Abstract 28: Cell penetrating proteins targeting Mcl-1 induce in vitro and in vivo on-target cancer cell killing of Mcl-1 dependent cell lines
Authors: Deroo Sabrina , Thiolloy Sophie , Desmet Johan , Baatz Franky , Vandenbroucke Karen , Lorent Eric , Henderikx Paula , Alard Philippe , Loverix Stefan , Lasters Ignace , McGrath Yvonne 
Published: July 2017

Abstract
The pro-survival protein Myeloid Cell Leukaemia-1 (Mcl-1) plays an essential role in survival of numerous cancers. MCL-1 gene amplifications occur in a variety of human cancers and overexpression of the Mcl-1 protein is often associated with chemotherapeutic resistance and disease relapse. Complix has developed Cell Penetrating Alphabodies (CPAB), a novel and unique therapeutic class of proteins engineered to efficiently enter cells and inhibit proteins including Mcl-1.
High affinity Alphabodies (ABs) targeting Mcl-1 were engineered by a combination of rational design and phage library screening. In affinity assays, these ABs were shown to bind to Mcl-1 with picomolar affinities whilst binding to Bcl-XL and Bcl-2 was below the detection limit of the assay. In vitro, CPAB uptake was shown to occur rapidly with cytosolic levels reaching up to 1 μM within 2 hours of CPAB exposure. Uptake was associated with cell death of the Mcl-1 dependent multiple myeloma (MM) cell line NCI-H929 (IC50=0.5 µM) and killing was correlated with caspase-3/7 activation. Anti-Mcl-1 CPAB were also shown to disrupt Mcl-1-Bak and Mcl-1-Bim complexes in H929 cells and induced dose-dependent Bak activation. In a panel of MM cell lines, anti-Mcl-1 CPAB induced cell death with a median IC50 of 0.96 μM and cell killing was not restricted to a specific subset of MM cell lines (CCDN1, MAF, MMSET). Gene expression analysis revealed that the anti-Mcl-1 CPAB cell killing potency correlates with MCL-1 gene expression but correlates best with the MCL-1:BCL-2 gene expression ratio. The same gene expression correlation analysis of the Bcl-2 targeting agent Venetoclax revealed an inverse pattern to that achieved with the Mcl-1 specific CPAB.
In vivo, CPAB conferred with an albumin binding domain for extension of half-life, showed a serum half-life in mice of more than 1 hour and associated tumor concentrations of more than 1 µM. Immunohistochemistry and direct detection of fractionated tumor tissue confirmed the intracellular presence of the CPAB in the tumor cells. When given daily IV at 20 mg/kg, anti-Mcl-1 CPAB induced tumor growth inhibition of 50% versus control in two MM xenograft models (H929 and MOLP-8). Tumor growth remained significantly inhibited even two days after the last treatment in the MOLP-8 model and tumor growth inhibition was associated with increased staining of cleaved caspase-3 as compared to vehicle treated tumors.
In summary, anti-Mcl-1 CPAB efficiently kill Mcl-1 dependent cancer cell lines by on-target effects as demonstrated by (1) disruption of Mcl-1-Bak and Mcl-1-Bim complexes, (2) Bak activation and (3) correlation of potency with MCL-1:BCL-2 gene expression ratio. These CPAB induced a robust reduction in tumor growth in mouse models and represent a best-in-class cell penetrating protein therapeutics for tackling intracellular PPI critical to diseases with unmet medical need.
Citation Format: Sabrina Deroo, Sophie Thiolloy, Johan Desmet, Franky Baatz, Karen Vandenbroucke, Eric Lorent, Paula Henderikx, Philippe Alard, Stefan Loverix, Ignace Lasters, Yvonne McGrath. Cell penetrating proteins targeting Mcl-1 induce in vitro and in vivo on-target cancer cell killing of Mcl-1 dependent cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 28. doi:10.1158/1538-7445.AM2017-28