biotinylated human her2 erbb2  (Sino Biological)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: Illustration of in silico design and experimental validation of HER2-binding proteins. (A) Structure of ZHER2:342 (green cartoon) in complex with HER2 (gray surface), utilized as a probe for structural profile detection. EvoDesign REMC simulations are performed to create sequence decoys as guided by the structural profile and physical force fields. (B) Left: histogram displaying EvoDesign sequence decoys ranked by EvoEF2 binding energy, folding integrity of I-TASSER model to the probe, and spacial aggregation property, respectively. Eleven top-ranking designs are highlighted in purple. Right: eleven designs are selected based on consensus scoring, with colors in the Venn diagram corresponding to those in left. (C) Left: The pCTCON plasmid vector is engineered to express the designed sequences (BindHer) with N-terminal HA and C-terminal cMyc epitope tags, fused to the yeast mating protein Aga2p on the yeast surface. Right: density plots of protein expression ( y -axis, Alexa Fluor 647 detected via anti-cMyc antibody) versus HER2 binding ( x -axis, Alexa Fluor 488 via Streptavidin–Alexa assay). Design-A: high HER2 affinity; Design-NA: low HER2 affinity.

Article Snippet: Recombinant Human HER2 ECD (1004-HCCH, Sino Biological) was immobilized (∼2000 resonance units) on a CM5 sensor chip (BR-1000-12, GE Life Sciences) and analyzed using a Biacore instrument (Biacore X100, GE Life Sciences).

Techniques: In Silico, Biomarker Discovery, Binding Assay, Sequencing, Plasmid Preparation, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: Experimental characterization of designed HER2-binding (BindHer) proteins. (A) Real-time binding profile of computationally designed proteins with the HER2 ECD through surface plasmon resonance (SPR), where designed proteins were injected as analytes in a 2-fold dilution series ranging from 100 to 3.125 nmol/L. K D is the equilibrium dissociation constant. (B) Denaturation curves obtained using differential scanning fluorimetry (DSF), where designed proteins were subjected to thermal scans from 25 to 95 °C at a heating rate of 1 °C/min with signals recorded at wavelengths of 350/330 nm. Melting temperature ( T m ) is marked by vertical lines. (C) Circular dichroism (CD) spectra of designed proteins under controlled temperature and timing conditions. (D) Flow cytometry assessing HER2 binding to MDA-MB-231, BT-474, and SK-BR-3 cells using FITC-conjugated designed proteins. (E) Quantification of the signal transformation in terms of molar residue ellipticity (MRE) at a wavelength of 222 nm for the designed proteins in CD spectra. (F) Quantification of fluorescence intensity performed in D. (G) Evaluation of trypsin resistance, where designed proteins are incubated with trypsin concentrations in 0.01–10 μmol/L, followed by the analyses using SDS-PAGE gel electrophoresis (top) and gray intensity analysis comparison (bottom). (H) In vivo living imaging from 1 to 8 h after FITC-labelled designed proteins administration via tail vein in HER2-overexpressing xenograft tumor-bearing mice; where other two designs (Design.01 and Design.03) which failed to target HER2 tumors well in vivo , are not shown. (I) Semiquantitative ex vivo biodistribution in tumors and organs post-sacrifice, with values expressed as means ± SD ( n = 3); where ∗ P < 0.05, ∗∗ P < 0.01, ns non-significant.

Article Snippet: Recombinant Human HER2 ECD (1004-HCCH, Sino Biological) was immobilized (∼2000 resonance units) on a CM5 sensor chip (BR-1000-12, GE Life Sciences) and analyzed using a Biacore instrument (Biacore X100, GE Life Sciences).

