recombinant human epha2 receptor extracellular domain  (R&D Systems)

Journal: Clinical Cancer Research

Article Title: A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2–Bispecific Antibody for Combination Cancer Therapy

doi: 10.1158/1078-0432.CCR-22-2535

Figure Lengend Snippet: The roadmap of the proposed unbiased strategy and characterization of the anti-EPHA2 antibody BMX-066. A, Workflow for identifying biologically optimal target combinations for bispecific therapeutic reagents. B, ELISA analysis of anti-EphA2 antibody (BMX-066) binding to a soluble His-tagged rhEPHA2 protein, representing EPHA2 extracellular portion. BMX-066 was titrated at the indicated concentrations over ELISA plates coated with 100 ng/well of rhEPHA2, and staining was performed with goat anti-human IgG(Fc)-HRP. C, Stable expression of EPHA2 in HEK-293 cells transfected with the EPHA2-encoding pcDNA3 expression vector (HEK-EPHA2). Mock-transfected cells (HEK-pcDNA3) are shown as a control. D, Flow cytometry analysis of HEK-EPHA2 and HEK-pcDNA3 cells stained with BMX-066 and R-phycoerythrin (PE) conjugated anti-human IgG Fc fragment (anti–hIgG-PE). Staining with nonspecific hIgG was used as a specificity control. E, EPHA2 levels in the indicated human cancer cell lines. F, Staining of the indicated cancer cells with BMX-066 or nonspecific human IgG (hIgG) and anti–hIgG-PE analyzed by flow cytometry. G, Human triple-negative breast cancer cells, MDA-MB-231, were injected into the mammary fat pad region of NOD-SCID mice (1.5–2×10 6 cells/mouse, with equal cell numbers used within each individual experiment). After initial tumor development, mice with detectable tumors were treated with 2 mg/kg of anti–EphA2-BMX, or nonspecific human IgG (hIgG) per week, given in two intraperitoneal injections. Tumor volumes were measured each 3–4 days. The graph summarizes two independent experiments ( n = 11 for hIgG and n = 12 for BMX-066 groups). Data are shown as means ± SD; *, P < 0.05, Student t test. Western blotting images were optimized using PowerPoint software where required.

Article Snippet: Recombinant human EPHA2 receptor extracellular domain (sEphA2; R&D Systems, #3035-A2–100) was immobilized to the chip ProteOn GLH (Bio-Rad, #176–5013) through amino groups.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Expressing, Transfection, Plasmid Preparation, Control, Flow Cytometry, Injection, Western Blot, Software

Journal: Clinical Cancer Research

Article Title: A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2–Bispecific Antibody for Combination Cancer Therapy

doi: 10.1158/1078-0432.CCR-22-2535

Figure Lengend Snippet: Identifying potential EPHA2 partners for combination therapies. A and B, MDA-MB-231 cells were injected into the mammary fat pad region of NSG mice (1.5×10 6 cells/mouse), and mice were treated as in for 27 days. Tumors were extracted, single-cell suspensions were prepared, and ex vivo cell cultures were established from BMX-066-conditioned and control hIgG-treated tumors (αEPHA2-Cond and cIgG-Tr cells, respectively). Proteome-wide phosphoprotein ( A ) and phosphopeptide ( B ) analyses revealed significant differences between αEPHA2-Cond and cIgG-Tr cells (shown in blue; t test, P < 0.05). C, The schematic representation of ex vivo shRNA-based genome-wide screening strategy for genes that when silenced, selectively suppress αEPHA2-Cond tumor cells. D, Volcano plot representing results of genome-wide pooled shRNA screening in αEPHA2-Cond and cIgG-Tr cells. The x -axis represents the fitness scores, and the y -axis represents negative log of the P value. The orange dots represent the hits that, when silenced, significantly ( P < 0.005) suppress αEPHA2-Cond and not cIgG-Tr cells, whereas inhibition of hits shown in blue provide significant ( P < 0.005) growth advantage to αEPHA2-Cond cells. E, HEK293 T-Rex stable cell lines express EPHA2-BirA*-FLAG bait or a YFP-BirA*-FLAG control. Confocal images display subcellular compartmentalization of BirA* fusion proteins (left; scale bar, 10 μm), and streptavidin Western blotting shows total endogenous protein biotinylation following addition of biotin for 24 hours to the cell culture medium (right). F, Cytoscape representation of the hits from the genetic (shRNA) screen and the BioID data that are enriched in multiple gene ontology processes according to the analysis with GSEA software.

