Journal: Cells

Article Title: Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

doi: 10.3390/cells10112904

Figure Lengend Snippet: αEGFR-E-P125A: Effects on angiogenesis and VM. ( A ) Schematic diagram of antibody–endostatin fusion proteins (αEGFR-E-P125A and Fc-E-P125A): Human endostatin-P125A domains are shown in orange, and the proline to alanine mutation at amino acid 125 of endostatin is indicated by a black line. Details in methods. ( B ) Inhibition of HUVEC CLS formation and TNBC VM. HUVEC, MDA-MB-231, MDA-MB231-4175, or MDA-MB-468 cells were resuspended in EBM2 and RPMI, plated on matrigel, and treated as indicated for 16 h. Tube formation was recorded through an inverted photomicroscope. Conditions: Media, endostatin-P125A (E-P125A, 90.92 nmol/L), Cetuximab (Cet, 45.46 nmol/L), Combination of 90.92 nmol/L E-P125A and 45.46 nmol/L cetuximab (E-P125A + Cet), Fc-E-P125A (45.46 nmol/L), and αEGFR-E-P125A (45.46 nmol/L). Note that αEGFR-E-P125A and Fc-E-P125A each contain two fused E-P125As per molecule of the fusion protein, so 2x the concentration of E-P125A (90.92 nmol/L) was compared to indicated fusion proteins (at 45.46 nmol/L). C-D. Vasculogenic mimicry by MDA-MB-231-4175 and individual cell migration paths using time-lapse photography. ( C ) tube formation (VM) by MDA-MB-231-4175 under indicated conditions was observed at 30-min intervals for 15 h. Migration path of ten randomly selected cells from 0–15 h. ( D ) Cumulative traversed distance by 10 randomly selected TNBC cells from 0–15 h in C. Data shown as the mean ± SEM. ( E ) Effects on HUVEC and MDA-MB-231-4175 migration: HUVEC (2 × 10 4 ) and MDA-MB-231-4175 cells (5 × 10 4 ) were incubated for 8 h in serum-free media containing an equimolar concentration (45 nM) of cetuximab, E-P125A, a combination of cetuximab and E-P125A, or αEGFR-E-P125A, in the top chamber of a transwell plate; the bottom chamber contained 10% FBS/EBM-2 as a chemoattractant. Migration-induced impedance changes were measured using xCELLigence RTCA-SP every 15 min. HUVEC and MDA-MB-231-4175 cells migrated to the bottom surface of the upper chamber within 3 h. HUVEC remained adherent for up to 8 h, while MDA-MB-231 cells gradually detached from the transwell bottom. Data are shown as the mean ± SEM.

Article Snippet: Anti-EGFR IgG1-huEndostatin-P125A heavy chain and kappa light chain gene coding sequences were cloned separately into SMART Expression pCT™ vectors (Celltheon) and co-transfected into CHO cells.

Techniques: Mutagenesis, Inhibition, Concentration Assay, Migration, Incubation

Journal: Cells

Article Title: Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

doi: 10.3390/cells10112904

Figure Lengend Snippet: αEGFR-E-P125A inhibition of ‘mosaic’ vessel formation: HUVEC and TNBC co-culture. Left Panel HUVECs labeled with CellTracker™ Red CMTPX and MDA-MB-468 with CellTracker™ Green CMFDA. HUVECs (left panel, top row) in serum-free EBM2 or MDA-MB-468 (left panel, second row) in serum-free RPMI cultured alone on matrigel for 16 as indicated: αEGFR-E-P125A, or controls (media, E-P125A, cetuximab, or the combination of E-P125A and cetuximab (E-P125A + Cet)). MDA-MB-468 and HUVEC in serum-free EBM2 and RPMI co-cultured on matrigel for 16 h with indicated treatment (Left panel bottom four rows). Fluorescent imaging is shown for HUVEC (red), MDA-MB-468 (green), or both (merged). Right Panel: HUVEC and MDA-MB-231-4175 co-culture (HUVEC labeled with CellTrace™ Calcein Red-Orange AM (red) or MDA-MB-231-4175 labeled with CellTrace™ calcein green AM (green) were cultured separately on matrigel for 16 h and treated with αEGFR-E-P125A or controls [media, E-P125A, cetuximab, or E-P125A and cetuximab combined (E-P125A + Cet)]. (Right panel, top two rows). Labeled MDA-MB-231-4175 and HUVEC cocultured on matrigel (Rightpanel, bottom 4 rows). Labelled MDA-MB-231-4175 and HUVEC co-cultured on matrigel 16 h and treated as indicated Images taken with a Leica fluorescence confocal microscope. (Left and Right bottom panels): Higher magnification views are provided of areas highlighted in yellow bordered squares below the coculture figures in the, demonstrating the alignment and coalescence of HUVEC (red) and TNBC cells (green) in mosaic vessels, as well as the more random distribution of HUVEC and TNBC in cocultures exposed to αEGFR-E-P125A.

