The development of antidrug antibodies (ADAs) is a common cause for the failure of biotherapeutic treatments and adverse hypersensitivity reactions. Here we demonstrate that poly(lactic-co-glycolic acid) (PLGA) nanoparticles carrying rapamycin, but not free rapamycin, are capable of inducing durable immunological tolerance to co-administered proteins that is characterized by the induction of tolerogenic dendritic cells, an increase in regulatory T cells, a reduction in B cell activation and germinal centre formation, and the inhibition of antigen-specific hypersensitivity reactions. Intravenous co-administration of tolerogenic nanoparticles with pegylated uricase inhibited the formation of ADAs in mice and non-human primates and normalized serum uric acid levels in uricase-deficient mice. Similarly, the subcutaneous co-administration of nanoparticles with adalimumab resulted in the durable inhibition of ADAs, leading to normalized pharmacokinetics of the anti-TNFα antibody and protection against arthritis in TNFα transgenic mice. Adjunct therapy with tolerogenic nanoparticles represents a novel and broadly applicable approach to prevent the formation of ADAs against biologic therapies.

A Fourier transform infrared spectroscopic method has been developed to analyze protein secondary structure by employing the amide III spectral region (1350–1200 cm−1)· Benefits of using the amide III region have been shown to be substantial. The interference from the water vibration (∼1640 cm−1) in the amide I region can be avoided when one is using the amide III band; furthermore, the amide III region also presents a more characterized spectral feature which provides easily resolved and better defined bands for quantitative analysis. Estimates of secondary structure are accomplished with the use of Fourier self-deconvolution, second derivatization, and curve-fitting on original protein spectra. The secondary structure frequency windows (α-helix, 1328–1289 cm−1; unordered, 1288–1256 cm−1; and β-sheets, 1255–1224 cm−1) have been obtained, and estimates of secondary structural contents are consistent with X-ray crystallography data for model proteins and parallel results obtained with the use of the amide I region. We have further applied the analysis to the structural change of calsequestrin upon Ca2+ binding. Treatment of calsequestrin with 1 mM Ca2+ results in the formation of crystalline aggregates accompanied by a 10% increase in α-helical structure, which is consistent with previous results obtained by Raman spectroscopy. Thus the amide III region of protein IR spectra appears to be a valuable tool in estimating individual protein secondary structural contents.

Zn2+-protease activity of botulinum neurotoxin causes the blockage of neurotransmitter release resulting in botulism disease. We have investigated the role of Zn2+ in the biological activity of type A botulinum neurotoxin by removing the bound Zn2+ by EDTA treatment, followed by monitoring its structure in terms of secondary and tertiary folding (second derivative UV, FT-IR, and circular dichroism spectroscopy) and function in terms of its effect on the release of norepinephrine from PC12 cells. The single Zn2+ bound to each neurotoxin molecule was reversibly removed by EDTA treatment, whereas the biological activity of the neurotoxin was irreversibly lost. Based on the Amide III IR spectral analysis, the alpha-helical content of neurotoxin increased from 29% to 42% upon removal of Zn2+, which reverted to 31% upon treatment with 1:5 molar excess of exogenous Zn2+. Second derivative UV spectroscopy revealed no change in surface topography of Tyr residues with removal of Zn2+. However, near-UV circular dichroism signals suggested significant alterations in the topography of Phe and Tyr residues that could be buried in the protein matrix. Thermal unfolding experiments suggested that removal of Zn2+ results in the formation of the molten globule-like structure of type A botulinum neurotoxin. Tertiary structural changes introduced by Zn2+ removal were irreversible, which correlated well with the irreversibility of the biological activity of the neurotoxin. On the basis of these results, we suggest that Zn2+ plays a significant structural role in addition to its catalytic role in Zn2+-protease activity of type A botulinum neurotoxin.