Journal: Cancer science

Article Title: Complement-mediated tumor-specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII).

doi: 10.1111/j.1349-7006.2011.02019.x

Figure Lengend Snippet: Fig. 1. Epidermal growth factor receptor variant III (EGFRvIII) recognition by different antibodies. (A) Lysates of EGFRvIII-transfected and parental A431 and HEK293T cells were analyzed by immunoblot using the stated antibodies for detection and b-actin as a loading control. Arrows indicate the expected molecular weights of wild-type (wt) EGFR and EGFRvIII, which appears as a double band. 13.1.2 exclusively detected EGFRvIII, whereas ch806 and MR1-1 also bound to wt EGFR on non- transfected cells. (B) Indirect immunofluorescence revealed a similar binding of EGFRvIII antibodies to EGFRvIII-expressing A431 (A431-vIII) and HEK293T (HEK293T-vIII) cells (lower panel). Additional binding to A431 cells was obtained with MR1-1 (grey line) and ch806 (black line). MR1-1 also displayed reactivity with HEK293T cells, whereas 13.1.2 (dotted line) only bound to EGFRvIII- transfected cells. Zalutumumab (black fill) recognized wt EGFR and EGFRvIII. An irrelevant IgG1 antibody (KLH) served as a control (grey fill). (C) Flow cytometric analyses relative to KLH binding (relative fluorescence intensity [RFI]; n = 3) yielded concentration-dependent antibody binding to HEK293T-vIII cells with the lowest EC50 value and a 95% confidence interval (CI) for zalutumumab (d), similar values for ch806 (4) and 13.1.2 ( ), and highest values for MR1-1 ( ).

Article Snippet: Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) and human embryonic kidney HEK293T cells (ATCC, Manassas, VA, USA) were kept in RPMI 1640- or DMEM-Glutamax-I medium (Invitrogen, Carlsbad, CA, USA) containing 10% FCS (Invitrogen), penicillin and streptomycin (both PAA, Pasching, Austria).

Techniques: Variant Assay, Transfection, Western Blot, Control, Binding Assay, Expressing, Concentration Assay

Journal: Cancer science

Article Title: Complement-mediated tumor-specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII).

doi: 10.1111/j.1349-7006.2011.02019.x

Figure Lengend Snippet: Fig. 2. Epidermal growth factor receptor variant III (EGFRvIII) antibodies mediated antibody-dependent cellular cytotoxicity (ADCC). The ADCC against non- transfected and stably transfected A431 (A431-vIII) cells was analyzed with isolated mononuclear cells (MNC) (A), monocytes (B) or polymorphonuclear cells (PMN) (C). Concentration-dependent lysis of A431-vIII cells was observed with zalutumumab (d), MR1-1 ( ), ch806 (4) and 13.1.2 ( ) with MNC and monocytes, whereas PMN-mediated killing was restricted to zalutumumab. MR1-1 and ch806 additionally triggered cytotoxicity of non- transfected A431 cells with MNC, and in the case of ch806, also with monocytes. KLH (e) served as a control and did not induce ADCC with either effector cell population. Data are presented as mean ± SEM from triplicates of at least four independent experiments with different donors. Significant lysis (P < 0.05) compared with the isotype control.

Article Snippet: Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) and human embryonic kidney HEK293T cells (ATCC, Manassas, VA, USA) were kept in RPMI 1640- or DMEM-Glutamax-I medium (Invitrogen, Carlsbad, CA, USA) containing 10% FCS (Invitrogen), penicillin and streptomycin (both PAA, Pasching, Austria).

