Expression of scFvFc on the surface of lentivirus transduced cells.

<p><i>Panel a</i> 293T cells were transduced with increasing dilutions (different MOIs as indicated) of lentivirus encoding the PS11-scFvFc-CD28-gp41-IRES-ZsGreen. Transduced cells were harvested, stained for Fc-surface expression, and analyzed by FACS. Expression of ZsGreen was measured to monitor levels of transduction. The graphs depict the percentage of transduced cells that express ZsGreen (blue diamonds) and the percentage values of transduced cells that express PS11-scFvFc as monitored by staining with APC-conjugated anti-human Fc IgG (pink squares). <i>Panels b</i> and <i>c</i>, cell -surface expressed scFvFc proteins are functional. 293T cells were transduced with a lentivirus encoding PS11-scFv-Fc-CD28-gp41-IRES-ZsGreen (<i>Panel b</i>) or 11A-scFvFc-CD28-gp41-IRES-ZsGreen (<i>Panel c</i>). Cells were incubated with biotinylated GD03-Fc, a specific antigen for 11A-scFvFc, and stained for streptavidin-APC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003181#s4" target="_blank">Methods</a>, and then analyzed by FACS. Note that two clusters of cells in <i>Panel c</i> represent high (R2 gated) and low (R3 gated) levels of 11A-scFvFc on their surface as measured by APC staining. These could reflect variations in cell-surface expression levels resulting from multiple integration events of the scFvFc cassette following transduction, or the difference in transgene integration site, <i>i.e.</i>, its proximity to active transcriptional units. R2 = 1390 and R3 = 220 are MFI values of 11A scFvFc expressing cells, where percentage of positive cells in each gate is 31% and 49% respectively. <i>Panel d.</i> a summary of specific antigen binding by the scFvFc displayed on the lentivirus transduced cells. The table shows the percentage of transduced cells expressing X48-scFvFc-CD28-gp41 or PS11 scFvFc-CD28-gp41 that bind to their cognate or irrelevant biotinylated antigens as visualized by APC staining and their corresponding MFI values.</p

Functional scFvFc are incorporated into lentivirus particles.

<p><i>Panel a.</i> An HIV-1 gp41 incorporation motif enhances scFvFc incorporation into viral particles-equal loads of lentiviruses encoding ZsGreen (lane 1), PS11-scFvFc-CD28-gp41-IRES ZsGreen (lane 2) or PS11-scFvFc-CD28-IRES ZsGreen (lane 3) were subjected to SDS-PAGE analysis, followed by western blotting using either HRP-conjugated anti-human Fc (upper panel) or anti-HIV-1 p24 (lower panel) antibodies. <i>Panel b.</i> Immunostaining of viruses. Viral particles were attached to Hela cells for 2 hour at 4°C. Cells were then fixed and stained with anti-HIV p24 antibody followed by Cy2-conjugated anti-mouse IgG or a rhodamine-conjugated anti-human Fc for surface Fc staining. Following these procedures, cells were washed and analyzed by confocal microscope. Shown separately are viruses stained for detection of p24 (image a) and scFvFc (image b) along with a merged image (image c). <i>Panel c.</i> Antigen specific capture of lentiviruses displaying corresponding scFvFc antibodies. Equal amounts of lentivirus particles expressing on their surface either the PS11-scFvFc or the 11A-scFvFc were loaded on a 96-well plate that was coated with the following specific antigens: TRM-peptide (PS11 specific), GD03-Fc (11A specific) or BSA. Following incubation to allow capture of the viruses to the antigens, wells were washed extensively and viral particles were eluted and quantitated by RT assay. Presented are normalized RT counts, where RT counts of particles bound to their antigens were divided by the RT counts of virus bound to the BSA control. Values are the average of duplicated samples and data are representative of two separate experiments.</p

Optimization of scFvFc cell-surface expression using different transmembrane domains.

<p>293T cells were transfected with the pcDNA 3.1 based constructs encoding PS11-scFvFc antibodies of different configurations as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003181#pone-0003181-g001" target="_blank">Figure 1</a> and labeled under each lane in Panels a and b. pcDNA3.1-CMV-GFP was co-transfected as an internal control for transfection efficiency. At 48 hours post transfection, cells were harvested and analyzed for GFP and scFv-Fc expression by FACS analysis. <i>Panels a</i> and <i>b</i>, represent results from FACS analysis of the percentage of cells that are positive for APC-anti-human Fc staining (<i>a</i>) and their respective MFI values (<i>b</i>). Error bars represent the standard deviation of the average of three experiments. <i>Panel c</i>. Cellular localization of the PS11-scFvFc-TM analyzed by confocal immunomicroscopy. 293T cells were transfected with either ZsGreen expression vector alone, or with a bicistronic vector expressing both the PS11 scFvFc-TM fusion proteins and ZsGreen. At 48 hours post transfection, cells were stained with a rhodamine-conjugated anti-human Fc for the detection of scFvFc expression as visualized by a confocal microscope. <i>Image a</i>, cells transfected with ZsGreen only vector; <i>Images b</i><i> and </i><i>c</i>, cells transfected with vectors expressing either PS11-scFvFc-gp41 (665–856)-IRES ZsGreen or PS11-scFvFc-CD28-gp41 (706–713)-IRES-ZsGreen, respectively. Absence of the ZsGreen fluorescence in some of the APC+ cells is likely the result of low level expression of ZsGreen from the second cassette of the bi-cistronic message. <i>Panel d</i>. PS11-scFv-CD28-gp41 is present as a dimer in transfected cells. 293T cells expressing pCDNA3.1-PS11-scFvFc-CD28-gp41 fusion protein were metabolically labeled with [³⁵S]-cysteine and [³⁵S]-methionine mixture. Cell lysates were immunoprecipitated with protein A sepharose beads, resuspended with 2× SDS non-reducing (lane 1) or reducing buffer (lane 2), and subjected to SDS-PAGE and autoradiogram.</p

