Enhanced targeting with heterobivalent ligands

Abstract

A novel approach to specifically target tumor cells for detection and treatment is the proposed use of heteromultivalent ligands, which are designed to interact with, and noncovalently crosslink, multiple different cell surface receptors. Although enhanced binding has been shown for synthetic homomultivalent ligands, proof of cross-linking requires the use of ligands with two or more different binding moieties. As proof-of-concept, we have examined the binding of synthetic heterobivalent ligands to cell lines that were engineered to coexpress two different G-protein-coupled human receptors, i.e., the human melanocortin 4 receptor (MC4R) expressed in combination with either the human delta-opioid receptor (deltaOR) or the human cholecystokinin-2 receptor (CCK2R). Expression levels of these receptors were characterized by time-resolved fluorescence saturation binding assays using Europium-labeled ligands; Eu-DPLCE, Eu-NDP-alpha-MSH, and Eu-CCK8 for the deltaOR, MC4R, and CCK2R, respectively. Heterobivalent ligands were synthesized to contain a MC4R agonist connected via chemical linkers to either a deltaOR or a CCK2R agonist. In both cell systems, the heterobivalent constructs bound with much higher affinity to cells expressing both receptors, compared with cells with single receptors or to cells where one of the receptors was competitively blocked. These results indicate that synthetic heterobivalent ligands can noncovalently crosslink two unrelated cell surface receptors, making feasible the targeting of receptor combinations. The in vitro cell models described herein will lead to the development of multivalent ligands for target combinations identified in human cancers.

Figure 1. MC4R and δOR saturation binding and competitive binding analysis

A. Saturation binding of Eu-DPLCE ligand to δOR in CHO/MC4R/δOR cells. The curve shows δOR specific binding only (total-nonspecific). From these data, the K_d = 10.5 ± 2.6 nM and B_max = 24,000 ± 2,000 AFU (R² = 0.91). Each data point indicates the average of four samples, with error bars indicating the standard error of the mean. B. Saturation binding assay of Eu-NDP-α-MSH ligand to MC4R in CHO/MC4R/δOR cells. The curve shows MC4R specific binding only. From these data, the K_d = 5.6 ± 2.7 nM and B_max = 7,700 ± 1,400 AFU (R² = 0.83). C-D. Competitive binding to CHO cells co-expressing MC4R and δOR. C. Increasing concentrations of Naloxone were added in the presence of 10 nM Eu-DPLCE. From these data, the IC₅₀ was 65 nM with R² = 0.90. D. Increasing concentration of NDP-α-MSH were added to cells in the presence of 10 nM Eu- NDP-α-MSH. From these data, IC₅₀ was 0.77 nM with R² = 0.89.

Figure 2. Immuno-labeling of MC4R and CCK2R and the distribution of heterobivalent ligand labeling in MC4R and CCK2R expressing cells

A & B. Images of Hek293 cells, that stably expressed both the CCK2R and MC4R, labeled with antibodies against the MC4R and CCK2R receptors. C & D. Images of cells incubated for 3 minutes with 0.8 nM of Cy5 labeled MSH-CCK ligand, then washed with ligand free media. The ligand distribution was determined immediately following the rinse (C), and 7 minutes thereafter (D). Scale bar = 20 μM for both images pairs.

Figure 3. MC4R and CCK2R saturation binding and Scatchard plot analysis

A. Saturation curve of Eu-NDP-α-MSH obtained from the MC4R and CCK2 dual expression cell line. The figure shows total binding (■) and binding in the presence of 10 μM NDP-α-MSH (▲). From these data, the K_d = 8.3 ± 1.9 nM, and B_max = 732,000 ± 59,000 AFU. Lines represent the computer modeled best fit of the data using GraphPad Prism software using the non-linear regression, one site-binding equation, with a R² value of 0.81. Each data point indicates the average of four samples, with error bars indicating the standard error mean. B. Saturation curve of Eu-CCK obtained from the MC4R and CCK2 dual expression cell line. The figure shows total binding (■) and binding in the presence of 1 μM CCK8 (▲). From these data, the K_d = 34.6 ± 3.9 nM, and B_max = 1,600,000 ± 83,000 AFU, with a R² value of 0.96.

Figure 4. Evaluation of heterobivalent ligand binding

There are two different approaches by which heterobivalent binding can be evaluated. One approach uses two different cell types (Figure A1 & A2); one that expresses a single complimentary receptor and another that expresses both of the complimentary receptors, while another approach uses the same cell type but blocks binding at one receptor by addition of an agent (Figure B1 & B2).

Figure 5. Representative competitive binding assay at the MC4R

A. Increasing concentrations of the MSH-Deltorphin heterobivalent ligand were added to CHO/MC4R/δOR cells in the presence of 10 nM Eu-DTPA-NDP-α-MSH and the absence (dimer) and presence (monomer) of competing naloxone. For dimer binding (absence of naloxone), the IC₅₀ = 3.2 nM with R² = 0.83; for monomer binding (presence of naloxone), the IC₅₀ = 134.0 nM with R² = 0.93. B. Increasing concentrations of the MSH-CCK heterobivalent ligand were added to cells in the presence of 10 nM Eu-DTPA-NDP-α-MSH. For dimer binding, Hek293/CCK2R/MC4R cells were used and for monomer binding, Hek293/MC4R cells were used. For dimer binding, the IC₅₀ = 4.5 nM with R² = 0.88; for monomer binding, the IC₅₀ = 349.5 nM with R² = 0.96.