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

<b>Background/Objectives:</b> 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. <b>Methods:</b> 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-<i>p</i>-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. <b>Results:</b> 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. <b>Conclusions:</b> 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.