Ultradilute Measurements of Self-Association for the Identification of Antibodies with Favorable High-Concentration Solution Properties.

There is significant interest in formulating antibody therapeutics as concentrated liquid solutions, but early identification of developable antibodies with optimal manufacturability, stability, and delivery attributes remains challenging. Traditional methods of identifying developable mAbs with low self-association in common antibody formulations require relatively concentrated protein solutions (>1 mg/mL), and this single challenge has frustrated early-stage and large-scale identification of antibody candidates with drug-like colloidal properties. Here, we describe charge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS), an affinity-capture nanoparticle assay that measures colloidal self-interactions at ultradilute antibody concentrations (0.01 mg/mL), and is predictive of antibody developability issues of high viscosity and opalescence that manifest at four orders of magnitude higher concentrations (>100 mg/mL). CS-SINS enables large-scale, high-throughput selection of developable antibodies during early discovery.

The Content of CpG-DNA in Antigen-CpG Conjugate Vaccines Determines Their Cross-Presentation Activity.

Cross-presentation, the process that facilitates display of exogenous antigens on MHC-I molecules, is a crucial step in the cascade of CD8 T cell activation. Potentiation of cross-presentation therefore represents an essential design criterion for development of subunit vaccines that target the induction of CD8 T cell immunity. Covalent conjugation of CpG-DNA to antigenic proteins has shown the potential to promote cross-presentation and has attracted great interest as a promising approach for vaccine development. However, heterogeneous product mixtures that result from typical conjugation schemes precluded identification of active conjugate species and impeded optimization of cross-presentation activity. In this report, we explore the effect of molecular composition of antigen-CpG conjugates on their cross-presentation activity using model Ovalbumin (OVA)-CpG conjugates. We developed a method to generate antigen-CpG conjugates with defined molecular compositions and leveraged this method to produce a series of OVA-CpG conjugates with one, two, and three CpG molecules linked to OVA. We observed that conjugates containing one CpG per OVA enhanced cross-presentation by 4-fold compared to native OVA, while conjugates with higher contents of CpG provided no cross-presentation enhancement. These differences are likely due to enhanced aggregation propensity observed for conjugates that carry more than one CpG per OVA. Our findings suggest that tuning molecular composition of antigen-CpG conjugates to maintain physical stability may be essential for achieving potent cross-presentation activity. Our method to generate defined conjugates could facilitate such molecular tuning and may be useful for continued development of antigen-CpG vaccines.

Size-Controlled Iron Oxide Nanoplatforms with Lipidoid-Stabilized Shells for Efficient Magnetic Resonance Imaging-Trackable Lymph Node Targeting and High-Capacity Biomolecule Display.

Nanoplatforms for biomolecule delivery to the lymph nodes have attracted considerable interest as vectors for immunotherapy. Core-shell iron oxide nanoparticles are particularly appealing because of their potential as theranostic magnetic resonance imaging (MRI)-trackable vehicles for biomolecule delivery. The key challenge for utilizing iron oxide nanoparticles in this capacity is control of their coating shells to produce particles with predictable size. Size determines both the carrier capacity for biomolecule display and the carrier ability to target the lymph nodes. In this study, we develop a novel coating method to produce core-shell iron oxide nanoparticles with controlled size. We utilize lipidlike molecules to stabilize self-assembled lipid shells on the surface of iron oxide nanocrystals, allowing the formation of consistent coatings on nanocrystals of varying size (10-40 nm). We further demonstrate the feasibility of leveraging the ensuing control of nanocarrier size for optimizing the carrier functionalities. Coated nanoparticles with 10 and 30 nm cores supported biomolecule display at 10-fold and 200-fold higher capacities than previously reported iron oxide nanoparticles, while preserving monodisperse sub-100 nm size populations. In addition, accumulation of the coated nanoparticles in the lymph nodes could be tracked by MRI and at 1 h post injection demonstrated significantly enhanced lymph node targeting. Notably, lymph node targeting was 9-40 folds higher than that for previously reported nanocarriers, likely due to the ability of these nanoparticles to robustly maintain their sub-100 nm size in vivo. This approach can be broadly applicable for rational design of theranostic nanoplatforms for image-monitored immunotherapy.