CD8 T cells transduced with the hHER2-CAR retrovirus were positive for the transgene as judged by flow cytometry analysis. B. B16F10 cells were transfected with mammalian human HER2 construct with lipofectamine. Cells were grown in the presence of G418 and sorted twice to generate single cell clones. C. CAR-T-mediated B16-HER2 cell killing assay. Apoptotic cells were stained for Annexin V and analyzed by flow cytometry. D. Whole mouse lung before (left) and after (right) CUBIC clearing.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: In Vivo, Transduction, Flow Cytometry, Transfection, Construct, Clone Assay, Staining

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Generation of GFP-ROSAβII-spectrin mouse model (Rosa26tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. B & C. Flow cytometry analysis of IFNγ and TNFα expression in hHER2-CAR transfected T cells from GFP-ROSAβII-spectrin mouse (TβII-spectrin) after treated with PBS or Tat-Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. D. CAR-mediated B16-HER2 cell killing assay with TβII-spectrin cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. E. Activated CD4+ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) or Gallein (30 min) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. F. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. G. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. H. Expression levels of βII-spectrin in human CD8 memory T cells (CD8+CD45RO+CD45RA–CD56–CD57–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, Transfection, Cell Culture, Staining, Migration, Injection, Activation Assay, Western Blot

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 1. Unique fragments of the CDRs within the heavy and light chains of the new mouse monoclonal anti-HER2 antibody. The amino acid sequence corresponds to the characteristic nucleotide sequence of (A) anti-human HER2/70.27.58 mAb and (B) anti-human HER2/70.21.73.67 mAb.

Article Snippet: Clones were screened in an ELISA assay using plates coated with a recombinant HER2 protein fragment spanning the N-terminal amino acids from Thr23–Ala510 that constitute the extracellular domain of a native HER2 receptor, and purified from the HEK293 expression system (ProSci, Fort Collins, CO, USA).

Techniques: Sequencing

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 2. Anti-HER2 monoclonal antibodies production and characterization. (A) Morphology of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 hybridoma cells photographed at 20 × and 40 × magnification. (B) FPLC chromatograms were recorded during the purification of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies using affinity chromatography on the Protein A resin. (C) SDS-PAGE analysis of the purified anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded at the amount of 1 µg/well on the 12% polyacrylamide gel under reducing conditions. (D) WB analysis of HER2 in whole cell lysates of the HER2 low expressing (MDA-MB-231) and HER2 high expressing (SK-BR-3, SK-OV-3) cells, probed with the home-made anti-human HER2/70.27.58 monoclonal antibody and detected with the secondary anti-mouse IgG-HRP (upper panel). The recombinant HER2 ECD protein was used as a reference. The loading control was performed with membrane probed with antibody binding β-actin (lower panel). (E) The formaldehyde-fixed SK-OV-3 cells were photographed in the bright field (BF) at the 40 × magnification. Immunofluorescence analysis was performed on cells stained with the commercial anti- HER2 ECD antibody followed by anti-mouse IgG-AlexaFluor594 (AF594) (red channel) and co-stained with the anti-HER2/70.27.58 or anti-HER2/70.21.73.67 antibodies detected with the AlexaFluor488-labeled (AF488) secondary antibody (green channel). Nuclei were stained with DAPI (blue channel) (F) Quantitative ELISA with the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded in a range of 0–5 µg/ ml on the plate coated with the recombinant chimera of the HER2 ECD-Fc protein. The signal generated from secondary antibody anti-mouse IgG-HRP was quantified by measuring absorbance at 450 nm and expressed after background subtraction (A450-A0).

Article Snippet: Clones were screened in an ELISA assay using plates coated with a recombinant HER2 protein fragment spanning the N-terminal amino acids from Thr23–Ala510 that constitute the extracellular domain of a native HER2 receptor, and purified from the HEK293 expression system (ProSci, Fort Collins, CO, USA).

Techniques: Bioprocessing, Purification, Affinity Chromatography, SDS Page, Expressing, Recombinant, Control, Membrane, Binding Assay, Immunofluorescence, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Generated

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 3. Parameters of the sandwich ELISA for HER2 detection. (A) HER2 binding kinetics in the standard curve concentration range of 0.156–10 000 ng/well (1.56–100 ng/ml). Results are expressed as absorbance at 450 nm after background subtraction (A450-A0). (B) Assay accuracy was tested by comparison of HER2 level measured by ELISA in the samples of the known antigen concentration (mock samples). Data were collected for 2, 5, 10, 30, and 50 ng/ml of HER2 (given concentration; x-axis), covering both physiological and increased concentrations. Experimentally measured concentration [ng/ml] is shown on the y-axis. Error bars indicate SD.

