Antibody-PROTAC Conjugates Enable HER2-Dependent Targeted Protein Degradation of BRD4

Abstract

Targeting protein degradation with Proteolysis-Targeting Chimeras (PROTACs) is an area of great current interest in drug discovery. Nevertheless, although the high effectiveness of PROTACs against a wide variety of targets has been established, most degraders reported to date display limited intrinsic tissue selectivity and do not discriminate between cells of different types. Here, we describe a strategy for selective protein degradation in a specific cell type. We report the design and synthesis of a trastuzumab-PROTAC conjugate (Ab-PROTAC 3) in which E3 ligase-directed degrader activity is caged with an antibody linker which can be hydrolyzed following antibody-PROTAC internalization, releasing the active PROTAC and inducing catalytic protein degradation. We show that 3 selectively targets bromodomain-containing protein 4 (BRD4) for degradation only in HER2 positive breast cancer cell lines, while sparing HER2 negative cells. Using live cell confocal microscopy, we show internalization and lysosomal trafficking of the conjugate specifically in HER2 positive cells, leading to the release of active PROTAC in quantities sufficient to induce potent BRD4 degradation. These studies demonstrate proof-of-concept for tissue-specific BRD4 degradation, overcoming limitations of PROTAC selectivity, with significant potential for application to novel targets.

Figure 1

(A) Proposed mode of action of an antibody–PROTAC conjugate, resulting in HER2-dependent protein degradation. (B) Structures of compounds 1 and 2. (C) Overall structure of Ab-PROTAC 3. (D) LC-MS analysis of trastuzumab-NGM-PROTAC conjugate (Ab-PROTAC 3) and the deconvoluted ion series mass spectrum. Observed mass of 152 010 corresponds to full antibody modified with four NGM-BCN-PROTAC molecules, where all NGM moieties are hydrolyzed to maleamic acids (expected: 152 008); 76 005 corresponds to the isomeric form in which the hinge cysteines are bridged in an intrachain manner (expected: 76 004). (E) Hydrolytic stability of Ab-PROTAC 3 determined by liquid chromatography–mass spectroscopy (LC-MS).

Figure 2

(A) Expression of HER2/neu receptor and BRD4 protein in the cell lines used in this study: Western blot analysis of HER2/neu, BRD4, and β-actin on cell lysates from MCF-7, MDA-MB-231, SK-BR-3, and BT-474 cells. (B) Representative frame from immunofluorescence microscopy images of HER2– MCF-7 and HER2+ SK-BR-3 cells labeled with a primary anti-HER2 antibody and stained with a secondary AF488-conjugated antibody (green). Nuclei were stained with DAPI (blue). (C) Monitoring of BRD4 degradation properties of PROTAC 1 in MCF-7, MDA-MB-231, SK-BR-3 and BT-474 cells. Western blot analysis of BRD4 and β-actin after treatment of cells with PROTAC 1, or control compounds. Cells were incubated with PROTAC 1 for either 4 h in the medium before harvesting cell lysates (“4 h incubation”), or for 1 h followed by washout (“washout”), which consisted of removal of the medium, washing with PBS, the addition of fresh medium, and harvesting 23 h later. (D) Ab-PROTAC 3 induces selective degradation of BRD4 in HER2+ cell lines, in a dose-dependent manner: Western blot analysis of BRD4 and β-actin following treatment of MCF-7, MDA-MB-231, SK-BR-3, or BT-474 cells with Ab-PROTAC 3, or control compounds. Cells were incubated with Ab-PROTAC 3 for either 4 h in the medium before harvesting cell lysates (“4 h incubation”), or for 1 h followed by washout (“washout”), consisting of removal of the medium, washing with PBS, the addition of fresh medium, and harvesting 23 h later. (E) Structure of BRD4 degrader 4 used as a positive control to gauge BRD4 degradation. (F) BRD4 degradation by Ab-PROTAC 3 is proteasome-dependent: Western blot analysis of BRD4 and β-actin after treatment of SK-BR-3 and BT-474 cells with Ab-PROTAC 3 or control compounds in the presence or absence of proteasome inhibitor bortezomib (BTZ). Cells were incubated for 2 h with BTZ or PBS (pH 6.9), followed by 1 h of treatment with Ab-PROTAC (3). A washout was performed on the wells treated with Ab-PROTAC 3, and cells were harvested 23 h after washout.

Figure 3

(A) HER2+ SK-BR-3 cells were treated with Alexa488-conjugated Ab-PROTAC (AF488-3) (100 nM) for 1, 4, 8, and 24 h (all post-1-h washout). Cells were then labeled with LysoTracker DeepRed (50 nM, 30 min). Shown is a representative frame from live imaging of cells via confocal microscopy. Scale bar = 20 μm. (B) HER2– MCF-7 cells were treated with AF488-3 (100 nM) for 30 min, followed by incubation with LysoTracker DeepRed (50 nM) for an additional 30 min. Shown is a representative frame from live imaging of cells via confocal microscopy. (C) Quantification of AF488-3 colocalization with LysoTracker DeepRed. The Pearson’s correlation coefficient is shown for various incubation time points in SK-BR-3 cells; data are mean + the standard error of measurement (SEM); n = 6 cells. Statistical analysis was performed by one-way ANOVA, followed by Tukey’s multiple comparison test (triple asterisk symbol (***) denotes P < 0.0001; “ns” = nonsignificant).