In Situ Ligation of High‐ and Low‐Affinity Ligands to Cell Surface Receptors Enables Highly Selective Recognition

Abstract

molecules that are highly expressed in tumor vasculature [11] are widely used to image breast, [15] brain, [16] and lung [17] cancers in small animal models; however, RGD peptides often provide poor imaging contrast because the integrins are also expressed on other endothelial cells. The RGD peptides act as "strongly" interacting integrin ligands that nonselectively bind to the various integrins and are rapidly captured by the cells via receptor-mediated endocytosis, which decreases the tumor/ background signal ratio.While most studies have focused on optimizing ligands with a high affinity and selectivity to the cell surface targets, we envisioned a new imaging approach that utilized one high-and one low-affinity ligand targeted to independent receptors on a target cell surface. This approach was inspired by the pretargeted method used frequently in the molecular imaging field. [18][19][20][21][22][23] The concept is schematically presented in Figure 1.A simplified model was proposed in which various surface receptors are expressed on cells A and B. Cell A may be selectively visualized by applying the fluorescently labeled probe C, which shows a high affinity toward a receptor overexpressed on the surface of A, i.e., at K D of nano molar level ( Figure 1A). The same receptor could also be expressed more This paper reports an entirely unexplored concept of simultaneously recognizing two receptors using high-and low-affinity ligands through ligating them in situ on the target cell surface. This de novo approach is inspired by the pretargeting strategy frequently applied in molecular imaging, and has now evolved as the basis of a new paradigm for visualizing target cells with a high imaging contrast. A distinct advantage of using a labeled low-affinity ligand such as glycan is that the excess labeled ligand can be washed away from the cells, whereas the ligand bound to the cell, even at the milli molar affinity level, can be anchored by a bioorthogonal reaction with a pretargeted high-affinity ligand on the surface. Consequently, nonspecific background is minimized, leading to improved imaging contrast. Importantly, despite previously unexplored for molecular imaging, a notoriously weak glycan/lectin interaction can now be utilized as a highly selective ligand to the targets. Cell ImagingMolecular imaging research has focused on noninvasively analyzing the molecular kinetics in small animals for use in diagnostic applications. [1][2][3][4][5][6][7] In vivo information about biologically active small molecules and biomolecules, such as the localization or expression levels of target receptors, may be readily imaged using fluorescence or radionuclide-based detection. Although many promising tracers can target specific organs or tumors, [8][9][10][11][12][13] the selectivity and specificity of these tracers toward target cells must be improved. For example, RGD peptides, highaffinity ligands of α V β 3 integrins, [9,14] which are cell adhesion

Cancer discrimination by on-cell N-glycan ligation

“…Both functionalized RGD peptide (strongly interacting ligands) and Nglycans (weakly interacting ligands) were designed and prepared according to a previously reported method ( Fig. 2 and Supplementary Methods) 8 . Thus, the azide function was introduced onto the cyclic RGDyK peptide ligands ( Fig.…”

“…At room temperature, the higher rate of integrinmediated internalization of the on-cell cyclic RGDyK reduced the efficiency of the on-surface click reaction when compared to the 4°C reaction conditions (Supplementary Figs. 16 and 17) 8 . After the cells were washed with medium, they were fixed with 4% paraformaldehyde prior to fluorescent microscopy analysis.…”

“…Therefore, the in situ ligation concept is very important for exploiting the advantages of both the strong and weak interactions in order to achieve clear cell imaging contrast. As we have investigated in the previous research 8 , the clicked products are efficiently internalized with the same efficiency as RGD peptide, while glycans are less efficiently internalized. Because the click reaction should occur on the cell surface, click ligation and subsequent internalization could be the key of the method to image the target cells.…”

“…In order to rapidly identify the optimal combination of both RGD and N-glycan ligands for imaging of the target cancer cells, the four RGD peptides and the five glycan ligands were screened simultaneously in a library fashion on cells in 96-well plates. We applied our previously optimized cell incubation conditions for pre-targeting and the strain-promoted click reaction to ensure that the azide group on the integrintargeted cyclic RGDyK had a sufficiently high clickable reactivity on the cell surface 8 . Namely, the cells (5 × 10 4 ) in each well were initially treated with the cyclic RGDyK peptides 1a-d (50 µM) for 15 min at room temperature (RT).…”

“…Recently, we proposed an approach for distinguishing normal HUVECs (human umbilical vein endothelial cells) from cancerous HeLa cells (human cervical cancer cells), both of which express common receptors such as α v β 3 integrins, by combining an RGD peptide with a glycan in a pre-targeted manner (Fig. 1c) 8 . In this approach, a cyclic RGDyK peptide with high affinity for α v β 3 integrins, prepared with an azide tag instead of an imaging label, was initially pre-targeted to both HUVECs and HeLa cells.…”