Article Snippet: Clones were screened in an ELISA assay using plates coated with a recombinant HER2 protein fragment spanning the N-terminal amino acids from Thr23–Ala510 that constitute the extracellular domain of a native HER2 receptor, and purified from the HEK293 expression system (ProSci, Fort Collins, CO, USA).

Techniques: Sandwich ELISA, Binding Assay, Concentration Assay, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 4. HER2 expression in tumors from mice with xenografted human cancer cells. (A) Immunohistochemistry staining using anti-HER2/70.27.58 mAb of the mouse tumors induced with the human ovarian cancer cells (SK-OV-3) overexpressing HER2 and (B) human epithelial breast cancer cells (MDA-MB-231) with low expression of HER.

Article Snippet: Clones were screened in an ELISA assay using plates coated with a recombinant HER2 protein fragment spanning the N-terminal amino acids from Thr23–Ala510 that constitute the extracellular domain of a native HER2 receptor, and purified from the HEK293 expression system (ProSci, Fort Collins, CO, USA).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: LegoBody: facile generation of bispecific and multi-specific antibodies

doi: 10.1101/2019.12.25.888586

Figure Lengend Snippet: ( A ) The scheme to produce bispecific antibodies. ( B ) Two bispecific antibodies, Ipili-Dara-KIH and Ipili-Her-KIH, were generated, both of which show a predominant component with a molecular weight of 150 kDa. A small fraction of contamination, likely caused by incorrectly paired homodimers, can be observed at the half size of an antibody (labeled with ‘*’). Under reducing condition, separated heavy and light chains, ∼50 kDa and ∼25 kDa respectively, were observed. ( C, D ) Bispecific Ipili-Dara-KIH binding to CTLA4-Fc ( C ) and CD38 ( D ), in comparison to the original monoclonal antibodies. ( E, F ) Bispecific Ipili-Her-KIH binding to CTLA4-Fc ( E ) and ErbB2-Fc ( F ), in comparison to the original monoclonal antibodies.

Article Snippet: Extracellular domain Fc fusions for human CTLA4 (CTLA4-Fc) and ErbB2 (ErbB2-Fc) were purchased from Abcam (ab180054 and ab168896).

Techniques: Generated, Molecular Weight, Labeling, Binding Assay, Comparison, Bioprocessing

Journal: Cancer medicine

Article Title: Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization.

doi: 10.1002/cam4.1099

Figure Lengend Snippet: Figure 2. Knockdown of SMYD3 attenuates HER2 activity. (A and B) Effects of SMYD3 knockdown on HER2 phosphorylation levels in ZR-75-1 cells (A) and MCF7 cells (B). Cells were transfected with one control siRNA (siNC) or either of two SMYD3 siRNAs (#1 and #2). After incubation for 96 h, cell extracts were fractionated into cytoplasmic and nuclear proteins, then samples were immunoblotted with anti-phospho HER2 (Tyr 1248) (#2247), anti-HER2 (29D8), anti-SMYD3 (D2Q4V), anti-ACTB (#4967), and anti-Histone H3 (ab1791). (C) The known three-dimensional structure of the ECD of HER2. Only part of domain I (blue) and domain II (orange) is shown. The side-chain amino group of Lys 175 in domain I makes a hydrogen bond with the backbone carbonyl group of Gly 223 in domain II. The drawing was prepared from the Protein Data Bank (entry code, 3WLW)[33] using Molecular Operating Environment (MOE), 2015.10 (Chemical Computing Group Inc.).

Article Snippet: Recombinant GST- HER2 (H00002064- P01, Novus biologicals, Littleton, CO) was incubated with SMYD3 enzyme and 2 μCi S- adenosyl- l- [methyl- [3]H]- methionine (SAM; PerkinElmer, Branchburg, NJ) in a mixture of methylase activity buffer (50 mmol/L Tris- HCl at pH 8.8, 10 mmol/L dithiothreitol (DTT), and 10 mmol/L MgCl2), for 3 h at 30°C.

Techniques: Knockdown, Activity Assay, Phospho-proteomics, Transfection, Control, Incubation

Journal: Cancer medicine

Article Title: Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization.

