recombinant human met ecd-fc  (R&D Systems)

Journal: Molecular Cancer Therapeutics

Article Title: Preclinical Profile of BYON3521 Predicts an Effective and Safe MET Antibody–Drug Conjugate

doi: 10.1158/1535-7163.MCT-22-0596

Figure Lengend Snippet: Competition experiments with rhHGF and MET ECD. A, BLI experiment to assess competition of rhHGF for MET ECD binding of SYD2884. Presented are the binding curve of SYD2884 visualized with an HRP-conjugated anti-hIgG (red line, solid squares) and rhHGF visualized using a polyclonal anti-HGF antibody (blue line, solid triangles). The detectable amount of SYD2884 binding to the recombinant human MET ECD, immobilized on the surface of the sensor tip, decreases whereas at the same time the detectable amount of rhHGF increases. The data are presented as the correlation between the measured signals versus the molar excess of fully activated rhHGF (left graph) and pro-HGF (right graph). B, Cell viability of EBC-1 cells treated with increasing concentrations of BYON3521 or nonbinding isotype control ADC in the presence or absence of 1 μg/mL MET ECD, either administered together directly, or after 30-minute preincubation of BYON3521 or isotype control ADC and MET ECD (Premix). (Approximate MW of BYON3521 is 149 kD, of SY2884 144 kD, and MET ECD 140 kD).

Article Snippet: In addition, the impact of exogenous recombinant human MET-ECD-Fc (approximate MW 130 kDa; HGFR, Sino Biological) on the binding of SYD2884 to the cells was studied.

Techniques: Binding Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor

doi: 10.3389/fimmu.2021.775151

Figure Lengend Snippet: Binding kinetics of RDO24_mIgG2a, RDO24_mFab2 and RDO24_mFab. Human MET ECD-Fc was immobilized on the sensor chip, and binding of increasing concentrations of engineered antibodies was determined by surface plasmon resonance.

Article Snippet: The kinetic constants of RDO24, Fab2, and Fab with recombinant human MET ECD-Fc (R&D) were measured using a Biacore T100 instrument (GE Healthcare) and the CM5 chip, following standard procedures.

Techniques: Binding Assay, SPR Assay

Journal: Frontiers in Immunology

Article Title: Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor

doi: 10.3389/fimmu.2021.775151

Figure Lengend Snippet: RDO24 mAb selectively binds the MET receptor. Binding of RDO24_mIgG2a (red line) to the extracellular domain (ECD)-fragment crystallizable region (Fc) of human MET (A) , RON (B) , and SEMA4D (C) was measured by enzyme-linked immunosorbent assay. The anti-RON and anti-SEMA4D antibodies were used as controls of respective ECD-Fc chimeras.

Article Snippet: The kinetic constants of RDO24, Fab2, and Fab with recombinant human MET ECD-Fc (R&D) were measured using a Biacore T100 instrument (GE Healthcare) and the CM5 chip, following standard procedures.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain

doi: 10.3390/ijms18091968

Figure Lengend Snippet: Binding properties of IRCR201. ( a ) Binding patterns of IRCR201 to the human c-Met extracellular domain (ECD)-fragment crystallizable region (Fc) and the human RON (recepteur d’origine nantais) ECD-Fc were measured by enzyme-linked immunosorbent assay (ELISA). IRCR201 binds to the human c-Met ECD-Fc with specificity and selectivity; ( b – d ) Surface plasmon resonance (SPR) sensorgrams binding with varying concentrations of IRCR201 to human c-Met, mouse c-Met, or bovine serum albumin (BSA) immobilized onto a CM5 Biacore TM sensor chip; ( e ) Cross-reactivity analysis of IRCR201 to human and mouse c-Met; ( f ) Analysis of c-Met expression in various types of cancer cell lines; ( g ) Binding analysis of IRCR201 to the cell surface c-Met through a flow cytometer (FACSAria™ III).

Article Snippet: Briefly, scFv displaying phage libraries were enriched on 1 µg immobilized human c-Met ECD-Fc (Sino Biological, 10692-H03H, Beijing, China) and mouse c-Met ECD-Fc (Sino Biological, 50622-M02H, Beijing, China) in MaxiSorp™ immune-tubes (Nunc, 444202, Roskilde, Denmark). c-Met specific binders were screened and selected by ELISA using produced scFvs in human and mouse c-Met-coated 96-well enzyme immunoassay/radioimmunoassay (EIA/RIA) plates (Costar, #3590, Corning, NY, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain

doi: 10.3390/ijms18091968

Figure Lengend Snippet: Kinetic constants and binding affinities of IRCR201.

