Antigen footprint governs activation of the B cell receptor.

Antigen binding by B cell receptors (BCR) on cognate B cells elicits a response that eventually leads to production of antibodies. However, it is unclear what the distribution of BCRs is on the naïve B cell and how antigen binding triggers the first step in BCR signaling. Using DNA-PAINT super-resolution microscopy, we find that most BCRs are present as monomers, dimers, or loosely associated clusters on resting B cells, with a nearest-neighbor inter-Fab distance of 20-30 nm. We leverage a Holliday junction nanoscaffold to engineer monodisperse model antigens with precision-controlled affinity and valency, and find that the antigen exerts agonistic effects on the BCR as a function of increasing affinity and avidity. Monovalent macromolecular antigens can activate the BCR at high concentrations, whereas micromolecular antigens cannot, demonstrating that antigen binding does not directly drive activation. Based on this, we propose a BCR activation model determined by the antigen footprint.

Tumor-targeted polydiacetylene micelles for in vivo imaging and drug delivery.

In vivo tumor targeting and drug delivery properties of small polymerized polydiacetylene (PDA) micelles (∼10 nm) is investigated in a murine MDA-MB-231 xenograft model of breast cancer. Three micelles with different surface coatings are synthesized and tested for their ability to passively target tumor through the enhanced permeability and retention effect. After injection (24 h), fluorescence diffuse optical tomographic imaging indicates a tumor uptake of nearly 3% of the injected dose for the micelles with a 2 kDa poly(ethylene glycol) (PEG)-coating (PDA-PEG2000). The uptake of PDA micelles in tumors is confirmed by co-localization with [(18) F]-fluorodeoxyglucose (FDG) positron emission tomography. Although FDG has a higher diffusion rate in tumors, 40 ± 19% of the retained micelles is co-registered with the tumor volume visualized by FDG. Finally, PDA-PEG2000 micelles are loaded with the hydrophobic anticancer drug paclitaxel and used in vivo to inhibit tumor growth. These findings demonstrate the potential of PDA-PEG2000 micelles for both in vivo tumor imaging and drug delivery applications.

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding.

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2'-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.

Biochemical properties of plasminogen activator inhibitor-1.

PAI-1 is a Mr ~50,000 glycoprotein, which is the primary physiological inhibitor of the two plasminogen activators uPA and tPA. PAI-1 belongs to the serpin protein family. Studies of PAI-1 have contributed significantly to the elucidation of the protease inhibitory mechanism of serpins, which is based on a metastable native state becoming stabilised by insertion of the RCL into the central beta-sheet A and formation of covalent complexes with target proteases. In PAI-1, this insertion can occur in the absence of the protease, resulting in generation of a so-called latent, inactive form of the protein. PAI-1, in its active state, also binds to the extracellular protein vitronectin. When in complex with its target proteases, it binds with high affinity to endocytosis receptors of the low density receptor family.