doi: 10.1002/cam4.1099

Figure Lengend Snippet: Figure 3. SMYD3-mediated methylation enhances the formation of HER2 homodimer. (A) HeLa cells were transfected with FLAG-HER2 and HA- HER2, with Mock vector or SMYD3-expressing vector. After 24 h of incubation, cells were treated with 0 or 100 ng/mL of EGF. Cell extracts were immunoprecipitated with anti-FLAG® M2 affinity gel, and immunoblotted with anti-HA (Y-11), anti-FLAG (F7425), anti-HER2 (29D8), anti-EGFR (D38B1), anti-SMYD3 (D2Q4V), and anti-ACTB (#4967). (B and C) 293T cells were transfected with HA-HER2-WT, and FLAG-HER2-WT or FLAG-HER2- K175A in the presence of SMYD3 expression vector and incubated for 48 h. Cell lysates were immunoprecipitated with anti-HA-agarose. (B) or anti- FLAG® M2 affinity gel. (C), then immunoblotted with anti-FLAG (F7425), anti-HA (Y-11), anti-SMYD3 (D2Q4V), and anti-ACTB (#4967). (D) 293T cells were cotransfected with FLAG-HER2-WT or FLAG-HER2-K175A, and Mock vector or SMYD3-expressing vector. After 48 h of incubation, cell lysates were immunoprecipitated with anti-FLAG® M2 affinity gel and immunoblotted with anti-FLAG (F7425), anti-phospho HER2 (Tyr 1248) (#2247), anti- SMYD3 (D2Q4V), and anti-ACTB (#4967). The signal intensities of phosphorylated HER2 were quantified, and normalized by each FLAG level.

Article Snippet: Recombinant GST- HER2 (H00002064- P01, Novus biologicals, Littleton, CO) was incubated with SMYD3 enzyme and 2 μCi S- adenosyl- l- [methyl- [3]H]- methionine (SAM; PerkinElmer, Branchburg, NJ) in a mixture of methylase activity buffer (50 mmol/L Tris- HCl at pH 8.8, 10 mmol/L dithiothreitol (DTT), and 10 mmol/L MgCl2), for 3 h at 30°C.

Techniques: Methylation, Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation

Journal: Cancer medicine

Article Title: Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization.

doi: 10.1002/cam4.1099

Figure Lengend Snippet: Figure 4. Effect of SMYD3-mediated HER2 methylation on downstream pathways. (A) HeLa cells were transfected with FLAG-HER2-WT or FLAG- HER2-K175A, and incubated for 48 h. Cell lysates were immunoblotted with anti-AKT (C67E7), anti-phospho AKT (Ser 473) (587F11), anti-PLCγ1 (D9H10, anti-phospho PLCγ1 (Tyr 783) (#2821), anti-ERK1/2 (#9102), anti-phospho ERK1/2 (Thr202/Tyr204) (D13.14.4E), anti-FLAG (F7425), anti- SMYD3 (D2Q4V), and anti-ACTB (#4967). (B) The schema of the effect of SMYD3-mediated methylation on HER2 function.

Article Snippet: Recombinant GST- HER2 (H00002064- P01, Novus biologicals, Littleton, CO) was incubated with SMYD3 enzyme and 2 μCi S- adenosyl- l- [methyl- [3]H]- methionine (SAM; PerkinElmer, Branchburg, NJ) in a mixture of methylase activity buffer (50 mmol/L Tris- HCl at pH 8.8, 10 mmol/L dithiothreitol (DTT), and 10 mmol/L MgCl2), for 3 h at 30°C.

Techniques: Methylation, Transfection, Incubation

Journal: Drug delivery

Article Title: Valency-affinity mapping of multivalent liposomes for tunable target cell discrimination.

doi: 10.1080/10717544.2025.2512950

Figure Lengend Snippet: Figure 3. Determination of kinetic rate constants via surface plasmon resonance. Sensorgrams were generated using immobilized HER2 panned with each of the DARPin variants. The sensorgrams were double referenced and fit to a 1:1 binding model from which the kinetic constants were derived. (a) G3 was panned at increasing concentrations (0, 1, 5, 10, 20, 30 nM) at a flow rate of 60 μL/min. (b) G3-V was panned at increasing concentrations (0, 1, 3, 5, 10, 20, 30 nM) at a flow rate of 60 μL/min. (c) G3-D was panned at increasing concentrations (0, 1, 5, 10, 20, 30, 40 nM) at a flow rate of 30 μL/min. (d) G3-HAVD was panned at increasing concentrations (0, 25, 50, 100 500, 1000, 2500 nM) at a flow rate of 30 μL/min.

Article Snippet: Biotin caPture Reagent was immobilized on the chip with a flow rate of 2 μl/min and contact time of 5 min. the ligand, avi-tag biotinylated heR2 (R&D systems, aVi11356), was reconstituted to 250 μg/ml in 1X PBs (cytiva, BR100672) per manufacturer instructions. immediately prior to use it was diluted in 1X hBs-P+ (cytiva, BR100827) to a working concentration of 75 nM. the prepared heR2 was then immobilized on the chip at a flow rate of 10 μl/min and contact time of 3 min. the DaRPin analytes (G3, G3-V, G3-D, G3-haVD) were diluted to their working concentrations, ranging from 0 to 40 nM, in 1X hBs-P+. the DaRPins were analyzed under two different run conditions: 1) with an association time of 10 min followed by a dissociation time of 1 h at a flow rate of 30 μl/min and 2) with an association time of 5 min followed by a dissociation time of 1 h at a flow rate of 60 μl/min. after each cycle, the chip was fully regenerated with three parts 8 M guanidine-hcl to one part 1 M NaOh for 2 minutes at a flow rate of 30 μl/min. hBs-P + was used as the running buffer and all runs were performed at 25 °c. the data were analyzed in BiacoretM insight evaluation software. all cycles were double-referenced against a blank run containing no analyte and a reference channel containing no immobilized ligand, and the data were fit to a 1:1 langmuir binding model.

