Sequences of RT-PCR primers.

<p>Sequences of RT-PCR primers.</p

Binding of (VH434)₂-Fc, (VH4127)₂-Fc, Fc and (VH4Sc)₂-Fc to the hLDLR.

<p>Representative confocal fluorescence micrograph of CHO-hLDLR-GFP cells incubated with 10 nM of (VH434)₂-Fc (a), (VH4127)₂-Fc (b) and Fc alone (c) used as a negative control. The Fc and Fc-fusion/conjugates were detected with an anti-hFc Alexa 594-conjugated antibody (red). Co-labeling of LDLR-GFP (green) and Fc fusion/conjugates appear in yellow in the merged pictures, as evidenced at higher magnification. (B) ELISA quantification of bound/endocytosed (VH4127)₂-Fc or (VH4Sc)₂-Fc conjugates to CHO-hLDLR-GFP cells 1 hr post-incubation at 10 nM (n = 3 per conjugate; ***p ≤ 0.001). Note that (VH4127)₂-Fc binding/endocytosis to the CHO-hLDLR-GFP cells is increased ≈300-fold compared to the (VH4Sc)₂-Fc control. (C) Representative sensorgrams of Fc-fusion/conjugate binding to the extracellular domain of hLDLR. A set of concentrations (0.5–80 nM for the Fc-conjugates or 5–80 nM for the Fc-fusion) was sequentially injected over immobilized hLDLR. The solid lines represent the specific binding of each Fc-fusion/conjugate and the dotted lines represent the fit of the data obtained with a Langmuir 1:1 model.</p

Membrane expression and functionality of hLDLR expressed by the CHO-hLDLR-EGFP cell line.

<p>(A) Representative confocal photomicrographs of immunostained CHO-hTfR-EGFP (negative control) and CHO-hLDLR-EGFP cells (green) with an anti-LDLR antibody diluted at 1/100 (red) following triton X100 permeabilization of the cell membrane. Cell nuclei are labeled with Hoechst#33258 at 0.5 μg/mL (blue). Co-labeling appears in yellow in the merged pictures. Only the cells with stable expression of the hLDLR-EGFP construct express the membrane receptor. (B) Representative confocal photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) after a 10 min incubation period with DiI-LDL 15 μg/mL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. DiI-LDL is essentially bound to the CHO-hLDLR-EGFP cells indicating that the hLDLR-EGFP chimera receptor binds its ligand. (C) Western blots performed on cell membrane preparations of CHO cells expressing hLDLR-EGFP, rLDLR-EGFP and mLDLR-EGFP compared to CHO WT, using anti-hLDLR (1/800) and anti-rat and mouse LDLR antibodies (1/1000). These bands are also labeled with an anti-EGFP antibody. Bands of 140 kDa and 190 kDa correspond respectively to the immature and mature LDLR-EGFP fusion proteins (arrows). Actin was used to check loading of equal amounts of protein.</p

Interaction of VH4127 conjugates with hLDLR and comparison with ApoB and ApoE peptides.

<p>Representative confocal fluorescence micrographs of CHO-hLDLR-GFP cells incubated with 10 μM of VH4127-Cy5.5 (light blue) and 20μg/mL of DiI-LDL (red). Insets correspond to higher magnification of boxed cells. (B) CHO-hLDLR-GFP cells incubated with 10 μM of VH4Sc-Cy5.5 and 20μg/mL of DiI-LDL (red). (C) CHO-hLDLR-GFP cells incubated with 10μM of VH4127-siGLO Cyclophiline B (red). (D) CHO-hLDLR-GFP cells incubated with 10μM of VH4127-S-Tag (light blue) following S-Tag detection with a goat anti-S-Tag and a secondary A647-conjugated anti-Goat antibody. Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. Note that all conjugates bind and largely co-localize with LDLR. (E) ELISA quantification of bound/endocytosed VH4127-S-Tag or VH4Sc-S-Tag conjugates to CHO-hLDLR-GFP cells 1 hr post-incubation at 10μM. The graph represents the mean of concentration in nM ± SD (n = 3 per conjugate, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (F) Image J quantification of the percentage of co-localization ± SD (n = 3 per peptide) between hLDLR-GFP (blue graph bars) or hTfR-GFP (orange graph bars) and ApoB, ApoE1, ApoE2 and VH4127 peptides conjugated to the S-Tag (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). The percentage of co-localization on the hLDLR-GFP (blue graph bars) between the ApoB, ApoE1 and ApoE2 peptides conjugated to the S-Tag was also compared to that of VH4127-S-Tag (### p ≤ 0.001).</p

Sequences of LDLR targeting peptides presented by phage.

