Towards Aptamer-Targeted Drug Delivery to Brain Tumors: The Synthesis of Ramified Conjugates of an EGFR-Specific Aptamer with MMAE on a Cathepsin B-Cleavable Linker

Abstract

Background/Objectives: Targeted delivery of chemotherapeutic agents is a well-established approach to cancer therapy. Antibody-drug conjugates (ADCs) typically carry toxic payloads attached to a tumor-associated antigen-targeting IgG antibody via an enzyme-cleavable linker that releases the drug inside the cell. Aptamers are a promising alternative to antibodies in terms of antigen targeting; however, their polynucleotide nature and smaller size result in a completely different PK/PD profile compared to an IgG. This may prove advantageous: owing to their lower molecular weight, aptamer-drug conjugates may achieve better penetration of solid tumors compared to ADCs. Methods: On the way to therapeutic aptamer-drug conjugates, we aimed to develop a versatile and modular approach for the assembly of aptamer-enzymatically cleavable payload conjugates of various drug-aptamer ratios. We chose the epidermal growth factor receptor (EGFR), a transmembrane protein often overexpressed in brain tumors, as the target antigen. We used the 46 mer EGFR-targeting DNA sequence GR-20, monomethylauristatin E (MMAE) on the cathepsin-cleavable ValCit-p-aminobenzylcarbamate linker as the payload, and pentaerythritol-based tetraazide as the branching point for the straightforward synthesis of aptamer-drug conjugates by means of a stepwise Cu-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Results: Branched aptamer conjugates of 1:3, 2:2, and 3:1 stoichiometry were synthesized and showed higher cytotoxic activity compared to a 1:1 conjugate, particularly on several glioma cell lines. Conclusions: This approach is convenient and potentially applicable to any aptamer sequence, as well as other payloads and cleavable linkers, thus paving the way for future development of aptamer-drug therapeutics by easily providing a range of branched conjugates for in vitro and in vivo testing.

Keywords: aptamers; brain tumors; branched conjugates; cytostatic payload; drug delivery.

Figure 1

Sketched structures of common antibody–drug conjugates (drug-to-antibody ratio may vary from 1 to 8) and aptamer–drug conjugates prepared in this study.

Scheme 1

Synthesis of 5′-azido- and 5′-AF488-modified oligonucleotides.

Scheme 2

Synthesis of 1:1 oligonucleotide-5′-payload conjugates; red—MMAE payload, gray—cathepsin-cleavable linker.

Scheme 3

CuAAC modification of 5′-alkyne-oligonucleotides with tetraazide.

Scheme 4

CuAAC modification of 5′-alkyne-oligonucleotides with tetraazide. For structures of the payload and the cleavable linker, see Scheme 2.

Figure 2

The 10% analytical denaturing PAGE. (A) 1—molecular weight markers bromophenol blue (low) and xylene cyanol (high), 2—alkyne-modified GR20, 3—the click reaction of alkyne–GR20 with 1,11-diazido-3,6,9-trioxaundecane, 4—the click reaction of alkyne–GR20 with an excess of tetraazide. (B) 1—the click reaction of alkyne–GR20 with tetraazide in a 3:1 molar ratio, 2—alkyne-modified GR20.

Figure 3

Typical HPLC profile of click reaction between alkyne–GR20 and tetraazide. A—HPLC profile of pure starting alkyne-modified aptamer GR20. B—HPLC profile of click reaction between tetraazide and alkyne-modified GR20. C—HPLC profile of click reaction of azido-modified GR20 mixture with alkyne-modified MMAE.

Figure 4

Flow cytometry (left panel) and MTT cell viability assay of conjugates (right panel) obtained in five cell lines—HCT116, U251, A431, U87, K562. (Left panel) blue—cell autofluorescence; AF488–oligonucleotide concentrations: red—1000 nM, orange—100 nM, green—10 nM, dark green—1 nM.

Scheme 5

Two-step CuAAC assembly of various aptamer–payload conjugates.