Techniques: Variant Assay, Transfection, Stable Transfection, Isolation, Concentration Assay, Lysis, Control

Journal: Cancer science

Article Title: Complement-mediated tumor-specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII).

doi: 10.1111/j.1349-7006.2011.02019.x

Figure Lengend Snippet: Fig. 3. Crossblocking experiments and C1q deposition. (A) Cross-competition studies were performed with epidermal growth factor receptor variant III (EGFRvIII)-expressing HEK293T cells stained with the FITC-labeled EGFRvIII antibody of interest in the presence of 200-fold excess of the indicated unconjugated antibodies. The percentage of maximal mean fluorescence intensity (MFI) was calculated as described in the Materials and Methods. None of the EGFRvIII antibodies demonstrated overlapping epitopes with any of the EGFR antibodies used. MR1-1 and 13.1.2 crossblocked each other, indicating an overlapping epitope, while ch806 did not compete with any other antibody. (B) C1q deposition on A431 and EGFRvIII-transfected A431 (A431-vIII) cells was analyzed in the presence of individual antibodies or combinations (10 lg ⁄ mL final mAb concentration). While zalutumumab plus HuMab-005 triggered efficient C1q deposition, none of the individual antibodies or the combination of zalutumumab and KLH (isotype control) was effective on either cell line. Zalutumumab induced significant C1q deposition in combination with each of the EGFRvIII antibodies selectively on A431-vIII (right panel), but not on A431 cells (left panel). Data are presented as mean ± SEM of at least three independent experiments. In (A), ( ) indicates significant (P < 0.05) binding differences of the FITC-labeled antibody in the presence of the unconjugated antibody vs KLH. In (B), ( ) marks significant differences in C1q binding after incubation with zalutumumab vs zalutumumab combinations. RFI, relative fluorescence intensity.

Article Snippet: Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) and human embryonic kidney HEK293T cells (ATCC, Manassas, VA, USA) were kept in RPMI 1640- or DMEM-Glutamax-I medium (Invitrogen, Carlsbad, CA, USA) containing 10% FCS (Invitrogen), penicillin and streptomycin (both PAA, Pasching, Austria).

Techniques: Variant Assay, Expressing, Staining, Labeling, Transfection, Concentration Assay, Control, Binding Assay, Incubation

Journal: Cancer science

Article Title: Complement-mediated tumor-specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII).

doi: 10.1111/j.1349-7006.2011.02019.x

Figure Lengend Snippet: Fig. 4. Epidermal growth factor receptor variant III (EGFRvIII)-specific complement-dependent cytotoxi- city (CDC) by combination of zalutumumab and EGFRvIII antibodies. Non-transfected and EGFRvIII- expressing A431 and HEK293T cells were incubated with antibody combinations in the presence of human serum. Zalutumumab plus 005 served as a control and mediated significant lysis of both A431 cell lines (upper panel) and EGFRvIII-expressing HEK293T cells (lower panel). No killing was observed by any individual antibody, or by any combination against HEK293T cells. Combinations of zalutumumab and the indicated EGFRvIII antibodies induced CDC selectively against cells expressing EGFRvIII. Data are presented as mean ± SEM of at least four independent experiments. ( ) Significant (P < 0.05) differences in lysis between zalutumumab and zalutumumab combinations.

Article Snippet: Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) and human embryonic kidney HEK293T cells (ATCC, Manassas, VA, USA) were kept in RPMI 1640- or DMEM-Glutamax-I medium (Invitrogen, Carlsbad, CA, USA) containing 10% FCS (Invitrogen), penicillin and streptomycin (both PAA, Pasching, Austria).

Techniques: Variant Assay, Transfection, Expressing, Incubation, Control, Lysis

Journal: Cancer science

Article Title: Complement-mediated tumor-specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII).

doi: 10.1111/j.1349-7006.2011.02019.x

Figure Lengend Snippet: Fig. 5. Fc engineering of MR1-1 improved complement-dependent lysis. (A) An Fc-engineered (K326A ⁄ E333A) variant of MR1-1 (MR1-1E3) alone triggered significant complement-dependent cytotoxicity (CDC) against epidermal growth factor receptor variant III (EGFRvIII)-expressing HEK293T cells (upper panel), but not against A431-vIII cells (lower panel). At 10 lg ⁄ mL final concentration, combinations of zalutumumab and MR1-1E3 were as effective as the combinations with MR1-1 on both cell lines (left panels). However, at lower concentrations, significantly enhanced killing was observed with MR1-1E3 (right panels). (B) Combinations of non-overlapping EGFRvIII antibodies induced CDC against HEK293T-vIII cells (right panel). Additionally, the combination of ch806 and MR1-1E3 mediated lysis of A431-vIII cells (left panel). Data are presented as mean ± SEM from triplicates of at least three independent experiments with different donors. ( ) Significant lysis (P < 0.05) and significantly increased lysis (#) by zalutumumab combination with MR1-1E3 vs MR1-1.

