Preferential Antibody and Drug Conjugate Targeting of the ADAM10 Metalloprotease in Tumours

Abstract

ADAM10 is a transmembrane metalloprotease that sheds a variety of cell surface proteins, including receptors and ligands that regulate a range of developmental processes which re-emerge during tumour development. While ADAM10 is ubiquitously expressed, its activity is normally tightly regulated, but becomes deregulated in tumours. We previously reported the generation of a monoclonal antibody, 8C7, which preferentially recognises an active form of ADAM10 in human and mouse tumours. We now report our investigation of the mechanism of this specificity, and the preferential targeting of 8C7 to human tumour cell xenografts in mice. We also report the development of novel 8C7 antibody-drug conjugates that preferentially kill cells displaying the 8C7 epitope, and that can inhibit tumour growth in mice. This study provides the first demonstration that antibody-drug conjugates targeting an active conformer of ADAM10, a widely expressed transmembrane metalloprotease, enable tumour-selective targeting and inhibition.

Keywords: ADAM metalloprotease; antibody–drug conjugate; brain cancer; colon cancer; therapeutic antibody.

Figure 1

Biodistribution of radiolabelled 8C7 in mice with subcutaneous LIM1215 tumours. (A–C) Mice (n = 5) were injected with a mix of ¹¹¹In- and ¹²⁵I-labelled 8C7, and blood and tissues were recovered after 2 h, 48 and 120 h (Day 0, 2 and 5) for determination of radioactivity, expressed as mean fraction of the injected dose/gram tissue (% ID/g, ±SD). (D) The data in A and B showing radiolabelled 8C7 retained in tumour versus normal tissues at Day 5 relative to Day 0. (E) Representative images of a mouse with subcutaneous tumour (circled), injected with ¹¹¹In-8C7 and imaged (Day 2) by surface rendered MRI and gamma camera SPECT scan.

Figure 2

Anti-ADAM10 mAb 8C7 preferentially binds human colon tumour cells compared to normal cells. (A) Single cell isolates from colon tumour and matched normal patient tissue were stained with the anti-ADAM10 antibodies 8C7 and 4A11, and with antibodies against CD45 and EpCam, and analysed by flow cytometry. Histograms show normalised staining of epithelial cell populations (EpCam positive, CD45 negative). (B) Tumours were recovered from mice bearing two human colorectal PDX lines, and single cell isolates were stained with 8C7, 4A11 or control IgG, before analysis by flow cytometry. Normal colon and spleen were also analysed for 8C7 binding (human-specific 4A11 was not used). Histograms show overlaid binding of ADAM10 antibodies with an isotype control. (C) Quantitation of 8C7 binding to tumour cell isolates, from multiple passages of six distinct colorectal PDX tumour lines (n = 14 tumours), compared to normal tissue (% positive cells). Binding of the anti-human ADAM10 mAb 4A11 to PDX tumour cells is also shown for comparison.

Figure 3

Binding of 8C7 to ADAM10 is sterically regulated by the metalloprotease domain obstructing the 8C7 epitope. (A) Crystal structure of the ADAM10 metalloprotease (MP), disintegrin (D) and cysteine-rich (C) domains [13]. (B) Crystal structure of the ADAM10 D and C domains in complex with the 8C7 Fab fragment [20]. (C) Cartoons of the structures in A and B, illustrating our approach to test whether removal of the MP domain allows binding of 8C7 to ADAM10, using ADAM10 D and C domains lacking the MP domain (ADAM10D + C), expressed on the cell surface. (D) HEK293 cells expressing endogenous ADAM10 were transfected with ADAM10D + C and compared to untransfected cells by flow cytometry for binding to the anti-ADAM10 mAbs 8C7 (blue) and 4A11 (yellow), or unstained cells (control, red). (A) and (B) were adapted with permission from Seegar et al., 2017, Cell [13].

Figure 4

8C7-ADCs specifically kill cells expressing ADAM10 with the exposed C domain epitope. (A) Structures of drugs (MMAE, PBD or DXd) and linkers conjugated to 8C7 and control IgG. (B) Cell viability of HEK293 cells expressing an ADAM10 construct lacking the metalloprotease domain (ADAM10D + C), or parental HEK293 cells, after treatment with the indicated doses of 8C7 ADCs or matched control IgG ADCs. Cell viability was tested after 3 days by MTS assay. Graphs show mean +/− SEM (n ≥ 4).

Figure 5

Treatment with 8C7-PBD inhibits tumour growth in mice. (A) Tumour volumes in mice bearing LIM1215 xenografts (n = 6) treated with 8C7-PBD (0.5 mg/kg), control IgG-PBD (0.5 mg/kg), 8C7 only (10 mg/kg) or vehicle control (PBS) at the indicated times (arrows). (B) Mice bearing U251 glioma xenografts (n = 5) were treated with 8C7-PBD, control IgG-PBD, control IgG (0.3 mg/kg) or 8C7 only (10 mg/kg) and tumour growth was measured. (C,D) LIM1215 tumours recovered from control- and 8C7-PBD-treated mice, and analysed via IHC staining of CD31 (C, brown) and immunofluorescence staining of phospho-Histone H2AX (D, red; nuclei, blue). Insets: secondary antibody-only controls. Representative zoomed images and quantitation of whole tumour sections are shown (scale bars in microns). (E) Tumour lysates were Western blotted for Notch 1, and actin was used as loading control. Graph shows quantitation of Notch1 expression relative to actin. All graphs show mean +/− SEM, * p < 0.05, ** p < 0.01.

Figure 6

Specificity of mAb 8C7 for an active conformer of the metalloprotease ADAM10 lies in its binding to an epitope in the C domain (green), which is obscured by the metalloprotease domain (MP) in the inactive conformer. Furthermore, 8C7 selectively targets ADAM10 in tumours, and shows improved anti-tumour activity when conjugated to cytotoxic payloads (red).