Article Snippet: Recombinant human EPHA2 receptor extracellular domain (sEphA2; R&D Systems, #3035-A2–100) was immobilized to the chip ProteOn GLH (Bio-Rad, #176–5013) through amino groups.

Techniques: Injection, Ex Vivo, Control, Phospho-proteomics, shRNA, Genome Wide, Inhibition, Stable Transfection, Western Blot, Cell Culture, Software

Journal: Clinical Cancer Research

Article Title: A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2–Bispecific Antibody for Combination Cancer Therapy

doi: 10.1158/1078-0432.CCR-22-2535

Figure Lengend Snippet: Co-expression of EPHA2 and EGFR in human tumors. EPHA2 and EGFR expressions in multiple tumor types analyzed using The Cancer Genome Atlas (TCGA) database. The x -axis represents the log 2 of RSEM normalized expression of EPHA2, and the y -axis represents the log 2 of RSEM normalized expression of EGFR. Each dot in the scatterplots represents individual patient data in the specific cancer type. Included are Spearman rank correlation and lines showing the linear best-fit trend.

Article Snippet: Recombinant human EPHA2 receptor extracellular domain (sEphA2; R&D Systems, #3035-A2–100) was immobilized to the chip ProteOn GLH (Bio-Rad, #176–5013) through amino groups.

Techniques: Expressing

Journal: Clinical Cancer Research

Article Title: A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2–Bispecific Antibody for Combination Cancer Therapy

doi: 10.1158/1078-0432.CCR-22-2535

Figure Lengend Snippet: Co-suppression of EPHA2 and EGFR enhances elimination of cancer cells. A, MDA-MB-231 were transduced with the CAS9-encoding pLv5 lentiviral vector (MDA-Cas9), subjected to selection with blasticidin, and CAS9 expression was assessed by Western blot (left). MDA-Cas9 cells were transduced with LV04 lentiviral vectors from Sanger human CRISPR library encoding EphA2-targeting sgRNAs, ID 2400992 and ID 2400602 (MDA-EphA2-KO) or control, non-targeting sgRNA Lenti CRISPR Universal Non-Target Control #2 Plasmid (LV04 vector; MDA-NTC) and subjected to puromycin selection. EPHA2 knockout was confirmed by Western blot (right). B, MDA-EphA2-KO and control cells were seeded at 1.6×10 3 cells per well (three wells per condition) in the MamoCult Basal Medium (Stemcell Technologies, Cat # 05621) into ultralow attachment 24-well plates and allowed to form tumorspheres for 8 days in the presence of 10 μmol/L of erlotinib or a matching concentration of DMSO. Tumorspheres in each well were trypsinized and individual cells counted. The graph represents abundance of the erlotinib-treated cells as percentages relative to matching DMSO controls. C, The indicated cells were seeded and allowed to form tumorspheres as in ( B ). Images of tumorspheres in each well were taken using an EVOS m5000 imager, and overall tumorsphere area was summarized using ImageJ software. The graph represents tumorsphere area of erlotinib-treated cells as a percentage of relative to the area in a matching DMSO control. D, Representative images of erlotinib-treated and DMSO-treated tumorspheres formed by MDA-EphA2-KO and MDA-NTC cells; scale bar, 1,000 μm. E, The indicated cells were seeded in DMEM medium with 1% FBS at 4.5×10 3 cells per well (five wells per condition) into 96-well plates to form monolayer cultures. Cells were treated for 72 hours with the indicated concentrations of erlotinib or DMSO concentration matching DMSO volume loaded with the highest erlotinib dose. Cell survival was quantified using the resazurin assay (R&D Systems, Cat# AR002). The graph represents survival of erlotinib-treated cells as percentages relative to matching DMSO controls. F, CAS9 was expressed in MIA PaCa-2 cells (MiaPaCa-Cas9) and EPHA2 knocked out (MiaPaCa-EphA2-KO) as in ( A ). Nontargeting sgRNA was used as a control (MiaPaCa-NTC), and CAS9 expression and EPHA2 knockout were confirmed by Western blot. G, The effect of erlotinib on tumorspheres formed by MiaPaCa-EphA2-KO and MiaPaCa-NTC cells was examined as in ( C ); cell seeding was done at 1×10 3 cells/well into ultralow attachment 24-well plates. Tumorsphere cell counting (as in B ) was not performed, as we could not get single-cell suspensions from these tumorspheres without damaging cells. H, Representative images of tumorspheres formed by the indicated cells in the presence of 10 μmol/L of erlotinib or matching DMSO control; scale bar, 1,000 μm. I, MiaPaCa-EphA2-KO and MiaPaCa-NTC cells were treated with erlotinib or DMSO in monolayer cultures, and cell survival was analyzed as in ( E ). J, Analysis of the AUC of PDX models representing non–small cell lung carcinoma and colorectal cancer. PDX models treated or not with cetuximab were classified on the basis of EPHA2 expression levels (top and low quartiles). The analysis reveals significantly stronger reduction in tumor size in response to cetuximab treatment in models with low EPHA2 levels compared with tumors with high EPHA2 expression. * , P < 0.05, Kolmogorov–Smirnov test. Data from monolayer ( E and I ) and tumorsphere ( B , C , and G ) experiments were analyzed using the Student t test, *, P < 0.01. Western blot images were optimized using PowerPoint software where required.

Article Snippet: Recombinant human EPHA2 receptor extracellular domain (sEphA2; R&D Systems, #3035-A2–100) was immobilized to the chip ProteOn GLH (Bio-Rad, #176–5013) through amino groups.

Techniques: Transduction, Plasmid Preparation, Selection, Expressing, Western Blot, CRISPR, Control, Knock-Out, Concentration Assay, Software, Resazurin Assay, Cell Counting

Journal: Clinical Cancer Research

Article Title: A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2–Bispecific Antibody for Combination Cancer Therapy

doi: 10.1158/1078-0432.CCR-22-2535

Figure Lengend Snippet: Structure and characterization of the bispecific anti-EGFR/EPHA2 antibody. A, An asymmetric monovalent bispecific anti-EphA2/EGFR antibody, BMX-661, composed of Fab fragment of BMX-066 and scFv derived from the anti-EGFR antibody cetuximab, fused to the human IgG1 Fc domain. Fc fragments of the antibody are heterodimerized by knob-in-hole mutations in the CH3 domains of the heavy chains. His5 amino acid tag is incorporated into the linker between scFv and the Fc region for purification purposes. B, Binding of BMX-661 and cetuximab to soluble immobilized rhEGFR (extracellular portion, top) or BMX-661 and BMX-066 to immobilized rhEPHA2 (bottom) in the ELISA assay. Soluble antigen proteins (2 μg/mL) were immobilized onto ELISA plates, and after incubation with the indicated concentrations of BMX-661, BMX-066, or cetuximab, bound antibodies were detected with goat anti-human IgG(Fc)-HRP. C, The surface plasmon resonance–binding curves for BMX-066 and BMX-661. Soluble rhEPHA2 was immobilized onto the ProteOn GLH chip through amino groups in the 150 mmol/L NaCl, 10 mmol/L HEPES pH 7.4, 0.05% Tween-20 buffer. After washing out the unbound protein, BMX-066 and BMX-661 were applied at 500 nmol/L (IgG) and the unbound antibodies were removed with the buffer. Following this, rhEGFR was introduced, as indicated (sEGFR), and the unbound ligand was removed after binding saturation. The measurements were done using Bio-Rad ProteOn XPR36, at 25°C. Bispecific BMX-661 bound both immobilized rhEPHA2 and soluble rhEGFR, whereas, as expected, monospecific anti–EPHA2 BMX-066 did not interact with rhEGFR. D, Dependence of integral volume of particles, V, from their sizes, d , for BMX-661 preparation measured by dynamic light scattering. BMX-661 (0.16 mg/mL) was analyzed in the 20 mmol/L l -histidine, 30 mmol/L citric acid, 32 mmol/L Na 2 HPO 4 , 1% trehalose, 0.05% Tween-20; pH = 6.0 buffer. The content of multimeric forms of the MABs was estimated using spectrometer ZS Zetasizer Nano (Malvern Instruments) at 25°C. E, HPLC analysis of BMX-661 solution, size exclusion chromatography. F, Stability of BMX-661 estimated by SDS-PAGE under reducing conditions. Freshly isolated antibodies BMX-066 (lanes 1 and 2) and BMX-661 (lanes 4 and 5); BMX-661 stored for 200 days at 4°C (lanes 6 and 7) or incubated at 37°C for 3 days (lanes 8 and 9) in the formulation buffer were loaded at 11 (lanes 1, 4, 6, and 8) and 4 (lanes 2, 5, 7, and 9) micrograms per well. Lanes 3 and 10 molecular weight markers. The top band in the heavy chain region of BMX-661 represents the EGFR-binding scFV-Fc portion of the antibody, whereas the second band and the lower band represent heavy and light chains of the EPHA2-binding portion of bispecific antibody. G, Blood serum stability of BMX-661. ELISA titration curves before (yellow) and after (red) incubation of 2 mg/mL of BMX-661 in human blood serum (Sigma-Aldrich, Cat # H4522) for 7 days at 37°C. ELISA plates were coated with rhEPHA2 (left panel) or sEGFR (right), and blood serum samples with BMX-661 were applied to titration. Staining was performed with goat anti-human IgG(Fc)-HRP. H, Flow cytometry analysis of BMX-661 with HEK-EPHA2 and HEK-pcDNA3 cells. Anti-hIgG-PE was used as a secondary antibody. Staining with nonspecific hIgG was used as a specificity control. I, EGFR levels in HEK-293 cells stably transfected with the EGFR-encoding pCW45 expression vector (HEK-EGFR) or mock-transfected HEK-293 (HEK-pCW45). J, Flow cytometry analysis of BMX-661 with HEK-EGFR and HEK-pCW45 cells. Western blot images were optimized using PowerPoint software where required.