Techniques: Binding Assay, SPR Assay, Injection, Circular Dichroism, Flow Cytometry, Transformation Assay, Residue, Fluorescence, Incubation, SDS Page, Nucleic Acid Electrophoresis, Comparison, In Vivo, Imaging, Ex Vivo

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: In vitro pharmacodynamic characterization of BindHer. (A) Flow cytometry analysis of HER2 levels in SK-BR-3 cells treated with HER2 siRNA, using Trastuzumab-PE/Cy7 to confirm HER2 expression. (B) ELISA confirming BindHer's binding specificity to HER2 across concentrations from 100 to 0.098 nmol/L. (C) In vivo immunogenicity of BindHer and ABY-025 with hIgG used as a positive control. (D) Western blot of SK-BR-3 cell lysates following BindHer treatment at various concentrations, with EGF as a positive control. Equal protein amounts were loaded in each lane; SDS-PAGE gels were run, and membranes were sectioned by molecular weight for analysis of total HER2, phospho-HER2, and β -actin. Phospho-HER2/HER2 quantification is depicted in graphs, where ∗∗∗∗ P < 0.0001; ns denotes non-significant results.

Article Snippet: Recombinant Human HER2 ECD (1004-HCCH, Sino Biological) was immobilized (∼2000 resonance units) on a CM5 sensor chip (BR-1000-12, GE Life Sciences) and analyzed using a Biacore instrument (Biacore X100, GE Life Sciences).

Techniques: In Vitro, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, In Vivo, Immunopeptidomics, Positive Control, Western Blot, SDS Page, Molecular Weight

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: 99m TC-BindHer noninvasive SPECT imaging of HER2 expression and biodistribution in mice with breast cancer xenografts. (A) Schematic of 99m Tc-labelled BindHer structure, with a GGGC chelator at the C-terminus for 99m Tc complexation. (B) Radiochemical stability of 99m Tc-BindHer (red curve) and 99m Tc-ABY-025 (blue curve) is assessed in vitro in PBS (dot) and serum (square) at 37 °C. (C) SPECT/CT imaging of 99m TC-BindHer in tumour-bearing mice at 1, 2, and 4 h, the arrows indicate the location of the tumor. (D) Data for tumor uptake at different time points. (E) Data for tissue uptake at 2 h point. (F) SPECT images of HER2 tumor-bearing mice after tail vein injection of 99m TC-BindHer (top) or 99m Tc-ABY-025 (bottom) at 1, 2, and 4 h. Coronal imaging was performed to visualize the mice (top), while transverse imaging captured the tumor and liver regions (bottom). The arrows indicate significant radionuclide accumulation in various tissues, including liver (L), kidney (K), and tumor (T). (G) Quantification of SPECT signals in HER2 tumors (top) and liver (bottom). Values are expressed as the means ± SD ( n = 3). where ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns represents non-significant.

Article Snippet: Recombinant Human HER2 ECD (1004-HCCH, Sino Biological) was immobilized (∼2000 resonance units) on a CM5 sensor chip (BR-1000-12, GE Life Sciences) and analyzed using a Biacore instrument (Biacore X100, GE Life Sciences).

Techniques: Single Photon Emission Computed Tomography, Imaging, Expressing, In Vitro, Injection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: 68 Ga-NOTA-BindHer noninvasive PET imaging of HER2 expression and biodistribution in mice with breast cancer xenografts. (A) Synthesis flow chart of 68 Ga-NOTA-BindHer. The precursor protein was first formed through a Michael addition reaction at 25 °C overnight using BindHer and MAL-NOTA, followed by chelation of 68 Ga and precursors in sodium acetate buffer (pH 4.0) at 75 °C for 15 min which resulted in the production of 68 Ga-NOTA-BindHer. (B) SDA-PAGE analysis of ABY-025 and BindHer reaction products with MAL-NOTA at varying chelation ratios. (C) HPLC analysis of reaction products at various chelation ratios. (D, E) Stability of NOTA-ABY-025 and NOTA-BindHer at 85 °C for 60 min or 25 °C for 4 weeks, with SDA-PAGE on the left and gray-value analysis on the right. (F) PET/CT imaging of 68 Ga-NOTA-BindHer and 68 Ga-NOTA-ABY-025 at different time points in HER2-expression tumor bearing mice. L: liver, K: kidneys, and T: tumor. (G) Quantification of PET signal in tumor (left) and liver (right), respectively. (H) The PET/CT imaging of 68 Ga-NOTA-BindHer at different time points in tumor bearing mice. (I) Comparison of tumor absorptions of 68 Ga-NOTA-BindHer at different time points. (J) Comparison of tissues absorption of 68 Ga-NOTA-BindHer at 90 min. Values are means ± SD ( n = 3), where ∗∗∗∗ P < 0.0001, ns represents non-significant.