Article Snippet: Anti-EGFR IgG1-huEndostatin-P125A heavy chain and kappa light chain gene coding sequences were cloned separately into SMART Expression pCT™ vectors (Celltheon) and co-transfected into CHO cells.

Techniques: Inhibition, Co-Culture Assay, Labeling, Cell Culture, Imaging, Fluorescence, Microscopy

Journal: Cells

Article Title: Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

doi: 10.3390/cells10112904

Figure Lengend Snippet: ( A ) Effects of αEGFR-E-P125A on sprouting: Tube formation by HUVECs or MDA-MB-231-4175 plated on matrigel, untreated or treated with E-P125A (58 μg/mL), cetuximab (170 μg/mL, Cet), a combination of E-P125A (58 μg/mL) and cetuximab (170 μg/mL), or αEGFR-E-P125A (250 μg/mL) for 16 h. Sprouting indicated by black arrows. ( B ) Western blot analysis for vimentin and SER39-phosphorylated vimentin following αEGFR-E-P125A treatment: MDA-MB-231-4175 cells plated in matrigel were treated with E-P125A (58 μg/mL), cetuximab (170 μg/mL), or αEGFR-E-P125A (250 μg/mL) for 16 h and harvested; cytoplasmic extracts were prepared, and western blot performed for vimentin or SER39-phosphorylated vimentin. Relative quantification of vimentin-S39- P /vimentin using the NIH ImageJ program is shown. ( C ) αEGFR-E-P125A inhibits secretion of MMP2 and shedding of MT1-MMP: MDA-MB-231-4175 cells were plated on matrigel, treated with E-P125A (58 μg/mL), cetuximab (170 μg/mL), or αEGFR-E-P125A (250 μg/mL) for 16 h; supernatants were harvested and analyzed for secreted MMP2 or shed MT1-MMP by ELISA. Details in methods. ( D ) αEGFR-E-P125A treatment reduces β-catenin levels. MDA-MB-231-4175 cells were plated on matrigel, treated with E-P125A (58 μg/mL), cetuximab (170 μg, or αEGFR-E-P125A (250 μg/mL) for 16 h and then harvested; total, cytoplasmic, and nuclear extracts were analyzed by Western blotting. Tubulin was used for cytoplasmic and lamin B for nuclear normalization. The right histogram shows the relative quantification of β-catenin/(tubulin or lamin B) staining using NIH ImageJ software. ( E ) αEGFR-E-P125A inhibits TCF/LEF transcriptional activity. MDA-MB-231 (TCF/LEF Luc Reporter) cells were plated and treated as indicated ( A). TCF/LEF reporter assay was performed in 96-well plates without (2D) or containing matrigel (3D, shown in the bottom panel) at 16 h. Luciferase activity in treated cells relative to the medium control is indicated.

Article Snippet: Anti-EGFR IgG1-huEndostatin-P125A heavy chain and kappa light chain gene coding sequences were cloned separately into SMART Expression pCT™ vectors (Celltheon) and co-transfected into CHO cells.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Software, Activity Assay, Reporter Assay, Luciferase