Cell surface expressed scFvFc proteins bind their cognate antigens.

<p>293T cells were transfected with the same constructs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003181#pone-0003181-g002" target="_blank">Figure 2</a> and labeled under each lane in Panels a & b. Two additional constructs encoding antibodies against CXCR4, X20- and X48-scFvFc-CD28-gp41, were also transfected. pcDNA3.1-CMV-GFP was again co-transfected as an internal control for transfection efficiency. At 48 hours post transfection, cells were harvested and stained for biotinylated-TRM and streptavidin-APC, followed by FACS analysis. GFP expression was also analyzed to ensure equal transfection efficiencies. <i>Panel a</i> and <i>b</i>, depict the percentage of positive cells that express a functional PS11 scFvFc as determined by staining with streptavidin-APC (Panel a) and their respective MFI values (Panel b). Error bars represent the standard deviation of the average of three experiments. P values<0.05 above the designated bars, represent statistically significant difference in MFI values. <i>Panel c.</i> Post-translational sulfation occurs in selected surface displayed scFvFc antibodies. 293T cells expressing cell surface X48 or X20-scFcFc-CD28-gp41 fusion proteins (lanes 2 and 3, respectively) were labeled with [³⁵S]-cysteine and [³⁵S]-methionine mixture (upper panel; Cys/Met) or with [³⁵S]-sulfate (lower panel; SO₄) with or without 100 mM sodium chlorate treatment. Cell lysates were immunoprecipitated with protein A sepharose beads, washed and analyzed by SDS-PAGE and autoradiography. pcDNA3.1 backbone empty vector was also used as negative control (lane 1).</p

Selection of rare scFvFc expressing cells by a two-step magnetic bead and FACS sorting procedure.

<p>293T cells were transduced with either PS11-scFvFc-CD28-gp41-IRES-ZsGreen or 11A-scFvFc-CD28-gp41-IRES-ZsGreen encoding lentiviruses. These two transduced cell populations were mixed at different 11A- to PS11-scFvFc ratios with a total cell number of ∼10⁹. Mixed cells were incubated with the biotinylated GD03-Fc protein antigen and APC-streptavidin. APC-positive cells were subjected to an enrichment using anti-APC magnetic micro-beads followed by FACS sorting. Cells from either R2 gate (high APC expressing cells) or R3 gated (low APC expressing cells) were isolated, propagated, re-stained for biotin-GD03-Fc binding and also analyzed for their scFv gene content by PCR rescue and DNA sequencing. <i>Panel a</i>, the FACS dot-blot profiles of APC- and ZsGreen positive cells within the 11A∶PS11 scFvFc expressing cell population (initial mixing ratio at 1∶10⁶), before magnetic beads enrichment (left panel), following magnetic bead enrichment (middle panel), and the R2 gated cells after sorting and expansion (right panel). <i>Panel b</i>, DNA sequencing results of individual clones recovered from the R2 or R3 gated pools of cells sorted from different 11A∶PS11 scFvFc expressing cell ratios (1∶10³–1∶10⁶). Note that the irrelevant sequencing data are most likely originated from cloning background.</p

Neutralization of SARS-CoV TOR2 spike protein pseudotyped lentiviral infection of ACE2 expressing cells mediated by cell-surface displayed anti-TOR2 spike 80R-scFvFC antibodies.

<p>Single round, TOR2 spike protein pseudotyped luciferase expressing lentiviral particles were incubated with increasing concentrations of 293T cells expressing on their surface the 80R scFvFc (blue diamond) or the control PS11 scFvFc (pink circle). As a control for non-specific reporter virus absorption, a VSV-G pseudotyped luciferase reporter lentivirus was also incubated with 80R scFvFc (red triangle) or PS11 scFvFc expressing cells (brown square). Following incubation, the supernatant containing remaining lentivirus was used to infect permissive cells that express the ACE2 receptor for SARS CoV. At 48 hours post transduction, cells were harvested, luciferase activity was measured and relative inhibition of reporter virus infection was calculated. Asterisks in the designated points represent a statistical analysis that was performed to verify significant differences in % of inhibition between viral absorptions with the 80R- or PS11-scFvFc at a specific cell number point (P<0.05).</p

High-Affinity VEGF Antagonists by Oligomerization of a Minimal Sequence VEGF-Binding Domain

Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu²⁺ oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ∼50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ∼2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (∼5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets