Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein–protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.

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We have developed Cell Penetrating Alphabodies (CPABs), a novel and unique therapeutic class of proteins engineered to efficiently enter cells. In vitro, uptake in a range of tumor and non-tumor cell types occurs rapidly with cytosol levels of up to 1 μM concentration after 2 hours of CPAB exposure. Early forms of these CPABs suffered from rapid serum clearance, thereby limiting their efficacy in vivo and amenability to drug development. The incorporation of an albumin binding region in the body of the protein has allowed extension of serum half-life in mice from a few minutes to more than one hour. These CPABs have been shown to be efficiently delivered to xenograft tumors in mice after IV bolus injection by tissue ELISA and immunohistochemistry. CPABs can be used to target and interfere with intracellular protein-protein interactions involved in tumor survival in a highly specific way. The anti-apoptotic protein Myeloid Cell Leukaemia-1 (Mcl-1) promotes through its interaction with Bak, the survival of a range of different tumor types including myeloid leukemia, breast cancer and non-small cell lung cancer. Moreover, Mcl-1 overexpression is often associated with chemotherapeutic resistance and disease relapse. Mcl-1, however, has proven difficult to target using the conventional small molecule approach. Alphabodies which bind to Mcl-1 were engineered by a combination of rational design and phage display library screening. The affinities for Mcl-1 ranged between 18 pM and 750 pM with binding to the closely related proteins Bcl-2 and Bcl-XL being below the limit of detection for the assay. In a Mammalian Two Hybrid assay, these Alphabodies inhibited Bak-Mcl-1 but not Bak-Bcl-XL interactions. Anti-Mcl-1 CPABs were shown to efficiently kill the Mcl-1 dependent multiple myeloma cell line NCI-H929 with IC50s ranging from 0.5 μM to 2 μM as monitored in cell viability assays. The dose responsive cell killing correlated with caspase-3/7 activation in NCI-H929 cells. Other Mcl-1 dependent tumor cell types including non-small cell lung cancer (NCI-H23) and Burkitt's lymphoma (Raji) or tumor cell types with high Mcl-1 expression such as ovarian cancer (A2780) and colorectal adenocarcinoma (COLO-320DM) were also killed efficiently using anti-Mcl-1 CPABs. Despite its short half-life, daily intraperitoneal administration of a prototype Mcl-1 targeting CPAB (without half-life extension) at 30 mg/kg for 14 days resulted in tumor inhibition of 33% as compared to vehicle control. Experiments are underway in mouse models using the more optimal CPABs with extended serum half-life and tumor exposure. CPABs represent the best-in-class cell penetrating protein therapeutics both in terms of efficiency of uptake and amenability to conversion to viable drugs opening unprecedented opportunities to tackle intracellular protein-protein interactions critical to diseases with unmet medical need. Citation Format: Sabrina Deroo, Sophie Thiolloy, Johan Desmet, Franky Baatz, Stefan Loverix, Karen Vandenbroucke, Eric Lorent, Paula Henderikx, Irma Lemmens, Philippe Alard, Ignace Lasters, Yvonne McGrath. First-in-class cell-penetrating proteins targeting Mcl-1 induce tumor cell apoptosis and inhibition of tumor growth in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3850.

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.

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