<p>Sequences of LDLR targeting peptides presented by phage.</p

Characterization of VH411-S-Tag peptide conjugate binding to the CHO-hLDLR-EGFP cell line.

<p>(A) Representative confocal photomicrographs of CHO-hLDLR-EGFP and CHO-hTfR-EGFP cells (green) incubated 1 hr at 37°C with 10 μM of VH411-S-Tag detected post-fixation with an anti-S-Tag and A647-conjugated secondary antibody (cyan) and 10 μg/mL of DiI-LDL. Cell nuclei are labeled with Hoechst#33258 (blue) at 0.5 μg/mL. Co-labeling appears in blue-green/orange in the merged pictures. Note that VH411-S-Tag co-localizes with hLDLR and is internalized by CHO-hLDLR-EGFP cells. (B) Representative confocal photomicrographs of CHO-hLDLR-EGFP cells (green) incubated 1 hr at 37°C with a macromolecular complex (see scheme Fig 3B) resulting from the co-incubation and interaction of 10 μM VH411-S-Tag peptide or VH411Sc-S-Tag peptide with the anti-S-Tag antibody (1/200) and the Alexafluor 647-conjugated secondary antibody (1/800) (cyan). Cells were concomitantly exposed to 10 μg/mL of DiI-LDL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in blue-green/orange in the merged pictures.</p

Sequences and K_D of LDLR targeting peptides, free or conjugated/fused to different molecules.

<p>Sequences and K_D of LDLR targeting peptides, free or conjugated/fused to different molecules.</p

<i>In vitro</i> validation of VH411 phage binding.

<p>(A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10¹⁰ VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.</p

Identification of the hLDLR domain that interacts with VH0445-S-Tag.

<p>(A) A pull-down assay was performed in concentrated supernatant of HEK cells transiently transfected with p-Fc as control or with phLDLR-Fc vector, using epoxy beads coated with VH0445-S-Tag or with VH4Sc-S-Tag control peptide (scrambled version of VH445). Western blots were carried out using anti-Fc (A1) and anti-hLDLR antibodies (A2). Note that the hLDLR is pulled-down by the beads coated with the VH0445-S-Tag peptide but not with the VH4Sc-S-Tag peptide. (B) Schematic representation of truncated LDLR (hLDLR-FL, hLDLR-ΔLBD, hLDLR-ΔEGF and hLDLR-ΔECD, all encompassing a HA Tag to verify appropriate extracellular distribution of the extracellular domains) produced by cells transfected with different plasmid constructs derived from hLDLR-pEGFP. (C) VH445-S-Tag binding to the hLDLR EGF precursor homology domain. Representative fluorescence micrographs of CHO cells transiently expressing hLDLR-FL, hLDLR-ΔLBD, hLDLR-ΔEGF and hLDLR-ΔECD (all in green) incubated with 10 μM of VH0445-S-Tag, goat Anti-S-Tag (1/200) and anti-goat Alexa 594 (1/800) (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow/orange in the merged pictures.</p

Optimization and <i>in Vivo</i> Validation of Peptide Vectors Targeting the LDL Receptor

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., <i>J. Med. Chem.</i> <b>2012</b>, <i>55</i> (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL­[CMPRLRGC]_cDPR) and structure–activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]_c) which specifically binds hLDLR with a <i>K</i>_D of 76 nM and has an <i>in vitro</i> blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-“Pen”M“Thz”RLRGC]_c), which showed the highest <i>K</i>_D value of 9 nM. However, this latter analogue displayed the lowest <i>in vitro</i> blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 10⁶ s^–1.M^–1 range, and off-rates varying from the low 10^–2 s^–1 to the 10^–1 s^–1 range. Furthermore, all these new analogues showed increased blood half-lives <i>in vitro</i>, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM“Thz”RLRG“Pen”]_c), showing a <i>K</i>_D of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled ³H-VH4127 in wild-type or <i>ldlr</i> −/– mice confirmed their active LDLR targeting <i>in vivo</i>. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential <i>in vivo</i>