Article Snippet: Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) and human embryonic kidney HEK293T cells (ATCC, Manassas, VA, USA) were kept in RPMI 1640- or DMEM-Glutamax-I medium (Invitrogen, Carlsbad, CA, USA) containing 10% FCS (Invitrogen), penicillin and streptomycin (both PAA, Pasching, Austria).

Techniques: Lysis, Variant Assay, Expressing, Concentration Assay

Journal: Scientific Reports

Article Title: Behavioral, blood, and magnetic resonance imaging biomarkers of experimental mild traumatic brain injury

doi: 10.1038/srep28713

Figure Lengend Snippet: Rats given an mFPI had significantly increased levels of ceruloplasmin ( a ) and NF-H ( b ) in plasma on D1 compared to sham-injured rats. Additionally, mFPI rats had significantly decreased levels of tau on D7 ( c ), and VEGF on D7 and D30 ( d ) compared to their sham counterparts. There were no statistically significant differences between the groups on any of the other plasma biomarkers ( e–h ). Mean ± s.e.m., *mFPI group significantly different than sham group, p < 0.05.

Article Snippet: The primary antibodies were diluted in antibody incubation buffer (0.1% BSA, EDTA-free Halt Protease and Phosphatase Inhibitor Cocktail [Thermo Fisher Scientific], 1× TBS, and 0.5% Tween-20) and used in the following dilutions: ceruloplasmin (1:20; Santa Cruz Biotechnology, sc-21240), NF-H (1:100; Sigma–Aldrich, N4142), tau (1:100; Cell Signaling Technology, 4019), VEGF (1:50; Abcam, ab53465), 4-HNE (1:1000; EMD Millipore, 393207), NSE (1:50; Abcam, ab53025), GFAP (1:20; Abcam, ab48050), and S100β (1:20; Abcam, ab41548).

Techniques: Clinical Proteomics

Journal: Cancers

Article Title: Implication of COPB2 Expression on Cutaneous Squamous Cell Carcinoma Pathogenesis

doi: 10.3390/cancers14082038

Figure Lengend Snippet: Effect of COPB2 knockdown on biologic behavior of cutaneous squamous cell carcinoma (cSCC) cells: ( A ) (i) COPB2 protein expression decreased predominantly after COPB2 knockdown in both HSC-1 and A431 cells (Magnification, 400×; Scale bar, 50 μm); (ii) Proliferation ability of both HSC-1 and A431 cells decreased significantly after COPB2 knockdown at the indicated time points (Both p < 0.001, Mann-Whitney U-test). ( B ) (i) Representative patterns of cell invasion in each group of cSCC cells (Magnification, 100×; Scale bar, 200 μm). (ii) Invasion ability of both HSC-1 and A431 cells decreased significantly after COPB2 knockdown (Both p = 0.002, Mann-Whitney U-test). ( C ) Apoptotic cells increased predominantly after COPB2 knockdown in both HSC-1 and A431 cells. ( D ) (i) Tumor nodules of xenograft mouse models obtained from control and shCOPB2 groups of A431 cells (Scale bar, 1 cm). (ii) Volume of the tumor nodules increased significantly in control group compared to that in the COPB2 knockdown group at the indicated days (* p = 0.008, Mann-WhitneyU-test). (iii) Representative patterns of apoptotic cells in tumor nodules from each group of A431 cells-xenograft models (Magnification, 400×; Scale bar, 50 μm). Apoptotic cells were predominantly higher in the COPB2 knockdown group than in the control group in A431 cells ( p < 0.001, Mann-Whitney U-test).

Article Snippet: COPB2 knockdown stable HSC-1 and A431, as well as related control cells, were established using pGFP-C-shCOPB2 lentiviral particles (OriGene, Rockville, MD, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Waltham, MA, USA) and 1% streptomycin/penicillin (Gibco, Waltham, MA, USA).

Techniques: Expressing, MANN-WHITNEY