Ronald M Lynch
Work Summary
Precise diagnosis and treatment of disease requires an ability to target agents to specific tissues and cell types within those tissues. We are developing agents that exhibit cell type specificity for these purposes.
Precise diagnosis and treatment of disease requires an ability to target agents to specific tissues and cell types within those tissues. We are developing agents that exhibit cell type specificity for these purposes.
A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.
The synthesis, characterization, and use of Eu-DTPA-PEGO-Trp-Nle-Asp-Phe-NH2 (Eu-DTPA-PEGO-CCK4), a luminescent probe targeted to cholecystokinin 2 receptor (CCK2R, aka CCKBR), are described. The probe was prepared by solid phase synthesis. A Kd value of 17±2nM was determined by means of saturation binding assays using HEK-293 cells that overexpress CCK2R. The probe was then used in competitive binding assays against Ac-CCK4 and three new trivalent CCK4 compounds. Repeatable and reproducible binding assay results were obtained. Given its ease of synthesis, purification, receptor binding properties, and utility in competitive binding assays, Eu-DTPA-PEGO-CCK4 could become a standard tool for high-throughput screening of compounds in development targeted to cholecystokinin receptors.
Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.
To achieve early detection and specific cancer treatment, we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (MSH) ligands, our aim is to target melanoma cells which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated, leading to more insights into the effects of multivalent compounds on transduction mechanisms.