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: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: Inhibitory effects of AQP3 and PLD2 siRNA sequences on the target gene. siRNA transfection and reverse transcription-quantitative polymerase chain reaction analyses were performed. The groups were as follows: CK group, normal cultured A431 cells; NC group, A431 cells were transfected with NC siRNA; 1, AQP3-homo-612 siRNA group; 2, AQP3-homo-363 siRNA group; 3, AQP3-homo-360 group; 4, PLD2-homo-602 group; 5, PLD2-homo-1352 group; 6, PLD2-homo-5338 group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Culture, Small Interfering RNA, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: Inhibitory effect of siRNA transfection on the proliferation of A431 cells. Cell proliferation was analyzed and the results are representative of three independent experiments. The statistical significance between two groups was determined using a paired-samples t-test. **P<0.01, compared with the NC group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Small Interfering RNA, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: AQP3 siRNA and PLD2 siRNA increase the apoptosis of A431 cells. Cell apoptosis was determined; results are representative of three independent experiments. The statistical significance between two groups was determined using a paired-samples t-test. **P<0.01, compared with the NC group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Small Interfering RNA, Control, Negative Control

Journal: bioRxiv

Article Title: Design of High Affinity Binders to Convex Protein Target Sites

doi: 10.1101/2024.05.01.592114

Figure Lengend Snippet: a . Model of 5HCS_PDL1_1 (cartoon) binding to PD-L1 (PDB ID: 3BIK), with 5HCS_PDL1_1 colored by Shannon entropy from site saturation mutagenesis results. b. Circular dichroism spectra from 25 °C to 95 °C for 5HCS_PDL1_1. c. Biolayer interferometry characterization of 5HCS_PDL1_1. Biotinylated PD-L1 was loaded to Streptavidin (SA) tips and these were incubated with 8 nM, 2.7 nM and 0.9 nM of 5HCS_PDL1_1 to measure the binding affinity. d. The increase of TCR activation induced signal (via NFAT pathway) from engineered PD-1 effector cells lines by 5HCS_PDL1_1 (green), control antibody (gold) is shown. The mean values were calculated from triplicates for the cell signaling inhibition assays measured in parallel, and error bars represent standard deviations. Color schemes and experimental details are as in Fig3. e. Heat map representing the log enrichments for the 5HCS_PDL1_1 SSM library selected with 6 nM PD-L1 at representative positions. The annotated amino acid in each column indicates the residue from the parent sequence. f. WT A431 and PD-L1 KO A431 cell lines were stained with fluorophore labled 5HCS_PDL1_1 and anti-PD-L1 antibody respectively and then analyzed through FACS. g,h. Unbound crystal structure of 5HCS_PDL1_1 and designed interactions between 5HCS_PDL1_1 (green) and PD-L1 (white). Color schemes are the same as .

Article Snippet: For electroporation, 2×105 cells of each cell type in 20 ul of electroporation buffer (Lonza, cell line SF for A431 and HEK293T, cell line SE for Jurkat) were mixed with 1 ul of electroporation enhancer (IDT cat. 1075916) and 2 ul of assembled RNPs prior to loading 20 ul into an electroporation cuvette strip (Lonza cat. V4XC-2032).

Techniques: Binding Assay, Mutagenesis, Circular Dichroism, Incubation, Activation Assay, Inhibition, Residue, Sequencing, Staining