Characteristics of constructs.

<p>Characteristics of constructs.</p

Binding of CD96-phages to CD96-positive HSB-2 cells.

<p><b>A</b>) In a polyclonal whole cell-phage ELISA preferential binding of phage preparations to CD96-positive cells was analyzed. HSB-2 (CD96⁺, CD7⁺) and CEM cells (CD96^-, CD7⁺) were incubated with phages from the original library (1), after the first screening round (2), after the second screening round (3), and after third screening round (4), helper phages (5), TH-111 antibody (6), TH-69 antibody (7), no primary antibody (8) <b>B</b>) Whole cell phage ELISA with monoclonal phage preparations. HSB-2 and CEM cells were incubated with phages from 10 single clones (1–10), helper phages (11), TH-111 antibody (12), TH-69 antibody (13), no primary antibody (14). An anti-M13-specific HRP-conjugated antibody was used for detection of phages; HRP-conjugated goat-anti-mouse-IgG antibodies for antibody stainings. □ =  CEM cells; ▪ =  HSB-2 cells.</p

Comparison of ADCC activity at saturating antibody concentrations.

<p>ADCC experiments with HSB-2 cells were performed using mononuclear cells as effector cells. (1, <b>▪</b>): CD96-S32F-scFv-IgG1-Fc-eng, (2, <b>▪</b>): CD96-wt-scFv-IgG1-Fc-eng, (3, <b>▪</b>): CD96-wt-scFv-IgG1-Fc, (4, <b>□</b>): TH-111. E/T ratio (80∶1). Antibody concentration 200 µg/ml. Data are presented as mean values +/− SEM of 5 independent experiments. (#) extend of lysis significantly different from lysis obtained with the murine TH-111 antibody. (*) extend of lysis significantly different between the two mini-antibodies (p<0.05).</p

Binding specificity of CD96-S32F-scFv-IgG1-Fc to CD96.

<p><b>A</b>) HSB-2 cells or CEM cells were incubated with CD96-S32F-scFv-IgG1-Fc (black) or CD20-scFv-IgG1-Fc (white). <b>B</b>) Competition binding assay. Mini-Ab = CD96-S32F-scFv-IgG1-Fc. <b>C</b>) Proteins CD96-wt-scFv-IgG1-Fc-eng (left panel) and CD96-S32F-scFv-IgG1-Fc-eng (right panel) were analyzed by size exclusion chromatography directly after affinity chromatography (black lines). Peak fractions containing the proteins with the expected molecular mass were collected and reanalyzed (red lines). <b>D</b>) Dose dependent binding of mini-antibodies containing (left panel) or devoid (right panel) of aggregated material was analyzed by flow cytometry using HSB-2 cells. (○): CD96-wt-scFv-IgG1-Fc-eng, (•): CD96-S32F-scFv-IgG1-Fc-eng. Data are presented as mean values +/− SEM from 7 and 3 independent experiments. (*) indicates significant difference in binding between the two binding curves at the indicated antibody concentration (p<0.05).</p

Dose dependent killing of Fc-engineered CD96-mini-antibodies.

<p>ADCC with HSB-2 cells. ADCC with KG1 cells. (○): CD96-wt-scFv-IgG1-Fc-eng, (•): CD96-S32F-scFv-IgG1-Fc-eng, (<b>□</b>):CD20-scFv-IgG1-Fc-eng, (<b>•</b>): TH-111. E/T ratio (80∶1). Data are presented as mean values +/− SEM of 4 and 5 independent experiments. (*) indicates statistically significant difference in lysis between CD96-wt-scFv-IgG1-Fc-eng, CD96-S32F-scFv-IgG1-Fc-eng.</p

Sequence alignment of the VH and VL domains of mutated CD96-scFv library.

<p>After the fifth round of screening, 30 clones were randomly selected and sequences were compared to same number of clones from the original library. The CDR regions are defined according the Kabat numbering scheme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042426#pone.0042426-Kabat1" target="_blank">[53]</a>. <b>A</b>) Light chains from original library and after the fifth round of screening. <b>B</b>) Heavy chains from original library and after the fifth round of screening.</p

Calculated homology model of wild type CD96-scFv.

<p><b>A</b>) A homology model of the wild type CD96-scFv was calculated using the Rosetta Antibody: Fv Homology modelling server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042426#pone.0042426-Sircar1" target="_blank">[46]</a> and displayed as a surface model using the Accelrys DS Viewer software. <b>B</b>) Top view of the model displayed in (A). The 6 loops defining the CDRs were assigned according to the Kabat numbering scheme. Heavy chain CDRs (green), light chain CDRs (yellow), heavy chain frameworks (white), light chain frameworks (blue), positions found mutated after screening (red), mut1 = S32F and mut2 = N97D.</p

Expression and binding specificity of CD96-wt-scFv-IgG1-Fc (mini-antibody).

<p><b>A</b>) Scheme of mini-antibody (upper panel). Coomassie blue stained SDS–PAGE gel (middle panel). Western blot (lower panel). Lanes 1 and 2: purified CD96-wt-scFv-IgG1-Fc protein, lane 3: molecular mass marker, lanes 4–6∶5, 2.5 and 1.25 µg of BSA. Data are representative of at least three experiments that were performed. <b>B</b>) Flow cytometric analysis: HSB-2, KG1 or CEM cells were incubated with 1) CD96-scFv-IgG1-Fc, 2) CD7-scFv-IgG1-Fc, and 3) TH-111. irrelevant mini-antibodies served as controls. <b>C</b>) Competition binding assay: 1) CD96-wt-scFv-IgG1-Fc, 2) CD96-wt-scFv-IgG1-Fc and TH-111, 3) CD96-wt-scFv-IgG1-Fc and TH-69. Representative experiments are presented.</p