Journal: Cells

Article Title: Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

doi: 10.3390/cells10112904

Figure Lengend Snippet: αEGFR-E-P125A effects on angiogenesis, as well as vasculogenic mimicry, tumor growth, and pulmonary metastasis of MDA-MB-468. ( A ) Tumor growth: MDA-MB-468 cells (5 × 10 6 /mouse) were injected into the mammary fat pad (mfp) of female NSG mice ( n = 8). Starting day 5 post tumor implantation, mice were treated with PBS, equimolar cetuximab (170 μg/injection), Fc-E-P125A (102 μg/injection), or αEGFR-E-P125A (250 μg/injection) at the indicated intervals (black arrows). Tumor size assessed by digital calipers. Data are shown as the mean ± SEM. ( B ). Primary tumor growth and pulmonary metastasis: In a separate experiment from A, MDA-MB-468 cells (5 × 10 6 /mouse) were implanted in the mammary fat pad (mfp), and mice were treated intravenously starting day 5 post implantation with PBS, cetuximab (170 μg/injection), or αEGFR-E-P125A (250 μg/injection) at the indicated intervals (black arrows). On day 30, mice were euthanized, and primary mammary tumor growth was measured by calipers ( B); lungs were harvested for further analysis of pulmonary metastases ( E,F). Data are shown as the mean ± SEM. ( C ) Immunohistochemical staining of MDA-MB-468 tumor for angiogenesis and VM. Primary tumor sections from B stained for murine CD31 (mCD31, the upper panel) or for human laminin (the bottom panel) and counterstained with hematoxylin on day 30. Representative sections from a tumor selected for ‘average’ size are shown. Relative positive optical density/total optical density of the immunohistochemical staining was analyzed using NIH ImageJ software for individual mouse lung sections and represented as yellow squares (PBS treated), blue triangles (cetuximab treated) or red circles (αEGFR-E-P125A treated). Details in Methods. ( D ) Immunofluorescence staining for mCD31 (red) and anti-human laminin (green) day 30 post tumor implantation. Vessels staining green only for human laminin correspond to areas of VM, while vessels staining red mixed with green represent mCD31+/huLaminin+ ‘mosaic ‘ vessels. Details in Methods. ( E , F ). αEGFR IgG1-huEndo-P125A inhibits pulmonary metastasis: ( E ) Formalin-fixed paraffin-embedded lung tissues from representative mice from the 10 mice/treatment group with near ‘average’ growth of a primary mfp implanted MDA-MB-468, stained with hematoxylin and eosin. ( F ) Metastatic burden in the lung sections (5 out of 10 mice) for the experiment represented in B,C. Relative percent metastasis containing area/total lung area in individual murine lung sections was analyzed using NIH ImageJ software, and relative percent metastases for individual mouse lung sections are represented as yellow squares (PBS treated), blue triangles (cetuximab treated) or red circles (αEGFR-E-P125A treated). Details in Methods.

Article Snippet: Anti-EGFR IgG1-huEndostatin-P125A heavy chain and kappa light chain gene coding sequences were cloned separately into SMART Expression pCT™ vectors (Celltheon) and co-transfected into CHO cells.

Techniques: Injection, Tumor Implantation, Immunohistochemical staining, Staining, Software, Immunofluorescence, Formalin-fixed Paraffin-Embedded

Journal: Cells

Article Title: Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

doi: 10.3390/cells10112904

Figure Lengend Snippet: ( A – F ) αEGFR IgG1-huEndo-P125A inhibits pulmonary tropic MDA-MB-231-4175 lung metastasis. ( A ) Pulmonary metastasis: 5 × 10 4 MDA-MB-231-4175 cells were injected via the tail vein (green arrow). Mice ( n = 8) were treated starting day 5 with PBS, cetuximab (170 µg/injection), or αEGFR-E-P125A (250 µg/injection) 2×/week (black arrows, 9 injections). Metastases were monitored by IVIS at intervals indicated by red arrows. Experiment terminated on day 36. A single mouse was lost in the cetuximab group on day 35 (a red blank in C). ( B , C ) Pulmonary average photon flux graph ( B ) and relative photon flux/2-min exposure on day 36 ( C ) The αEGFR-E-P125A-treated group showed reduced total body tumor burden and lung metastasis. Average photon flux on day 36 is shown in a graph. Data shown as the mean ± SEM. ( D , E ) αEGFR-E-P125A inhibits mCD31+ angiogenesis (D) and huLaminin+ TNBC VM (E) of MDA-MB-231-4175. Immunohistochemical staining of tumor sections on day 36 from mice in B and C. Cryosections from representative treated mice were stained with anti-murine CD31 ( D ) or anti-human laminin antibody ( E ) and counterstained with hematoxylin. Relative positive optical density/total optical density of the immunohistochemical staining was analyzed using NIH ImageJ. Each dot represents an immunohistochemically stained cryosection from a tumor selected for near ‘average’ photon flux intensity. Details in Methods. ( F ) αEGFR-E-P125A inhibits vimentin-SER39 phosphorylation: On day 36, lung sections containing metastatic MDA-MB-231-4175 from sacrificed mice ( B,C) were analyzed by IHC. Cryosections from mice with near average relative photon flux intensity stained for human vimentin (brown, Vim, the upper panel ) or human vimentin phosphorylated on serine 39 (brown, VimS39-P, bottom panel ). Hematoxylin was used for nuclear counter-staining. Representative tumor cryosections from mice treated with PBS, cetuximab, or αEGFR-E-P125A are presented. Relative vimentin+ or SER39 vimentin+ staining optical density/total optical density of IHC staining was quantified using NIH ImageJ. ( G ) Survival of mice following αEGFR-E-P125A treatment. MDA-MB-231-4175 cells (5 × 10 4 ) were injected via the tail vein and survival was measured. Nine mice/group were treated starting day 5 with PBS, cetuximab (68 µg/injection), or αEGFR-E-P125A (100 µg/injection) 2×/week (10 injections) per schedule shown above the survival curve. Survival was analyzed using the survival analyses and one-way ANOVA test (GraphPad Prism 7.03). Green arrow, tumor inoculation; black arrow: treatment administration ( PBS, cetuximab, or αEGFR-E-P125A as indicated), blue arrow: experiment termination.

