Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting

Abstract

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold’s termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.

The positive regulatory loop of TCF4N/p65 promotes glioblastoma tumourigenesis and chemosensitivity

“…TAT peptides can efficiently aid protein crossing biological membranes and promote the uptake of macromolecular cargo. 55 The scrambled sequence of first 12 amino acids of TCF4N (Nsc) was designed as a control protein. To obtain a nuclear‐localization control protein, a tandem repeat of the nuclear localization signal (NLS) was fused at C‐terminus to induce nuclear translocation of Nsc (Figure 7A ).…”

“…Considering recombinant proteins do not readily enter cells, the amino acid of TCF4N was designed to be fused to a TAT peptide at the N‐terminal. TAT peptides can efficiently aid protein crossing biological membranes and promote the uptake of macromolecular cargo 55 . The scrambled sequence of first 12 amino acids of TCF4N (Nsc) was designed as a control protein.…”

Understanding MCL1: from cellular function and regulation to pharmacological inhibition

“…The beta-helix of the CMPX-321A and CMPX-383B Alphabodies bind to the MCL1 BH3 groove to induce selective high-affinity MCL1 inhibition. As a result, CMPX-321A displayed cell-penetrating capacity and induced BAK-mediated cell death in MCL1-dependent cancer cell lines, while CMPX-383B possessed an extended serum half-life and exhibited a potent antitumor activity that reduced tumor burden in MM xenograft mice [172]. While most of the described strategies targeting MCL1 modulate its anti-apoptotic function, there remains the potential to compromise the non-apoptotic functions of MCL1, which remain poorly understood.…”

“…The beta‐helix of the CMPX‐321A and CMPX‐383B Alphabodies bind to the MCL1 BH3 groove to induce selective high‐affinity MCL1 inhibition. As a result, CMPX‐321A displayed cell‐penetrating capacity and induced BAK‐mediated cell death in MCL1‐dependent cancer cell lines, while CMPX‐383B possessed an extended serum half‐life and exhibited a potent antitumor activity that reduced tumor burden in MM xenograft mice [ 172 ].…”