Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly

Abstract

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory halflife. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fasthydrolyzing monobromomaleimide (MBM) linker and nucleaseresistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation

“…Increased recycling was observed for EgA1 and Atto647 functionalized assemblies compared to the non-functionalized designs. We have previously also observed increased cellular recycling of a fluorophore functionalized albumin-duplex assembly . It is speculated that the inclusion of fluorophores could mediate hydrophobic interactions with the cell membrane and that these additional cellular interactions could increase the internalization and subsequent recycling of the functionalized construct.…”

“…Previous work using a two-strand system showed high stability even in 50% serum. 11 However, disassembly of the annealed HJ construct under physiological conditions in vivo cannot be discounted and may have contributed to the shorter than expected half-life extension, as discussed above. Future work could address inclusion of other stabilized nucleotide analogues or different sizes and architectures of the assembly.…”

“…10 However, utilizing this for conjugation of the shorter and highly modified 12-mer Q2 oligonucleotide strand presented issues in the ionexchange (IEX)-HPLC purification, with excess unconjugated oligonucleotides overlapping with the rHA-Q2 conjugate peak (Figure S1), resulting in poor annealing efficiency of the rHA-HJ assembly (data not shown). The conjugation and purification was improved by employing a recently published stable monobromomaleimide (MBM)-bicyclo[6.1.0]nonyne (BCN) linker, 11 which afforded pure rHA-Q2 fractions (Figure 2 fraction 1−7 pooled in frame C).…”

“…We have previously introduced a conjugated albumin-oligonucleotide modular assembly based on a simple oligonucleotide duplex design to incorporate aptamers, fluorophores, , and cytotoxic drug molecules . This was performed by annealing a functionalized oligonucleotide module to a complementary oligonucleotide site-selectively conjugated to Cys34.…”

Preparation of Maleimide-Modified Oligonucleotides from the Corresponding Amines Using N-Methoxycarbonylmaleimide

“…Albumin was chosen as the model protein, as it contains one free thiol available for selective modification using thiol-selective reagents . Furthermore, it is highly relevant, as conjugation of albumin is a tool for half-life extension of drugs and biomacromolecules. , For the experiments we prepared maleimide, MBM and SMCC modified DNA on a 20 nmol scale and purified the conjugates by reverse-phase HPLC. Analytical HPLC of the crude products of the maleimides showed only one major peak (Figure A, Figure S14), and LC-MS analysis of the purified DNA confirmed that the desired products were formed (Figure B, Figure S15).…”