Article Snippet: Recombinant Human HER2 ECD (1004-HCCH, Sino Biological) was immobilized (∼2000 resonance units) on a CM5 sensor chip (BR-1000-12, GE Life Sciences) and analyzed using a Biacore instrument (Biacore X100, GE Life Sciences).

Techniques: Imaging, Expressing, Positron Emission Tomography-Computed Tomography, Comparison

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: 18 F-NOTA-BindHer noninvasive PET imaging of HER2 expression and biodistribution in breast cancer xenograft mice. (A) Synthesis of 18 F-NOTA-BindHer through Al18F chelation in sodium acetate buffer (pH 4.0) at 100 °C for 15 min, followed by impurity removal via gel filtration chromatography. (B) Coronal and transverse Micro-PET/CT imaging of breast cancer xenograft mice treated with 18 F-NOTA-BindHer and 18 F-NOTA-ABY-025, showing the presence of HER2 positive breast cancer tumors (T), liver (L) and kidneys (K) at various time points. (C) Quantitative time–radioactivity curves of 18 F-NOTA-BindHer (blue circle) and 18 F-NOTA-ABY-025 (red tirangle) in tumors and liver, based on dynamic PET/CT imaging over 0–1 h. (D) Static Micro-PET/CT scanning of both HER2 + SK-BR-3 (blow, blue circle) (Imaging is shared with the image in B), HER2 + SK-BR-3+Blocking (orange square) and (MDA-MB-3) (blue circle) tumor-bearing nude mice models at 10, 30, and 60 min. Arrows indicate tumor locations. (E) Tumor absorption of 18 F-NOTA-BindHer at different time points. (F) Tissue absorption of 18 F-NOTA-BindHer at 60 min. Values are means ± SD ( n = 3), where ∗∗∗∗ P < 0.0001, ns represents non-significant.

Article Snippet: Recombinant Human HER2 ECD (1004-HCCH, Sino Biological) was immobilized (∼2000 resonance units) on a CM5 sensor chip (BR-1000-12, GE Life Sciences) and analyzed using a Biacore instrument (Biacore X100, GE Life Sciences).

Techniques: Imaging, Expressing, Filtration, Chromatography, Micro-PET, Radioactivity, Positron Emission Tomography-Computed Tomography, Blocking Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Evolution-guided design of mini-protein for high-contrast in vivo imaging

doi: 10.1016/j.apsb.2025.07.015

Figure Lengend Snippet: Characteristics of designed BindHer protein. (A) Sequence alignments of BindHer (Design.05) with ZHER2:342, ABY-025, and wild-type Z-domain. Conserved residues are indicated by “:”, yellow highlights mutated residues against Z-domain, and blue stars mark the core interface binding residues (R10, Y13, W14, R28, R32, Y35) with HER2. (B) Structural alignment of the I-TASSER (green) and AlphaFold2 (orange) prediction of BindHer. (C) Structure overlay of AlphaFold2 model of BindHer on ABY-025 in complex with HER2. The right panel shows the zoom-in of the ABY-025 structure, where the core interface residues to HER2 ECD are displayed in sticks. For reference, the Fab fragments of therapeutic monoclonal antibodies trastuzumab (red) and pertuzumab (cyan) are shown binding HER2 epitopes, which are distant from the binding regions of BindHer and ABY-025.Right showed that the core interface binding residues (R10, Y13, W14, R28, R32, Y35) with HER2. (D) Distribution of surface hydrophobic networks, with orange indicating hydrophobic residues of ABY-025 and BindHer. (E) Bis-ANS fluorescence of ABY-025 and BindHer at different concentration, with error bar representing 95% confidence interval (CI) from three technical replicates, ∗∗∗∗ P < 0.0001. (F) The ABY-025 and BindHer structures are depicted in the electrostatic surface view, with red representing potentials of –5 KTe –1 and blue representing potentials of 5 kTe -1 . The electrostatic potentials were computed using PyMol's APBS module. (G) Comparison of positive, negative and net charges of ABY-025 and BindHer. The secondary structure, surface hydrophobicity, and surface electrostatic potentials of each protein in the plot were visualized using PyMol and UCSF ChimeraX.

