Tozorakimab (MEDI3506): an anti-IL-33 antibody that inhibits IL-33 signalling via ST2 and RAGE/EGFR to reduce inflammation and epithelial dysfunction.

Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33) and oxidized IL-33 (IL-33) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products/epidermal growth factor receptor (RAGE/EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 10 M s, to effectively neutralize IL-33 following rapid release from damaged tissue.

NTF2 monomer-dimer equilibrium.

Nuclear transport factor 2 (NTF2) mediates nuclear import of RanGDP, a central component of many nuclear trafficking pathways. NTF2 is a homodimer and each chain has independent binding sites for RanGDP and nuclear pore proteins (nucleoporins) that contain FxFG sequence repeats. We show here that the monomer-dimer dissociation constant for NTF2 obtained by sedimentation equilibrium ultracentrifugation is in the micromolar range, indicating that a substantial proportion of cellular NTF2 may be monomeric.

Dissecting the interactions between NTF2, RanGDP, and the nucleoporin XFXFG repeats.

We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial for nuclear trafficking. Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the interaction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 microM range. Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP.

Influence of the P5 residue on alpha1-proteinase inhibitor mechanism.

The reactive center loop of native alpha1-proteinase inhibitor has been reported to be in a helical conformation and in a beta-strand conformation by two different studies. In the beta-strand loop structure the P5 glutamic acid plays a unique role by stabilizing the loop in the predicted optimal conformation for the interaction with target proteinases and insertion into beta-sheet A. We hypothesize here that disrupting the interactions that stabilize the beta-strand conformation of the loop would result in changes in the inhibitory properties of the serpin.

The P6-P2 region of serpins is critical for proteinase inhibition and complex stability.

Two of the prototypic serpins are alpha1-proteinase inhibitor and ovalbumin. alpha1-Proteinase inhibitor is a rapid inhibitor of a number of proteinases and undergoes the characteristic serpin conformational change on cleavage within the reactive center loop, whereas ovalbumin is noninhibitory and does not undergo the conformational change. To investigate if residues from P12 to P2 in the reactive center loop of ovalbumin are intrinsically incapable of being in an inhibitory serpin, we have made chimeric alpha1-proteinase inhibitor variants containing residues P12-P7, P6-P2, or P12-P2 of ovalbumin and determined their inhibitory properties with trypsin and human neutrophil elastase.