Adapting Ferritin, a Naturally Occurring Protein Cage, to Modulate Intrinsic Agonism of OX40

Abstract

Ferritin is a multivalent, self-assembling protein scaffold found in most human cell types, in addition to being present in invertebrates, higher plants, fungi, and bacteria, that offers an attractive alternative to polymer-based drug delivery systems (DDS). In this study, the utility of the ferritin cage as a DDS was demonstrated within the context of T cell agonism for tumor killing. Members of the tumor necrosis factor receptor superfamily (TNFRSF) are attractive targets for the development of anticancer therapeutics. These receptors are endogenously activated by trimeric ligands that occur in transmembrane or soluble forms, and oligomerization and cell-surface anchoring have been shown to be essential aspects of the targeted agonism of this receptor class. Here, we demonstrated that the ferritin cage could be easily tailored for multivalent display of anti-OX40 antibody fragments on its surface and determined that these arrays are capable of pathway activation through cell-surface clustering. Together, these results confirm the utility, versatility, and developability of ferritin as a DDS.

Figure 1

(A) Deconvolution from LC/MS analysis after digestion using TEV protease, with a zoomed-in view in the bottom panel. Identified masses have been assigned. (B) Size exclusion chromatography in-line with a quasi-elastic light scattering detector (SEC-QELS) of rFer₂₄ with His-tag (red trace) and after TEV protease digestion (blue trace), as well as rFer₂₄-anti-OX40 Fab conjugate (1) and unconjugated Fab (2) from reaction 1 (green trace), overlaid with measured R_H values across each peak. Averaged R_H values are also reported in the legend. (C) Physical stability profiles for rFer₂₄ and rFer₂₄-anti-OX40 Fab₂₄ were measured in phosphate-buffered saline (PBS) at 25 °C over 14 days.

Figure 2

(A) Reaction scheme showing rFer₂₄ in magenta (Q-tag in red), anti-OX40 Fab (K-tag in cyan), and assembled rFer₂₄ + 24 Fabs. (B) Chromatogram of absorbance at 280 nm demonstrating resolution by reverse phase (RP) between unreacted anti-OX40 Fab (1.7 min), rFer_m-anti-OX40 Fab (2 min), rFer_m (2.3 min), and rFer_m with N-term His-tag (2.9 min) using a molar ratio of 1:0.3. (C) Representative deconvolution (top panel) with a zoomed-in view (bottom panel) around the mass of the rFer_m-anti-OX40 Fab conjugate and other minor conjugate masses.

Figure 3

PYMOL rendering of rFer₂₄-anti-OX40 Fab₂₄ from the homology model PDB file, where the rFer₂₄ cage is in magenta and 24 repeats of the Fab moiety are in green.

Figure 4

(A) Representative 2D class averages of negatively stained rFer-OX40 and Fab₂₄ conjugates. (B) Representative 2D class averages of rFer-OX40 Fab particles were imaged using cryo-EM. (C) Back projections of the homology model are presented in Figure 3.

Figure 5

OX40+ Jurkat reporter assay. RLU denotes relative luminescence. Molecule concentrations (nM) were calculated as follows: rFer₂₄-Fab conjugate and aOX40 Fab concentrations were based on Fab molar mass, OX40L-Fc XL concentration was based on OX40L-Fc XL molar mass, aOX40 IgG1 concentration was based on the antibody molar mass, and rFer₂₄ concentration was based on the molar mass of the assembled ferritin cage.