Article Snippet: Anti-EGFR IgG1-huEndostatin-P125A heavy chain and kappa light chain gene coding sequences were cloned separately into SMART Expression pCT™ vectors (Celltheon) and co-transfected into CHO cells.

Techniques: Injection, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Cells

Article Title: Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

doi: 10.3390/cells10112904

Figure Lengend Snippet: αEGFR-E-P125A effects on angiogenesis and VM lead to decreased tumor growth and metastasis: αEGFR-E-P125A reduces TNBC motility and migration, leading to decreased circulating tumor cells. Assembly of tumor blood vessels (angiogenesis) by endothelial precursors and tumor cell-lined vascular channels (VM) are both inhibited, reducing tumor growth and metastasis.

Article Snippet: Anti-EGFR IgG1-huEndostatin-P125A heavy chain and kappa light chain gene coding sequences were cloned separately into SMART Expression pCT™ vectors (Celltheon) and co-transfected into CHO cells.

Techniques: Migration

Journal: PLoS Pathogens

Article Title: Antibody recognition of the Pneumovirus fusion protein trimer interface

doi: 10.1371/journal.ppat.1008942

Figure Lengend Snippet: (A) Epitope mapping of the hMPV F-specific mAb panel. Epitope control mAbs include 101F (site IV), DS7 and MPV196 (DS7 epitope), and MPV364 and MPE8 (site III). MPV465 and MPV458 do not compete with known mAbs, and compete with each other for binding, suggesting both mAbs bind at a previously undiscovered antigenic site. Data indicate the percent binding of the competing antibody in the presence of the primary antibody, compared with the competing antibody alone. Cells filled in black indicate full competition, in which ≤33% of the uncompeted signal was observed; cells in gray indicate intermediate competition, in which the signal was between 33% and 66%; and cells in white indicate noncompetition, where the signal was ≥66%. Antigenic sites are highlighted at the top and side based on competition binding with the control mAbs. (B) Plaque neutralization curves for MPV458 and MPV465 with controls. Both MPV458 and MPV465 are neutralizing, while MPV458 has neutralizing properties similar to MPE8 and 101F. IC 50 values are inlaid in each curve. The pneumococcal-specific antibody Ply34 was used as a negative control. Data points are the average of three replicates and error bars are 95% confidence intervals. Data are shown from one experiment and are representative of two independent experiments. A mAb was considered neutralizing if >50% plaque reduction was observed at the highest concentration.

Article Snippet: For recombinant mAbs, plasmids encoding cDNAs for the heavy and light chain sequences of 101F [ ], MPE8 [ ], and DS7 [ ] were synthesized (GenScript), and cloned into vectors encoding human IgG1 and lambda or kappa light chain constant regions, respectively. mAbs were obtained by transfection of plasmids into Expi293F cells as described above.

