Fig. 2. CPAB-formatted CMPX-321A Alphabody efficiently enters MM cell lines to elicit MCL-1 inhibition.

(A) Addition of an N- and C-terminal arginine-proline ([RP]₇) tag allows bilayer phospholipid crossing of CMPX-321A, the CPAB-formatted successor of CMPX-152A. (B) Western blot of the rest fraction (RF) and cytosolic fraction (CF) of NCI-H929 cells after 2 hours of incubation with 2 μM of the CPAB CMPX-321A. (C) Overlay of immunofluorescence experiments confirming the cell-penetrating capacity of CMPX-321A (green) in HeLa cells, after 30 min of incubation with 1 μM Alphabody. Nuclei are stained with Ruby stain (red). (D) RNA expression data (41, 42) show that MCL-1 is substantially more expressed in MM cells compared to other cancer cell types, indicating that this cancer cell type might be MCL-1 dependent. (E) The cell killing potential of CMPX-321A was evaluated via a panel of 33 MM cell lines. Thirteen MM cell lines were shown to be very sensitive toward MCL-1 inhibition (IC₅₀ < 1 μM), 10 cell lines were moderately sensitive (IC₅₀ = 1 to 2 μM), and 6 cell lines were not sensitive to unresponsive (IC₅₀ > 2 μM). (F) The cell killing potency of CMPX-321A cannot be predicted on the basis of the classical molecular signature of MM cell lines (CCDN1, MAF, MMSET, and others). FPKM, fragments per kilobase million.