Article Snippet: Briefly, scFv displaying phage libraries were enriched on 1 µg immobilized human c-Met ECD-Fc (Sino Biological, 10692-H03H, Beijing, China) and mouse c-Met ECD-Fc (Sino Biological, 50622-M02H, Beijing, China) in MaxiSorp™ immune-tubes (Nunc, 444202, Roskilde, Denmark). c-Met specific binders were screened and selected by ELISA using produced scFvs in human and mouse c-Met-coated 96-well enzyme immunoassay/radioimmunoassay (EIA/RIA) plates (Costar, #3590, Corning, NY, USA).

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain

doi: 10.3390/ijms18091968

Figure Lengend Snippet: Epitope mapping of IRCR201. ( a , b ) The reactivity of IRCR201 against a panel of overlapping peptides representing the c-Met extracellular domain was determined by peptide array. A peptide library spanning amino acids 1–932 of the c-Met extracellular domain was synthesized (JPT Peptide Technologies GmbH, Berlin, Germany). The library was prepared as overlapping linear peptides covalently bound to a cellulose membrane. The results show dot blots of specific epitope sequences for IRCR201 (yellow frame). The quantified levels of dot intensity were determined by Multi Gauge V3.0 program; ( c ) Binding pattern analysis using domain proteins of c-Met; ( d ) Hepatocyte growth factor (HGF)/c-Met competitive ELISA. After the HGF (2.5 μg/mL) was pre-treated with the human c-Met protein-immobilized plates, IRCR201 or huOA5D5.v2 was added to confirm whether HGF and each antibody were competitively bound. IPT: immunoglobulin–plexin–transcription; PSI: plexin-semaphorin-integrin domain.

Article Snippet: Briefly, scFv displaying phage libraries were enriched on 1 µg immobilized human c-Met ECD-Fc (Sino Biological, 10692-H03H, Beijing, China) and mouse c-Met ECD-Fc (Sino Biological, 50622-M02H, Beijing, China) in MaxiSorp™ immune-tubes (Nunc, 444202, Roskilde, Denmark). c-Met specific binders were screened and selected by ELISA using produced scFvs in human and mouse c-Met-coated 96-well enzyme immunoassay/radioimmunoassay (EIA/RIA) plates (Costar, #3590, Corning, NY, USA).

Techniques: Peptide Microarray, Synthesized, Binding Assay, Competitive ELISA

Journal: International Journal of Molecular Sciences

Article Title: Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain

doi: 10.3390/ijms18091968

Figure Lengend Snippet: Docking of IRCR201 to human c-Met. ( a ) A three-dimensional (3D) model of IRCR201 variable domains was generated in a single-chain variable fragment (scFv) format by Rosetta computational homology modeling. The complementarity determining regions (CDRs) of the V H and V L domains are shown in blue and red, respectively. The framework regions of the V H and V L domains are represented in dark grey and light grey, respectively. The CDR sequences of IRCR201 are represented according to the Kabat numbering scheme; ( b , c ) IRCR201 was docked to c-Met (PDB accession: 1SHY) using the ZDOCK docking program. Epitope was mapped on to the 3D structure of c-Met (25–567), incorporating the Sema domain and plexin-semaphorin-integrin (PSI) domain. The figures were drawn with PyMOL (DeLano Scientific LLC, Palo Alto, CA, USA). The V H and V L domains of IRCR201 are shown in blue and red, respectively. Yellow = IRCR201 binding site (SAPPFVQ). Light grey = Sema domain. Dark grey = PSI domain. Green = serine protease homology domain (SPHD) of HGF.

Article Snippet: Briefly, scFv displaying phage libraries were enriched on 1 µg immobilized human c-Met ECD-Fc (Sino Biological, 10692-H03H, Beijing, China) and mouse c-Met ECD-Fc (Sino Biological, 50622-M02H, Beijing, China) in MaxiSorp™ immune-tubes (Nunc, 444202, Roskilde, Denmark). c-Met specific binders were screened and selected by ELISA using produced scFvs in human and mouse c-Met-coated 96-well enzyme immunoassay/radioimmunoassay (EIA/RIA) plates (Costar, #3590, Corning, NY, USA).

Techniques: Generated, Binding Assay