Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET

Abstract

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non-small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain-Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.

Keywords: EGFR exon 20 insertion; MET amplification; amivantamab; bispecific anti-EGFR×MET antibody; combinatorial screening; crystal structure; epitope mapping; non–small cell lung cancer (NSCLC).

Figure 1

Procedure for generating BsAbs panel and strategy for identification of lead BsAb.A, the blue panel represents an array of five IgG1 anti-MET and one control anti-gp120 (b12) mAbs with the leucine to phenylalanine substitution at position 405 (F405L). The green panel represents an array of eight IgG1 anti-EGFR and one control anti-gp120 mAbs with the lysine to arginine substitution at position 409 (K409R). Upon separate cell-culture growth, each mAb was purified using protein A chromatography. The grid of BsAbs was generated by cFAE for hit selection. B, elimination strategy for the identification of optimal lead BsAb. A schematic flowchart that outlines the selection of the lead bivalent EGFR×MET bispecific antibody. The steps include binding of EGFR/MET monovalent BsAbs by flow cytometry to NCI-H441, H1975, and A549 cells; confirmation of the absence of MET phosphorylation in A549 cells; determination of the minimal proliferation in H1975, KP4, and NCI-H441 cells; and absence of cross phosphorylation of EGFR. BsAb, bispecific antibody; EGFR, epidermal growth factor receptor; MET, mesenchymal–epithelial transition factor.

Figure 2

Selection phase 1.A, binding activity of functionally monovalent BsAb to NCI-H441, H1975, and A549 cells. Monovalent BsAbs (b12×EGFR and MET×b12) were screened for binding to NCI-H441, H1975, and A549 cell lines. The binding EC₅₀ values are shown as red, green, and blue dots for A549, H1975, and H441 cells, respectively. Monovalent EGFR C and EGFR D molecules had EC₅₀ values greater than 1 μg/ml (marked by dotted line). Monovalent EGFR F and EGFR G BsAbs did not show appreciable binding, indicated as NB. B, identification of agonistic EGFR×MET BsAbs via MET phosphorylation in A549 cells. MET agonism was measured via MET phosphorylation in A549 cells incubated with MET×EGFR BsAbs. Equal amounts of sample on the Western blot were confirmed by the equivalent levels of total MET. The absence (similar to negative control antibody and untreated cell-only control) or low levels of MET phosphorylation levels were shown as dark green triangles with a score of 1. Low levels of MET phosphorylation levels (just above the detection limit) were shown as blue triangles with a score of 2. Medium levels of MET phosphorylation levels (like MET B IgG1 mAb) were shown as orange triangles with a score of 3. High levels of MET phosphorylation levels (like control MET 5D5 IgG1 mAb) were shown as red triangles with a score of 4. C, overview of MET antagonism score for all assessed BsAb. The same color scheme was used as in B. BsAb, bispecific antibody; EGFR, epidermal growth factor receptor; MET, mesenchymal–epithelial transition factor.

Figure 3

Effect of EGFR×MET BsAbs on cell proliferation.A, inhibition of HGF binding-driven proliferation of KP4 cells with an autocrine HGF/MET loop. The bivalent MET B IgG1 mAb served as a positive control. BsAb concentrations of 10 μg/ml (green), 0.37 μg/ml (black), and 0.014 μg/ml (gray) were used. B, inhibition of EGF-dependent EGFR based on proliferation of H1975 cells. Bivalent EGFR H IgG1 was used as positive control and b12 IgG1 as negative control. MET 5D5 IgG1 was used as agonistic control mAb. The best proliferation inhibitors that had activity better than the negative control are indicated by green bars. C, identification of agonistic BsAb-based proliferation of NCI-H441 cells. Same controls as in B. BsAbs that induced proliferation are indicated by red bars. D, EGFR phosphorylation in A549 cells. Unstimulated A549 cells were treated with BsAbs to identify agonistic molecules. None of the BsAbs induced EGFR phosphorylation. In parallel, cells were stimulated with EGF and clear induction of EGFR phosphorylation by EGF was observed, which was effectively blocked by BsAbs containing EGFR arm H and partially by the BsAb with EGFR arm E. Positive control IgG1 EGFR H was fully blocking, and no inhibition was observed with the controls b12 IgG1and monovalent MET A×b12. All samples were loaded to have similar levels of total EGFR (bottom panels). BsAb, bispecific antibody; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MET, mesenchymal–epithelial transition factor.

Figure 4

Epitope of anti-MET arm of amivantamab based on the crystal structure of MET Sema-PSI–Fab complex.A, representation of the crystal structure. The Fab light (LC, blue) and heavy (HC, green) chains bound to the Sema domain (pink) of MET. The numbers in the center of Sema refer to the seven blades that form Sema β-propeller structure (red numbers for Fab-binding blades). The epitope, LC paratope, and HC paratope regions are shown in red, purple, and yellow, respectively. The binding CDRs and MET loops are labeled. B, interactions between amivantamab and MET. The left panel shows a close view of the combining site with hydrogen bond interactions indicated as dashed lines. The right panel shows an interaction map with hydrogen bonds as solid lines, van der Waals interactions as dashed lines, MET, LC and HC residues in gray boxes, white boxes, and ovals, respectively. C, open book view of the interface between MET (left) and Fab (right). Residues at the interface were labeled and colored according to their buried surface area (red for residues that bury greater than 75% of their total area, yellow for 45%–74% range, and gray for 15%–44% range). Light chain residues are underlined. D, overlay of MET crystal structures bound to either amivantamab or onartuzumab + HGF. The HGF region that clashes (distance between atoms < 1.5 Å) with the LC of amivantamab is shown in red. HGF, hepatocyte growth factor; MET, mesenchymal–epithelial transition factor.

Figure 5

Epitope mapping of the anti-EGFR arm of amivantamab.A, differences in affinity of EGFR H (amivantamab arm) and M225 (mAb containing cetuximab Fv) to single-point mutants of EGFR in reference to wildtype EGFR. The right panel shows the location of the mutations in the crystal structure of EGFR bound to ligand TGFα (PDB code 1MOX; reference (48)). TGFα (brown) is shown at its binding pocket on EGFR domain III (blue). Mutations S418G and S474D (white regions) do not impact EGFR H and M225. Mutations K465E, I467M, K443R, and S468N (green regions) impact both EGFR H and M225, although with different strengths. Mutations G471A and N743K (yellow regions) impact only M225. B, possible partial epitope (residues K443, K465, I467, and S468; shown in red) of the anti-EGFR arm of amivantamab mapped on the crystal structure of EGFR bound to cetuximab (Protein Data Bank 1YY9, (48)). EGFR domains and cetuximab are indicated. EGFR, epidermal growth factor receptor; TGFα, transforming growth factor alpha.

Figure 6

Amivantamab showed superior efficacy in the HCC827-HGF model. Animals (groups of nine) were treated with agents listed above for the first 21 days of study. Subcutaneous tumors were measured twice weekly and the results plotted as the mean tumor volume, expressed in mm³ + standard error of the mean (SEM), of each group. The plots are labeled PBS control as black circles, crizotinib vehicle and erlotinib vehicle as green circles, 25 mg/kg erlotinib as purple triangles, 30 mg/kg crizotinib as white rhombi, 25 mg/kg erlotinib and 30 mg/kg crizotinib as blue circles, and 10 mg/kg amivantamab as red triangles.