Article Snippet: Recombinant human EPHA2 receptor extracellular domain (sEphA2; R&D Systems, #3035-A2–100) was immobilized to the chip ProteOn GLH (Bio-Rad, #176–5013) through amino groups.

Techniques: Derivative Assay, Purification, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, SPR Assay, Size-exclusion Chromatography, SDS Page, Isolation, Formulation, Molecular Weight, Titration, Staining, Flow Cytometry, Control, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Western Blot, Software

Journal: Clinical Cancer Research

Article Title: A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2–Bispecific Antibody for Combination Cancer Therapy

doi: 10.1158/1078-0432.CCR-22-2535

Figure Lengend Snippet: BMX-661 suppresses tumors in xenograft models. A, EGFR and EPHA2 levels in the indicated human malignant cell lines. B, Flow cytometry analysis of BMX-661 with the indicated cell lines. C, Xenograft tumors were developed in NSG mice by injecting MDA-MB-231 cells stably expressing EGFP, as described previously in . Flow cytometry inset shows EGFP expression in MDA-MB-231. Tumors were allowed to achieve a measurable size, and mice were treated by twice weekly intraperitoneal injections of BMX-661 or nonspecific hIgG ( n = 5 per treatment). Tumor growth was monitored as in , and upon experiment termination, lungs were extracted and imaged with the IVIS Spectrum CT Imaging System to assess metastasis. Fluorescence is shown in red; contrast was adjusted in PowerPoint to optimize the image. The graph on the right represents the mean fluorescence intensity of lungs in each group as quantified for the total surface area of each sample using Living Image software. D, MDA-MB-231 cells were injected into the mammary fat pad of NSG mice (1.5×10 6 cells/mouse) as in . Mice with measurable tumors were randomly assigned into four groups (G1–G4) and treated with intraperitoneal injections of BMX-661 (1.5 mg/kg/wk, split into two injections 3–4 days apart), cisplatin (2.5 mg/kg/wk), a combination of both, or a nonspecific human IgG and a solvent control matching cisplatin solvent (PBS) in a 2×2 design. Tumor growth was monitored as in . E, Triple-negative breast cancer patient-derived xenograft cells, HCI-010, were introduced into the mammary fat pad region of 4- to 6-week-old female NSG mice (1 × 10 6 cells per mouse), and tumors were allowed to develop to a measurable size. The mice were treated intraperitoneally twice a week by 1 mg/kg injection of hIgG, cetuximab, or BMX-661, and tumor growth was monitored as in . The graph summarizes three independent experiments ( n = 14 per group). F, MIA PaCa-2 cells were mixed with Corning Matrigel Matrix (4 × 10 6 cells in 50 μL of PBS mixed with 50 μL of the Matrigel) and injected subcutaneously into the right flanks of 5- to 6-week-old NSG male mice. Once tumors reached a measurable size, mice were treated twice a week by intraperitoneal injections of 1 mg/kg of hIgG, cetuximab or BMX-661. Tumor growth was monitored as in . The graph summarizes two independent experiments ( n = 10 for hIgG, and n = 11 for anti-EGFR and anti-EGFR/EPHA2–treated groups). Western blot images were optimized using PowerPoint software where required. Data in the graphs are shown as means ± SD; *, P < 0.05, treatment with hIgG and cetuximab versus BMX-661, Student t test.

Article Snippet: Recombinant human EPHA2 receptor extracellular domain (sEphA2; R&D Systems, #3035-A2–100) was immobilized to the chip ProteOn GLH (Bio-Rad, #176–5013) through amino groups.

Techniques: Flow Cytometry, Stable Transfection, Expressing, Imaging, Fluorescence, Software, Injection, Solvent, Control, Derivative Assay, Western Blot