Article Snippet: Recombinant Human HER2 ECD (1004-HCCH, Sino Biological) was immobilized (∼2000 resonance units) on a CM5 sensor chip (BR-1000-12, GE Life Sciences) and analyzed using a Biacore instrument (Biacore X100, GE Life Sciences).

Techniques: Sequencing, Binding Assay, Bioprocessing, Fluorescence, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis

doi: 10.1101/2025.01.28.635284

Figure Lengend Snippet: (A & B) Deparaffinized tumor sections were stained with indicated antibodies. Representative immunohistochemistry staining images of ECD and ERBB2 are shown. (C) Representative images of hematoxylin and eosin-stained sections. Three independent tumors from each Tg mice are shown. Magnification of images are 400x and Scale bar, 50 µm.

Article Snippet: The empty vectors or ECD/ERBB2 constructs were transiently transfected into Plat-GP packaging cell line (cat# RV-103, Cell BIOLABS) grown in DMEM with 10% FBS (fetal bovine serum) and retroviral supernatants were used for transduction of cells in the presence of 10 μg/ml Polybrene (cat# TR-1003-G, Sigma-Aldrich).

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis

doi: 10.1101/2025.01.28.635284

Figure Lengend Snippet: (A-F) Western blotting of 76NTERT (A-C) and 70NTERT (D-F) expressing vector control, ECD, ERBB2, and ECD+ERBB2. Representative densitometries of ECD and ERBB2 are shown in respect to vector control and ERBB2 overexpressing cells respectively after normalization to loading control β-actin. Band intensities of three biological experiments were measured using ImageJ software and plotted. Data represents mean +/− SEM with two-tailed un-paired t test. n = 3; ns (not significant), Three technical replicates from three independent biological experiments.

Article Snippet: The empty vectors or ECD/ERBB2 constructs were transiently transfected into Plat-GP packaging cell line (cat# RV-103, Cell BIOLABS) grown in DMEM with 10% FBS (fetal bovine serum) and retroviral supernatants were used for transduction of cells in the presence of 10 μg/ml Polybrene (cat# TR-1003-G, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Software, Two Tailed Test

Journal: bioRxiv

Article Title: ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis

doi: 10.1101/2025.01.28.635284

Figure Lengend Snippet: (A) Principal component analysis (PCA) analysis of RNA-seq data shows clustering of vector, ECD, ERBB2, and ECD+ERBB2 overexpressing 76NTERT cells. (B, C) Bar plots display enriched hallmark gene sets including unfolded protein response (UPR), and glycolysis. X-axis represents normalized enrichment scores (NES) of the signaling pathways with significant nominal p-values (NOM p-val). Blue arrows represent pathways emphasized in this study. (D) GSEA (Gene set enrichment analysis) plots display upregulation of UPR and glycolysis pathways genes in ECD+ERBB2 cells, as compared to ERBB2 or ECD overexpressing cells. NES, nominal p-values and false discovery rate (FDR) are indicated on the plot. (E) Heatmap shows upregulated (yellow) and downregulated (blue) key UPR genes ( E ) and glycolytic genes ( F ). qRT-PCR analyses of glycolytic genes HK2 ( G ), LDHA ( H ), SLC2A1 (GLUT1) ( I ), ENO1 ( J ) and PDK1 (K) in 76NTERT transductants are shown. mRNA quantitation data represents mean ± SEM with two-tailed unpaired t test. n = 3; ns, P > 0.05; **, P < 0.01; ***, P < 0.001. Relative UPR related mRNA levels of HSPA5 ( GRP78 ) ( L ), ATF6 ( M ), EIF2AK3 (PERK) ( N ) are shown in indicated cells. Relative levels of each transcript are expressed as fold change with respect to vector control cells after normalizing with the house keeping gene, β-actin using the ΔΔCT method. Each bar graph indicates mean fold change +/− SEM from three experiments, each with three technical replicates (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: The empty vectors or ECD/ERBB2 constructs were transiently transfected into Plat-GP packaging cell line (cat# RV-103, Cell BIOLABS) grown in DMEM with 10% FBS (fetal bovine serum) and retroviral supernatants were used for transduction of cells in the presence of 10 μg/ml Polybrene (cat# TR-1003-G, Sigma-Aldrich).

Techniques: RNA Sequencing Assay, Plasmid Preparation, Quantitative RT-PCR, Quantitation Assay, Two Tailed Test, Control

Journal: bioRxiv

Article Title: ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis

doi: 10.1101/2025.01.28.635284

Figure Lengend Snippet: Transcripts per million (TPM) values for 76NTERT transductants RNA samples used for RNA-sequencing and analysis. Vector is shown in blue, ECD in red, ERBB2 in green, and ECD+ERBB2 in purple. ECD TPM ( A ) and ERBB2 TPM ( B ) displayed for validation of samples used in analyses. ( C ) Bar graphs display enriched GSEA hallmark pathways including unfolded protein response (UPR), glycolysis, protein secretion, mTORC1 signaling, hypoxia signaling in ECD OE (overexpression) vs. Vector ( C ) and ERBB2 OE vs. Vector ( D ). X-axis represents normalized enrichment scores (NES) of the signaling pathways with significant nominal p-values (NOM p-val). Heatmap shows upregulated (yellow) and downregulated (blue) key EMT genes ( E ). TPM values of key EMT genes VIM (F) and TWIST2 (G) are displayed. TPM values of key UPR-related genes HSPA5 (H), HSP90B1 (I), ATF6 (J), EIF2AK3 (K) and glycolytic genes LDHA (L), PGK1 (M), HK2 (N), IDH1 (O), SOD1 (P), BIK1 (Q) are shown as bar graphs.

Article Snippet: The empty vectors or ECD/ERBB2 constructs were transiently transfected into Plat-GP packaging cell line (cat# RV-103, Cell BIOLABS) grown in DMEM with 10% FBS (fetal bovine serum) and retroviral supernatants were used for transduction of cells in the presence of 10 μg/ml Polybrene (cat# TR-1003-G, Sigma-Aldrich).

Techniques: RNA Sequencing Assay, Plasmid Preparation, Over Expression

Journal: bioRxiv

Article Title: ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis

doi: 10.1101/2025.01.28.635284

Figure Lengend Snippet: (A, B) Western blotting of 76NTERT transductants grown in serum-free DFCI-3 medium at indicated time points revealed prolonged expression of GRP78 and XBP-1s (spliced form of XBP-1). ECD and ERBB2 expression confirms the overexpression of ECD and ERBB2 in transductants. Densitometries are in respect to no starvation of vector cells after normalizing with β-actin. Glucose uptake in 76NTERT (C) and 70NTERT (D) transductants. The values were normalized with respective to cell counts and depicted as compared with vector control. Quantification of results from three biological experiments, each with four technical replicates is shown as a bar graph. Data represents as mean ± SEM and two-tailed unpaired test with Welch correction (*p<0.05, **p<0.01, ***p<0.001). (E-L) Seahorse glycolytic rate was assessed in 76NTERT (E) and 70NTERT (F) transductants. Cells were seeded in 96-well plates and exposed to Rot/AA (rotenone and antimycin cocktail) and 2-DG to measure proton eflux rate (PER, E & F) and extracellular acidification rate (ECAR, G & H) . One representative PER and ECAR plots from three independent experiments are displayed ( E-H ). Basal glycolysis (I & J) and compensatory glycolysis (K & L) glycoPER in pmol/min are presented as bar graphs calculated from subtracting ECAR (extra cellular acidification rate) from PER (proton efflux rates) ( I-L ). Quantitation data represents mean ± SEM with two-tailed unpaired t test. n = 3; ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The empty vectors or ECD/ERBB2 constructs were transiently transfected into Plat-GP packaging cell line (cat# RV-103, Cell BIOLABS) grown in DMEM with 10% FBS (fetal bovine serum) and retroviral supernatants were used for transduction of cells in the presence of 10 μg/ml Polybrene (cat# TR-1003-G, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, Control, Two Tailed Test, Quantitation Assay