Techniques: Control, Binding Assay, Neutralization, Negative Control, Concentration Assay

Journal: PLoS Pathogens

Article Title: Antibody recognition of the Pneumovirus fusion protein trimer interface

doi: 10.1371/journal.ppat.1008942

Figure Lengend Snippet: (A) ELISA binding curves for hMPV F-specific mAbs against a panel of hMPV F proteins including purified monomeric and trimeric hMPV B2 F protein that was treated with trypsin. Binding curves and EC 50 values are colored according to the legend. MPV458 and MPV465 have lower EC 50 values (higher affinity) for monomeric hMPV B2 F than trimeric hMPV B2 F. MPV458 and MPV465 bind to hMPV F proteins from all four subgroups. Data points are the average of four replicates and error bars are 95% confidence intervals. > indicates the calculated EC 50 was either not in the range of the curve (due to an overall low signal) or a signal above 0.5 absorbance units was not detected at the highest concentration of 20 μg/mL. Each data point is the average of four replicates and error bars represent 95% confidence intervals. Data are representative of one experiment from two independent experiments. (B) Binding curves from biolayer interferometry. Biotinylated monomeric hMPV B2 F protein coated streptavidin biosensors were exposed to Fabs (MPV458 and MPV465: 10/5/1/0.5 μg/mL, 101F: 100/75/50/25 μg/mL) for 300 s before dissociating in buffer for 600 s. Binding constants are displayed underneath each graph and were averaged from the four replicates.

Article Snippet: For recombinant mAbs, plasmids encoding cDNAs for the heavy and light chain sequences of 101F [ ], MPE8 [ ], and DS7 [ ] were synthesized (GenScript), and cloned into vectors encoding human IgG1 and lambda or kappa light chain constant regions, respectively. mAbs were obtained by transfection of plasmids into Expi293F cells as described above.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, Concentration Assay

Journal: PLoS Pathogens

Article Title: Antibody recognition of the Pneumovirus fusion protein trimer interface

doi: 10.1371/journal.ppat.1008942

Figure Lengend Snippet: (A) The X-ray crystal structure of pre-fusion hMPV F is shown with the 66–87 epitope colored in red (PDB ID: 5WB0). (B) The corresponding 66–87 epitope is colored on the X-ray crystal structure of post-fusion hMPV F (5L1X). The 66–87 epitope is surface exposed on trimeric post-fusion hMPV F. (C) Structural overlay of the 66–87 region between pre-fusion (cyan) hMPV F from the hMPV B2 F + MPV458 Fab complex and post-fusion (red) hMPV F (PDB ID: 5L1X). Conserved amino acid residues between the B2 and A1 subgroups are listed in black, while residues that have mutations or shift positions are colored according to the corresponding structure. (D) Structural overlay of MPV458 on the hMPV F protein with previously structurally characterized hMPV F-specific mAbs. MPE8 (site III, orange) and 101F (site IV, red) were aligned onto hMPV F by aligning the corresponding RSV F residues onto hMPV F from the co-complex structures with RSV F (PDB ID: 5U68 and PBD ID: 3O45). DS7 was aligned from PDB 4DAG. (E) The structure overlay in (D) is rotated 90 degrees to view the hMPV F protein from the top down.

Article Snippet: For recombinant mAbs, plasmids encoding cDNAs for the heavy and light chain sequences of 101F [ ], MPE8 [ ], and DS7 [ ] were synthesized (GenScript), and cloned into vectors encoding human IgG1 and lambda or kappa light chain constant regions, respectively. mAbs were obtained by transfection of plasmids into Expi293F cells as described above.

Techniques:

Journal: Structure (London, England : 1993)

Article Title: Vaccine-elicited murine antibody WS6 neutralizes diverse beta-coronaviruses by recognizing a helical stem supersite of vulnerability

doi: 10.1016/j.str.2022.06.004

Figure Lengend Snippet: Spike-mRNA-immunized mice elicit antibodies against diverse regions of spike, several of which bound diverse beta-coronavirus spikes and one of which, WS6, neutralized them (A) Immunization scheme. NatSP is the full transmembrane-containing native sequence of spike WA-1 strain; S-dTM is the soluble spike protein residues 1–1,206 of wild-type WA-1 strain. (B) Binding of isolated antibodies by ELISA to subdomains of the SARS-CoV-2 spike. Purified monoclonal antibodies from hybridoma supernatants were analyzed for binding to SARS-CoV-2 S-dTM, S1 (S1-short and S1R), RBD, and NTD by ELISA. (C) Binding of isolated antibodies assessed by ELISA to diverse beta-coronavirus pre-fusion-stabilized spikes (S2Ps). (D) Neutralization assessment of hybridoma antibodies against SARS-CoV-2 WA-1 and SARS-CoV pseudoviruses on 293T-ACE2 cells. See also Figure S1 .

Article Snippet: Select hybridomas were sent to GenScript (Piscataway, NJ 08854, USA) for hybridoma heavy and light chain variable sequences and IMGT ( ) analysis.

Techniques: Sequencing, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay, Purification, Bioprocessing, Neutralization