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. 2012 Dec 26;109(52):21295-300.
doi: 10.1073/pnas.1211762109. Epub 2012 Dec 10.

Heterobivalent Ligands Target Cell-Surface Receptor Combinations in Vivo

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Free PMC article

Heterobivalent Ligands Target Cell-Surface Receptor Combinations in Vivo

Liping Xu et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Chemical structures of labeled compounds and their molecular dynamic profiling for assessing ligand mobility. (A) Heterobivalent ligands containing melanocortin (brown) and cholecystokinin (green) pharamacophores connected via a PEGO-[PG]3-PEGO linker (cyan) that bears a lysine handle for incorporation of Cy5 and Eu-DTPA tags (blue). (BD) Molecular dynamic study of Cy5 labeled ligand 1 (htMVL 1). (B) Illustration of one of the conformers with various functionalities and atom labels that were monitored for distances during the simulation is shown in C, and energy plot of various conformations (distance mapped from atoms 48–496) against temperature is shown in D. The study reveals significant linker mobility with 30–70 Å spacing between the two motifs and an average distance range of 40 Å (see SI Materials and Methods for more details).
Fig. 2.
Fig. 2.
Live cell imaging of Cy5 htMVL 1 specifically binding to target cells. (A) Four types of cells were treated with htMVL 1 at the indicated concentrations for 15 min. Representative live cell fluorescence images of htMVL 1 binding are shown. Red, Cy5; blue, Hoechest. (B) The percentage of Cy5-positive cells was determined after htMVL 1 treatments in the target cells (red bars) and control cells (black and blue bars). The data are the mean ± SEM (n = 3). A total of ∼800 cells were counted for each condition. (C) Representative images of blocking study conducted in the target cells. Control (-), no htMVL 1 treatment; Control (+), 10 nM htMVL 1; Block 1: 10 nM htMVL 1 + 10 µM NDP-α-MSH; Block 2: 10 nM htMVL 1 + 10 µM CCK8; Block 3: 10 nM htMVL 1 + 10 µM CCK8 + 10 µM NDP-α-MSH. (D) A total of ∼600 cells were counted for each condition. The data are shown as mean ± SEM (n = 3), *P < 0.05. (E) htMVL 1 ligand was processed and internalized over time after incubation in the target cells. (AF) Cy5 fluorescence images (red). (GL) Cy5 fluorescence images merged with a visible light image. (Scale bars: 25 µm.)
Fig. 3.
Fig. 3.
In vivo fluorescence imaging of Cy5 htMVL 1 specifically retained in target tumors. (A) Representative time course of fluorescence images of the Cy5 htMVL 1 retained in mice bearing xenograft tumors. R flank, target tumor; L flank, control tumor (MC1R control). (B) Mean surface radiance of htMVL 1 (2.5 nmol per mouse) in the target tumors (red bars) and control tumors (black bars) was quantified at the indicated postinjection time points. Fold increase of mean surface radiance in target relative to control tumors (number shown above asterisks) is shown. Error is expressed as SEM (n = 4). (C) Representative images of blocking experiments. Control (+) (n = 4): 2.5 nmol htMVL 1 per mouse; Block 1 (n = 3): 2.5 nmol htMVL 1 + 50 nmol NDP-α-MSH; Block 2 (n = 5): 2.5 nmol htMVL 1 + 50 nmol CCK8; Block 3 (n = 3): 2.5 nmol htMVL 1 + 50 nmol CCK8 + 50 nmol NDP-α-MSH. (D) Mean surface radiance for each target tumor (R flank) under each condition. Error is reported as SEM. Block 2 and block 3 agents lead to significantly decreased signal compared with control tumors (without blocking). (E) Representative ex vivo image of tumors and organs excised 4 h after injection of 2.5 nmol htMVL 1. Tumor (R): target tumor; Tumor (L): control tumor. (F) Mean surface radiance for each organ. Error is expressed as SEM (n = 6), *P < 0.05.
Fig. 4.
Fig. 4.
In vivo quantification of Eu htMVL 2 retained in target tumors. (A) Eu content (fmol/mg of tumor) was measured for each tumor from mice injected with 2.5 nmol Eu htMVL 2 as determined by time-resolved fluorometry. The Eu content in target tumors was significantly higher than that in MC1R control tumors. n = 3 for each time point. (B) Eu content in target tumors versus MC1R control tumors from mice treated with a lower dose (0.5 nmol per mouse). n = 3 for each time point. (C) Eu content in target tumors versus CCK2R control tumors with 2.5 nmol Eu htMVL 2 treatments. n = 3 for each time point. (D) Eu content in target tumors versus CCK2R control tumors with 0.5 nmol Eu htMVL 2. n = 8 for each time point. (E) Eu content was measured in the target tumors under the following conditions: Control (+) (n = 4): 0.5 nmol htMVL 2 per mouse; Block 1 (n = 6): 0.5 nmol htMVL 2 + 50 nmol NDP-α-MSH; Block 2 (n = 5): 0.5 nmol htMVL 2 + 50 nmol CCK8; Block 3 (n = 5): 0.5 nmol htMVL 2 + 50 nmol CCK8 + 50 nmol NDP-α-MSH. (F) Eu content was measured for the kidney and liver from mice injected with 2.5 nmol of htMVL 2. n = 3 for each time point. (G) Eu content was also measured with a lower dose (0.5 nmol per mouse) in the kidneys and livers. n = 3 for each time point. *P < 0.05.

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