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>

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in purified muPECAM.

<p>Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in purified muPECAM.</p

Anti-muPECAM-1 [125I]-mAb binding in cellular homogenates of live cells is enhanced by paired mAb.

<p>Binding parameters of anti-muPECAM-1 mAbs (A) Mec13.3 and (B) 390 in cellular homogenates of live cells stably expressing muPECAM. Membrane preparations were added to antibody cocktail [125I]-mAb solo or with paired mAb and incubated for 2h, followed by filtering through Millipore Multiscreen Filter Plates using vacuum manifold. The results are presented as total binding corrected for nonspecific binding on membrane preparations of REN-WT cells. Kd of Mec13.3 sees 0.77 ± 0.02-fold change in solo vs paired. Notably, Bmax values increase for both mAbs Mec13.3 and 390 co-incubated with a paired mAb. The insets show Scatchard analysis of experiments.</p

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in cellular homogenates of live cells stably expressing muPECAM-1.

<p>Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in cellular homogenates of live cells stably expressing muPECAM-1.</p

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb 390 in live cells.

<p>Binding curves of anti- [125I]-mAb 390 alone (“solo”) or in the presence of unlabeled mAb Mec13.3 to paired epitope (”paired”) in live cells stably expressing recombinant muPECAM-1 (RmP) (A) and mutant form of muPECAM-1 (RmPK89A) (B) determined by RIA-based method. Increasing concentrations of [125I]-mAb treatments solo or with a paired mAb were added to cells and incubated at 4°C for 2h. The results are presented as total binding corrected for nonspecific binding on REN-WT cells. The effect of co-incubation with paired mAb vs solo was not significant. Yet, Kd changed 0.86 ±0.07-fold in RmP and 0.83±0.05 in RmPK89A. The insets show Scatchard analysis of experiments.</p

Single-Walled Carbon Nanotubes Deliver Peptide Antigen into Dendritic Cells and Enhance IgG Responses to Tumor-Associated Antigens

We studied the feasibility of using single-wall carbon nanotubes (SWNTs) as antigen carriers to improve immune responses to peptides that are weak immunogens, a characteristic typical of human tumor antigens. Binding and presentation of peptide antigens by the MHC molecules of antigen presenting cells (APCs) is essential to mounting an effective immune response. The Wilm’s tumor protein (WT1) is upregulated in many human leukemias and cancers and several vaccines directed at this protein are in human clinical trials. WT1 peptide 427 induces human CD4 T cell responses in the context of multiple human HLA-DR.B1 molecules, but the peptide has a poor binding affinity to BALB/c mouse MHC class II molecules. We used novel, spectrally quantifiable chemical approaches to covalently append large numbers of peptide ligands (0.4 mmol/g) onto solubilized SWNT scaffolds. Peptide-SWNT constructs were rapidly internalized into professional APCs (dendritic cells and macrophages) within minutes <i>in vitro</i>, in a dose dependent manner. Immunization of BALB/c mice with the SWNT–peptide constructs mixed with immunological adjuvant induced specific IgG responses against the peptide, while the peptide alone or peptide mixed with the adjuvant did not induce such a response. The conjugation of the peptide to SWNT did not enhance the peptide-specific CD4 T cell response in human and mouse cells, <i>in vitro</i>. The solubilized SWNTs alone were nontoxic <i>in vitro</i>, and we did not detect antibody responses to SWNT <i>in vivo</i>. These results demonstrated that SWNTs are able to serve as antigen carriers for delivery into APCs to induce humoral immune responses against weak tumor antigens

Anti-muPECAM-1 [125I]-mAb binding to purified muPECAM is enhanced by paired mAb.

<p>Binding parameters of anti-muPECAM-1 mAbs (A) Mec13.3 and (B) 390 with recombinant purified muPECAM-1. Millipore Multiscreen filter plates were coated with 5 μg/ml of recombinant muPECAM-1 and incubated for 2 h at 37°C. Radioligand binding assay was performed as described for cellular homogenates. The results are presented as total binding corrected for nonspecific binding on Filter Plates coated with 1% BSA. Kd of Mec13.3 sees 0.53 ± 0.03-fold change in solo vs paired. Again, Bmax values increase for both mAbs Mec13.3 and 390 co-incubated with a paired mAb. The insets show Scatchard analysis of experiments.</p

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in live cells stably expressing full-length (RmP) and mutant forms (RmP_K89A) of muPECAM-1.

<p>Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in live cells stably expressing full-length (RmP) and mutant forms (RmP_K89A) of muPECAM-1.</p