Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Design of FR-engineered e23sFv derivatives. (A) Amino acid sequence alignment of the VL and VH domains of mouse anti-HER2 single-chain variable fragment, e23sFv, and their five most homologous counterparts identified in the National Centre for Biotechnology Information protein database. L1-L5 represent VL homologous sequences and H1-H5 represent VH homologous sequences. CDRs and FRs are indicated in columns. The residues that are identical to those of e23sFv are indicated with dashed lines, and missing residues in the CDRs are indicated with asterisks. Non-identical FR residues in e23sFv and all their five homologs in the VL or VH collection are in red. Introduced site-directed mutations are indicated by blue triangles, above which are the corresponding substituted residues. (B) The schematic structure of three e23sFv derivatives. EMEY includes 11 mutated residues in the FRs of e23sFv, as indicated by triangles. EX1 and EX2 represent CDR grafts of e23sFv in the L1-H1 and L2-H2 FR scaffolds, respectively. FR, framework region; VL, light-chain variable region; VH, heavy-chain variable region; CDR, complementarity-determining region.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: In vitro binding of the e23sFv-derived scFvs to recombinant HER2. (A) Affinity measurement by ELISA. HER2-coated microplates were incubated with the e23sFv derivatives at various concentrations, and the bound scFvs were detected using an anti-His antibody. scFv15 served as the negative control. (B) One-shot kinetics of SPR. Five sensorgrams indicated the response of HER2-immobilized sensor chips with five diluted concentrations of the e23sFv derivatives. (C) Comparison of K on , K off and K D of the e23sFv derivatives calculated from SPR sensing. scFv, single-chain variable fragment; SPR, surface plasmon resonance; RU, resonance unit; K on , association rate constant; K off , dissociation rate constant, K D , equilibrium constant; Chi 2 , goodness-of-fit between the binding model and theoretical affinity; OD, optical density.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: In Vitro, Binding Assay, Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control, SPR Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Binding of the e23sFv-derived single-chain variable fragments to HER2 on the cell surface. HER2-positive cells (BT-474 and SKOV-3 cells) and HER2-negative cells (MCF-7 cells) were incubated with FITC-labelled e23sFv derivatives and subjected to flow cytometry analysis. (A) Representative dataset of one-parameter histograms. For the isotype control, FITC-labelled scFv15 against HBsAg was used. For the positive control, a commercial FITC-conjugated anti-HER2 antibody was used. (B) Statistical analysis from three independent parallel experiments. * P<0.05; ** P<0.01 and *** P<0.001. ns, non-significant.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Binding Assay, Derivative Assay, Incubation, Flow Cytometry, Positive Control

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Internalization of the e23sFv-derived single-chain variable fragments by HER2-positive cells. Following incubation with FITC-labelled e23sFv derivatives, BT-474, SKOV-3 and MCF-7 cells were observed under fluorescence microscopy. MCF-7 cells served as the HER2-negative cell controls and scFv15 was the non-specific binding control. Scale bar, 100 µm. The data are representative of at least three independent experiments.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Derivative Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Docking mechanism of the enhanced EX1-HER2 interaction. (A-D) In silico docking of e23sFv, EMEY, EX1 and EX2 and their interactions with the predicted surface models of the HER2 ECD. All the scFv fragments form distinct but overlapping interfaces with domain IV of the HER2 ECD. The 3D structures of (A) e23sFv, (B) EMEY, (C) EX1 and (D) EX2 are presented as coloured ribbons. (E) Binding energy with HER2 and the predicted binding epitopes of all the scFv fragments. ECD, extracellular domain; scFv, single-chain variable fragment.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: In Silico, Binding Assay

Journal:

Article Title: Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector

doi: 10.1128/JVI.76.20.10497-10502.2002

Figure Lengend Snippet: Estimation of vector gene copy number in AAV-transduced livers by quantitative PCR (3 months after vector administration). A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems. Template for PCR was as follows: 100 ng of genomic DNA extracted from several random pieces of liver and subsequently combined for each individual animal. Each sample column represents an individual animal. standards, linearized plasmid pAAV-EF1α-hF.IX (0.01, 0.1, or 1 pg) mixed with 100 ng of genomic mouse DNA (extracted from untransduced animal); NC, negative control (template, genomic DNA from untransduced animal). Bands were analyzed by densitometric scanning, and intensities were quantitated with NIH Image 6.16 software. Shown is one representative blot. Ratios of band intensities (hF.IX band/mAAT band) are for the blot shown. Gene copy number estimates are average for two experiments.

Article Snippet: A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems.

Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Software

Journal: Cancer Research

Article Title: AREG and EREG Are Predictive Biomarkers of Response to EGFR Inhibition in Gastroesophageal Cancer

doi: 10.1158/0008-5472.CAN-25-0073

Figure Lengend Snippet: A subset of gastroesophageal tumors not displaying EGFR gene amplification is sensitive to anti-EGFR drugs. A, Heatmap showing the viability (in percentage vs. untreated) of 27 primary cell lines treated for 6 days with the indicated doses of the mAb cetuximab. The heatmap represents the average of three independent experiments. B, Boxplot showing the sensitivity of the 27 primary cells (divided into resistant and sensitive groups on the basis of the response to cetuximab) to the indicated drugs. Each dot represents the mean AUC computed by the mean of three independent experiments. Statistical significance was calculated using the Student t test. The labeled dot indicates a HER2-amplified cell line (GTR0233). C, Boxplots showing the percentage of nonviable cells (positive for trypan blue staining), untreated (−) cells, or cells treated (+) with cetuximab (CTX; 5 µg/mL for 6 days). The average of four independent experiments performed on three cetuximab-sensitive (GTR0539, GTR0640, and GTR0247) and three cetuximab-resistant cell lines (GTR0125, GTR0498, and GTR0687) is shown. Statistical significance was calculated using the ratio paired t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: The following primary antibodies were used: HER3 (Cell Signaling Technology #12708 RRID: AB_2721919), HER2 (Cell Signaling Technology #2165 RRID: AB_10692490), vinculin (Millipore 05-386 RRID: AB_11212640), and human EGFR (described in ref. ).

Techniques: Amplification, Labeling, Staining