Previously, we developed Alphabodies that act as potent antagonists of the proinflammatory cytokine interleukin-23 (IL-23) ( 7, 8), an extracellular protein target of clinical importance ( 8, 9). This all-Ile core endows the Alphabody with exceptional thermal stability ( T M of about 120☌), while allowing for up to 25–amino acid positions that can be varied for the purpose of constructing a binding site to the intended molecular target ( 7). The three helices are each composed of four heptad repeats with tightly packed isoleucines at core positions ( a and d) in a regular “knobs-into-holes” configuration. Alphabodies are based on a single-chain non-Ig protein scaffold featuring an antiparallel triple-helix coiled-coil fold ( Fig. In this study, we sought to develop the Alphabody as a non-Ig protein scaffold that may help to address these challenges in drug discovery and development. However, despite successes in preclinical studies, the progression of nonantibody scaffolds to clinical development is often hampered by the limited serum half-life of these small-sized protein scaffolds and, similar to antibodies, their inability to reach intracellular targets ( 4– 6). Interest in non-immunoglobulin (Ig) protein scaffolds has been particularly intense because of the ease of screening and selection of high-affinity binders, exquisite stability, scalability, and low cost of production. To overcome some of these impediments, alternative approaches emerged, including engineered antibody fragments, such as the recently Food and Drug Administration–approved caplacizumab ( 3), as well as nonantibody protein scaffolds ( 4, 5). In addition, they impose high production or formulation costs due to low stability and the need for essential posttranslational modifications and harbor complexities in drug administration and patient care.
Although therapeutic monoclonal antibodies are endowed by many favorable characteristics that are important for their use as therapeutic agents, such as long serum half-life, high specificity, and immunological effector functions, they are also critically limited by low tissue penetration and inability to directly address intracellular drug targets. Monoclonal antibodies are currently at the forefront of clinical treatments for various cancers, infections, and (auto)immune diseases and represent 7 of the top 10 best-selling drugs worldwide in 2018 ( 1, 2). 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. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. 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. 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. The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets.
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