Internalisation of anti-MUC1 HT186-D11 and huHMFG1 IgGs.

<p>Internalisation was analysed by incubation of MCF-7 cells with anti-MUC1 antibodies at 37°C or on ice (on ice or 4°C, should be consistent throughout the paper) for 1h. Remaining cell surface-associated mAbs were detected by staining with PE-conjugated mouse anti-human IgG mab.</p

Flow cytometry analysis of anti-MUC1 IgG and scFv-Fc antibodies on different tumour cell lines HEK293T (A), SKOV3 (B) and MCF-7 (C).

<p>Different anti-MUC1 antibody concentrations were incubated on two different MUC1 positive cell lines and one MUC1 negative cell line. Detection was performed as given for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015921#pone-0015921-g008" target="_blank">figure 8B</a>.</p

Overview about the constructed IIB6 mutation libraries.

<p>Overview about the constructed IIB6 mutation libraries.</p

Activity studies performed in xenograft models.

<p>(A) Mean tumour volume curves observed in the MCF model. Starting on day 27, mice were IP injected with irrelevant antibody LFB-R297 or HT186-D11 at 10 mg/kg weekly and for a total of 4 weeks. These treatments were performed alone or in combination with IV injections of Taxol at 7.5 mg/kg injected once every week for 3 consecutive weeks. (B) Survival curves observed in the NIH:OVCAR-3 model. Starting on day 13, mice were IP injected with irrelevant antibody LFB-R297 or HT186-D11 at 10 mg/kg weekly and for a total of 4 weeks. These treatments were performed alone or in combination with IV injections of Taxol at 7.5 mg/kg injected once every week for 3 consecutive weeks.</p

Analysis of the IHC staining with non tumour tissues.

<p>Eight slides of each tissue type were analysed.</p><p>*suspected reaction of the detection system with endogenous biotin.</p

Size exclusion chromatography analysis to analyse the dimerisation tendency of the anti-MUC1 scFvs.

<p>80 µg purified scFv fragments were separated on a Superdex200 10/300 column using PBS as running buffer with a flow rate of 0.5 mL min⁻¹. The UV-absorption (A280) was drawn against the retention volume.</p

IIB6 binding to MUC1. MUC1 negative fibroblast, MUC1 positive T47D cells and MUC1 preparations (BMA) were separated by 7.5% SDS-PAGE and Western-blotted.

<p>The blot was stained with 5 µg/ml IIB6, mouse anti-his tag (1∶1000) and goat anti-mouse IgG (Fab specific) HRP conjugate (1∶5000)</p

Epitope mapping.

<p><b>A</b> The epitope mapping membrane (15mer oligopeptide, 1 amino acid overlap) was stained with 30 µg scFvs. The bound scFvs were detected with mouse anti-c-myc IgG (9E10) (1∶500) and a goat anti-mouse IgG (Fab spec.) AP conjugate (1∶2000). NWS  =  detection antibodies only. <b>B</b> Sequence overview. Amino acid sequences of the single spots on the nitrocellulose membrane. Immunostained spots were marked in grey. Amino acids forming the minimal epitope are given in bold. The sequence of one complete VNTR repetitive region is underlined.</p

Germinality index and humanness (Z-score) for the variable region of the anti-MUC1 scFvs.

<p>Germinality index is calculated by aligning the amino acid sequence of the variable region to the next human germline sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015921#pone.0015921-Pelat3" target="_blank">[62]</a>. Z-scores were calculated using the SHAB web interface (<a href="http://www.bioinf.org.uk/abs/shab/" target="_blank">http://www.bioinf.org.uk/abs/shab/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015921#pone.0015921-Abhinandan1" target="_blank">[61]</a>.</p

Comparison of scFv amino acid sequences of IIB6 and affinity matured anti-MUC1 scFvs.

<p>Differences in amino acid sequences are given by the corresponding amino acid in the table (single letter code). * represents a silent point mutation leading to no change in amino acid sequence. The upper panel shows the VH alignment, the middle pannel shows the VL alignment and the bottom pannel shows the alignment of the linker sequences between VH and VL, consisting of the N-terminal part of CH1 and the yol epitope, and the N-terminal part of CL downstream of VL. s.a.  =  see above.</p