Techniques: SPR Assay, Generated, Binding Assay, Derivative Assay

Journal: Drug delivery

Article Title: Valency-affinity mapping of multivalent liposomes for tunable target cell discrimination.

doi: 10.1080/10717544.2025.2512950

Figure Lengend Snippet: Figure 5. HEK293T HER2-eGFP Binding metrics. (A) (top panel) Flow cytometry scatter data for HEK293T cells transfected with HER2-EGFP and incubated with Cy5-PEG liposomes (conjugated with G3-D at 3 µM in this representative example) were fitted to a Hill function (red curve) and plotted with HER2αMAX (red dashed line) to note the peak binding selectivity. The Cy5 threshold for positive binding (horizonal blue dashed line) was based on no-protein control particle binding signal, and HER2OPT (vertical blue dashed line) denotes the HER2 threshold which maximizes BPI (iv/(i + iv))×(ii/(ii + iii)). X-axes for all three plots indicate log-transformed HER2-EGFP signal scaled from 0-1 and y-axis for top panel indicates log-transformed Cy5 signal scaled from 0-1. The binding selectivity (middle panel) was calculated by determining the slope of the fitted Hill curve across all HER2 values and was overlayed with HER2αMAX (red dashed line) and HER2OPT (vertical blue dashed line). BPI (bottom panel) was determined across the range of normalized HER2 expression values from 0.1 to 0.9 to find BPIMAX (vertical blue dashed line). (B) HER2αMAX and (C) HER2OPT for all DARPin types tethered at concentration between 0.5 and 5.0 μM of protein (light to dark bars, respectively) demonstrate similar behavior due to changes in DARPin valency and affinity, as increases in both tend to shift optimal binding conditions to lower receptor values. The magnitudes of (E) αMAX and (F) BPIMAX also follow similar trajectories across various DARPin affinities and valences. When examining the contributions of (D) on-target binding and (G) off-target binding to BPIMAX, DARPins achieved similarly high on-target binding across various tethering densities and all except G3-V demonstrated similar magnitude of off-target binding which increased for each DARPin as a function of tethering concentration. G3-V exhibited very strong off-target binding when tethered at high DARPin concentrations despite having a lower affinity than G3, suggesting some degree of nonspecific binding to cells. Two-way ANOVA without replication reveals that both DARPin type and tethering concentration significantly impact HER2OPT, HER2αMAX, and off-target binding but not on-target binding and BPI (DARPin type: p = 1.24E-05, 2.03E-05, 1.69E-03, 2.24E-01, and 1.74E-01, respectively; tethering concentration: p = 5.63E-04, 2.59E-02, 1.14E-02, 8.65E-01, and 6.17E-01, respectively).

Article Snippet: Biotin caPture Reagent was immobilized on the chip with a flow rate of 2 μl/min and contact time of 5 min. the ligand, avi-tag biotinylated heR2 (R&D systems, aVi11356), was reconstituted to 250 μg/ml in 1X PBs (cytiva, BR100672) per manufacturer instructions. immediately prior to use it was diluted in 1X hBs-P+ (cytiva, BR100827) to a working concentration of 75 nM. the prepared heR2 was then immobilized on the chip at a flow rate of 10 μl/min and contact time of 3 min. the DaRPin analytes (G3, G3-V, G3-D, G3-haVD) were diluted to their working concentrations, ranging from 0 to 40 nM, in 1X hBs-P+. the DaRPins were analyzed under two different run conditions: 1) with an association time of 10 min followed by a dissociation time of 1 h at a flow rate of 30 μl/min and 2) with an association time of 5 min followed by a dissociation time of 1 h at a flow rate of 60 μl/min. after each cycle, the chip was fully regenerated with three parts 8 M guanidine-hcl to one part 1 M NaOh for 2 minutes at a flow rate of 30 μl/min. hBs-P + was used as the running buffer and all runs were performed at 25 °c. the data were analyzed in BiacoretM insight evaluation software. all cycles were double-referenced against a blank run containing no analyte and a reference channel containing no immobilized ligand, and the data were fit to a 1:1 langmuir binding model.

Techniques: Binding Assay, Flow Cytometry, Transfection, Incubation, Liposomes, Control, Transformation Assay, Expressing, Concentration Assay