Analysis of binding of the SL ScBsDb/linker complex to DD2 positive Jurkat cells.

<p>(A), (a, black graph) IgM isotype negative control. (a, red graph) Binding of the anti-slan IgM mab DD2 to slan(+) positive Jurkat cells. (b, black graph) Anti-penta-HIS, isotype negative control. (b, red graph) Lack of binding of the monovalent SL scBsDb (SL) in the absence of a linker peptide. (c,d black graph) Isotype negative control. (c,d, red graph) Bivalent or trivalent anti-slan scaffolds consisting of the SL scBsDb and the linker peptide l-GFP-l (c, red graph) or l-l-GFP-l (d, red graph). (B), Detection of anti-slan scaffolds on the surface of Jurkat cells by epifluorescence microscopy. GFP (a), phase contrast (b), bar = 10 µm.</p

Analysis for proliferation of CD4+ T cells after delivery of the TT-derived T-cell epitope to slanDCs via the novel immuno targeting system.

<p>(A) CFSE labeled PBMCs were prepared and incubated at 37°C for eight days with either full length TT (b) or the linker module containing (d) or lacking (c) the TT-derived T-cell epitope TT_p and analysed by FACS. The data indicate that PBMCs of the selected donor contain anti-TT memory T cells that can be recalled by full length TT and to a less extent by the TT_p peptide linker but not by the linker peptide lacking the TT_p epitope. Untreated PBMCs (a). (B) CFSE labelled PBMCs were incubated with either the antigen-containing scaffold (c) or the single components (a,b) at 4°C. Unbound material was removed by washing.</p

Immuno targeting of slan(+) Jurkat cells with a multivalent anti-slan scaffold containing a TT-derived T-cell epitope.

<p>(a, black graph) Binding of the anti-slan mab DD2 to slan(+) Jurkat cells. (c,d, red graph) Binding of anti-slan scaffolds consisting of the SL scBsDb and trivalent linker peptides either containing (c) or lacking (d) theTT-derived peptide epitope TT_p. (b,c,d, black graph) Control binding of the single components of the anti-slan scaffolds including the SL scBsDb (b), the trivalent linker l-l-GFP-l (c), and the trivalent linker l- TT_p -l-GFP-l (d).</p

Characterisation of purified linker peptides.

<p>For the formation of bi- or trivalent anti-slan scaffolds peptide linker molecules were constructed containing the l-Tag twice (l-GFP-l) or three times (l-l-GFP-l; l-TT_p-l-GFP-l). The purified peptide linkers were analysed by SDS-PAGE/immunoblotting using the anti-La mab 7B6 directed to the l-Tag. (M) Marker proteins.</p

Construction and characterisation of the SL scBsDb.

<p>(A) A schematic view of the structure and the complete AA sequence of the SL scBsDb. (B) The SL scBsDb was expressed and purified as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016315#s4" target="_blank">Materials and Methods</a>. The flow through fractions, wash fractions (wash1, wash2) and the eluted fractions (elution 1, elution 2) containing the SL scBsDb were analyzed by SDS-PAGE (C) and immunoblotting (B). After SDS-PAGE separated protein(s) were stained with Coomassie brilliant blue (C, CBB), and after transfer the membrane was developed with an anti-penta-HIS antibody (B). M, marker proteins.</p

Binding of the SL scBsDb/linker complex to native slanDCs in PBMCs.

<p>(A) (a) Estimation of the amount of slan-DCs in a PBMC sample using the anti-slan IgM mab DD2. (d) Estimation of the amount of slanDCs in the same PBMC sample using the anti-slan scaffold consisting of the SL scBsDb and the l-l-GFP-l linker peptide. Control staining of the single components of the anti-slan scaffold including the SL scBsDb (b, SL) or the peptide linker module (c, l-l-GFP-l). (B) GFP-labelled anti-slan scaffolds identified by epifluorescence microscopy. GFP (a), phase contrast (b), bar = 10 µm.</p

Recombinant antibodies derived from IgM antibodies: Restoring avidity by oligomerisation on a modular peptide scaffold.

<p>(A) The IgM anti-slan mab DD2 is a pentameric molecule. Preparation of a scFv (B) resulted in a monovalent molecule which failed to bind to slanDCs (C, data not shown). For recovery of binding avidity a scBsDb (D, SL scBsDb) and suitable linking peptide molecules (F,G) were constructed (H,I). In the scBsDb the variable heavy and light chain domains of the two mabs (A, DD2, S; E, 7B6, L) are recombinantly fused via glycine serine linkers (D; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016315#pone-0016315-g003" target="_blank">Fig. 3</a>). This SL scBsDb is on the one hand directed to the slan epitope and on the other hand to a continuous peptide epitope (l-Tag). The linker peptide modules contain the l-Tag either two times (l-GFP-l, F) or three times (l-l-GFP-l, G(i); l-TT_p-l-GFP-l, G(ii)). Binding of the SL scBsDb to the respective linker peptides results in the formation of a divalent (H) or trivalent (I) anti-slan scaffold with increased avidity.</p

Binding of the SL scBsDb/linker complex to slan(+) Jurkat cells.

<p>(A) Anti-slan scaffolds were obtained by incubation of the SL scBsDb (SL) with the trivalent linker peptide l-l-GFP-l or the bivalent linker peptide (l-GFP-l, data not shown) at various ratios as indicated and analyzed for binding to slan(+) Jurkat cells. Binding of the maternal IgM anti-slan antibody DD2 was set as 100%. (B) After estimation of the optimal conditions, the binding of the bivalent (SL + l-GFP-l) and trivalent anti-slan scaffolds (SL+l-l-GFP-l) were compared to the binding of the maternal anti-slan IgM antibody DD2 (DD2 IgM). Moreover, the binding of the scaffolds in the presence of the maternal anti-slan IgM antibody DD2 was determined (DD2 IgM+SL+l-l-GFP-l).</p

Stability of immune complexes formed via the slan epitope on isolated slanDCs.

<p>(A), (a, black graph) Staining of slanDCs freshly isolated by magnetic anti-IgM/MDC8 anti-slan beads with a secondary FITC-conjugated anti-mouse IgM antibody. (a, red graph) Freshly isolated slanDCs that are stained in addition with the anti-slan IgM mab DD2 and the secondary FITC-conjugated anti-mouse IgM antibody. (b, black graph) Staining of slanDCs with a secondary FITC-conjugated anti-mouse IgM antibody four hrs after isolation and an incubation at 37°C. (b, red graph) Staining of slanDCs with the anti-slan IgM mab DD2 and the secondary FITC-conjugated anti-mouse IgM antibody four hrs after isolation and an incubation at 37°C. (B), (a, black graph) Binding of the multivalent anti-slan scaffold is blocked to freshly isolated slan DCs. (b, black graph) Binding of the multivalent anti-slan scaffold consisting of the SL scBsDb (SL) and the l-TT_p-l-GFP-l linker module regains four hrs after isolation and an incubation at 37°C. (c, black graph) Staining of slan DCs after an additional incubation of 2.5 h at 37°C. The scBsDb/peptide linker scaffold can no more be detected on the surface of slanDCs.</p