CD spectra of peptides 1, 2, LCP-1 and LCP-2 in the presence of 10% of TFE.

<p>Spectra were acquired at 1 nm intervals from 260 nm to 190 nm, were the average of 3 individual scan and are reported in units of mean residue ellipticity [θ].</p

Comparison of antibody responses against peptides with adjuvant vs. LCP-peptides alone.

<p>(a) LCP-2 (A₂₉₁Y-GCN4-LCP) is more immunogenic than peptide 2 (A₂₉₁Y-GCN4 without LCP) formulated with Freund’s adjuvants. Antibody responses are depicted as OD values at a serum dilution of 1∶100 where each serum was used to probe its cognate immunogen peptide or LCP. (b) Higher antibody titres were observed in mice immunised with 60 µg of <b>LCP-2</b> without Freund’s adjuvants (experiment 2). Total IgG binding to plated <b>LCP-2</b> from individual mice on day 42 serial dilution from 1∶100 to 1∶10000. Each symbol represents a single animal.</p

Neutralisation of the enzymatic activity of recombinant <i>Na</i>-APR-1 using purified IgG from mice immunized with LCP-2.

<p>Relative fluorescence units (RFU) are corrected to enzyme-free wells which contained substrate alone. Percent inhibition of enzymatic activity with each IgG was determined by establishing the baseline fluorescence using negative control IgG.</p

The structure of peptide epitopes (1, 2) and LCP constructs (LCP-1 and LCP-2).

<p>Peptide epitope <b>2</b> retains native epitope sequence <b>1</b> (regular fonts) flanked by helicity inducing sequence from GCN4 protein (italic fonts).</p

Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging

Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate ∼14 small-molecule fluorophores (AlexaFluor 488) and 3–4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo <i>in vitro</i> and <i>in vivo</i>

Vascular Accessibility of Endothelial Targeted Ferritin Nanoparticles

Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10–50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core–shell structure with sizes of 20–25 nm and ∼4–5 Ab (or ∼7–9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers