Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes.

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.

Trans-cyclooctene tag with improved properties for tumor pretargeting with the diels-alder reaction.

Radioimmunotherapy (RIT) of solid tumors is hampered by low tumor-to-nontumor (T/NT) ratios of the radiolabeled monoclonal antibodies resulting in low tumor doses in patients. Pretargeting technologies can improve the effectiveness of RIT in cancer therapy by increasing this ratio. We showed that a pretargeting strategy employing in vivo chemistry in combination with clearing agents, proceeds efficiently in tumor-bearing mice resulting in high T/NT ratios. A dosimetry study indicated that the chemical pretargeting technology, which centered on the bioorthogonal Diels-Alder click reaction between a radiolabeled tetrazine probe and a trans-cyclooctene-oxymethylbenzamide-tagged CC49 antibody (CC49-TCO(1)), can match the performance of clinically validated high-affinity biological pretargeting approaches in mice ( Rossin J Nucl Med. 2013 , 54 , 1989 - 1995 ). Nevertheless, the increased protein surface hydrophobicity of CC49-TCO(1) led to a relatively rapid blood clearance and concomitant reduced tumor uptake compared to native CC49 antibody. Here, we present the in vivo evaluation of a TCO-oxymethylacetamide-tagged CC49 antibody (CC49-TCO(2)), which is highly reactive toward tetrazines and less hydrophobic than CC49-TCO(1). CC49-TCO(2) was administered to healthy mice to determine its blood clearance and the in vivo stability of the TCO. Next, pretargeting biodistribution and SPECT studies with CC49-TCO(2), tetrazine-functionalized clearing agent, and radiolabeled tetrazine were carried out in nude mice bearing colon carcinoma xenografts (LS174T). CC49-TCO(2) had an increased circulation half-life, a 1.5-fold higher tumor uptake, and a 2.6-fold improved in vivo TCO stability compared to the more hydrophobic TCO-benzamide-CC49. As a consequence, and despite the 2-fold lower reactivity of CC49-TCO(2) toward tetrazines compared with CC49-TCO(1), administration of radiolabeled tetrazine afforded a significantly increased tumor accumulation and improved T/NT ratios in mice pretargeted with CC49-TCO(2). In conclusion, the TCO-acetamide derivative represents a large improvement in in vivo Diels-Alder pretargeting, possibly enabling application in larger animals and eventually humans.

Inhibitory effects of Schizandrae Fructus on eotaxin secretion in A549 human epithelial cells and eosinophil migration.

Eosinophilia have been implicated in a broad range of diseases, most notably allergic conditions (e.g. asthma, rhinitis and atopic dermatitis) and inflammatory diseases. These diseases are characterized by an accumulation of eosinophils in the affected tissue. Defining the mechanisms that control the recruitment of eosinophil is fundamental to understanding how these diseases progress and identifying a novel target for drug therapy. Accordingly, this study was conducted to evaluate the regulatory effect of Schizandrae Fructus (SF) on the expression of eotaxin, an eosinophil-specific chemokine released in respiratory epithelium following allergic stimulation, as well as its effects on eosinophil migration. To accomplish this, human epithelial lung cells (A549 cell) were stimulated with a combination of TNF-alpha (100ng/ml) and IL-4 (100ng/ml) for 24h. The cells were then restimulated with TNF-alpha (100ng/ml) and IL-1beta (10ng/ml) to induce the expression of chemokines and adhesion molecules involved in eosinophil chemotaxis for another 24h. Next, the samples were treated with various concentrations of Schizandrae Fructus (SF) (1, 10, 100, 1000microg/ml) or one of the major constituents of SF, schizandrin (0.1, 1, 10, 100microg/ml), after which following inhibition effect assay was performed triplicates in three independence. The levels of eotaxin in secreted proteins were suppressed significantly by SF (100 and 1000microg/ml, p<0.01) and schizandrin (10 and 100microg/ml, p<0.01). In addition, SF (1, 10, 100 and 1000microg/ml) decreased mRNA expression levels in A549 cells significantly (p<0.01). Eosinophil recruitment to lung epithelial cells was also reduced by SF, which indicates that eotaxin plays a role in eosinophil recruitment. Furthermore, treatment with SF suppressed the expression of another chemokine, IL-8 (0.1 and 1microg/ml SF, p<0.01), as well as intercellular adhesion molecule-1 (10 and 100microg/ml SF, p<0.01) and vascular cell adhesion molecule-1 (0.1 and 1microg/ml SF, p<0.05), which are all related to eosinophil migration. Taken together, these findings indicate that SF may be a desirable medicinal plant for the treatment of allergic diseases.