SUMMARY

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  • A summary of the information provided in this response is provided as a video that can be accessed by clicking the following link:
    • RYBREVANT - Mechanism of Action Video
• Amivantamab is a low fucose, fully human immunoglobulin G1 (IgG1)based bispecific antibody with immune celldirecting activity that targets activating and resistance epidermal growth factor receptor (EGFR) mutations and mesenchymal-epithelial transition (MET) mutations and amplifications.^1-4
  • EGFR is a transmembrane receptor tyrosine kinase (RTK) involved in normal cellular and pathological processes, including cell proliferation, invasion, survival, and angiogenesis.¹
  • MET receptors, which are RTKs expressed on epithelial cells, are activated by the binding of hepatocyte growth factor (HGF), resulting in downstream signaling processes that mediate invasive growth.¹
  • A preclinical study showed amivantamab blocked ligand-induced phosphorylation of EGFR and the MET receptor more potently than the combination of single receptorbinding antibodies.²
• Most of the EGFR mutations observed in non-small cell lung cancer (NSCLC) tumors comprise Exon 19 deletions (Exon19del) and L858R point mutations in Exon 21; the less common mutations include Exon 20 insertion (Exon20ins) mutations.^6,7
• Amivantamab shows pre-clinical and clinical activity against NSCLC tumors with Exon19del, L858R, and Exon20ins primary EGFR activating mutations, T790M and C797S secondary EGFR resistance mutations, and resistance due to activation of the MET pathway.^1-3,5
• In preclinical studies, amivantamab has shown antitumor activity in the EGFRmutated NSCLC setting through several distinct mechanisms of action²:
  • Dual inhibition of ligand-induced activation and downstream signaling of both EGFR and the MET receptor,
  • Downmodulation or degradation of these receptors, and
  • Targeting of tumor cells for destruction by immune effector cells, such as natural killer (NK) cells and macrophages, through antibody-dependent cellular cytotoxicity (ADCC) and trogocytosis mechanisms, respectively.^1,4
• In additional preclinical studies in NSCLC cell lines and xenograft mouse tumor models harboring EGFR Exon20ins mutations, amivantamab showed a dose-dependent reduction in cell viability, inhibited EGFR and MET receptor downstream signaling pathways, and decreased tumor volumes.³
  • Fc-mediated monocyte and macrophage interactions, and associated trogocytosis, were also identified as key mediators of receptor downmodulation and antibodymediated cancer cell apoptosis.⁴

BACKGROUND

Role of EGFR and MET in Non-Small Cell Lung Cancer

Disruption and aberrant activation of RTKs that are involved in essential cellular processes via transduction pathways have been linked to oncogenesis and tumor progression.⁹ MET receptors are RTKs activated by a single ligand, HGF. Binding of HGF to the MET receptor activates tyrosine residue autophosphorylation and downstream signaling processes, which trigger cell proliferation, cell survival, motility function, and morphogenesis. MET is overexpressed in NSCLC tissues in a majority of cases.¹⁰

The EGFR is a transmembrane protein receptor involved in normal cellular and pathological processes, including cell proliferation, survival, oncogenesis, and metastasis.¹¹ EGFR helps in potentiating MET-mediated cell proliferation, invasion, and survival.¹⁰ EGFR mutations in NSCLC tumors can lead to activation of EGFR signaling pathway in the absence of ligands and triggers downstream signaling pathways and anti-apoptotic signals such as phosphatidylinositol 3-kinase/protein kinase B (AKT) and extracellular signalregulated kinase (ERK)/mitogen-activated protein kinase also seen in MET receptor activation.^6,10,12 Exon 19 deletions and L858R point mutation in Exon 21 are the most common EGFR mutations observed in NSCLC and these mutations are sensitive to tyrosine kinase inhibitors (TKIs).^6,7 The less common EGFR mutations in NSCLC include Exon20ins mutations.⁷ EGFR Exon20ins mutations in NSCLC have been associated with insensitivity or resistance to currently available small-molecule EGFR TKIs.^13-16

MECHANISM OF ACTION

Amivantamab is a low fucose, fully human IgG1-based EGFR-MET bispecific antibody with immune cell-directing activity that targets activating and resistance EGFR mutations and MET mutations and amplifications. Amivantamab shows pre-clinical and clinical activity against NSCLC tumors with Exon19del, L858R substitution, and Exon20ins primary activating mutations, T790M and C797S secondary EGFR resistance mutations, and resistance due to activation of the MET pathway.^1-3,5 In preclinical studies, amivantamab has shown activity against tumors with aberrant EFGR and MET signaling through several distinct mechanisms: dual inhibition of ligand-induced activation and downstream signaling of EGFR and MET receptors, downmodulation or degradation of these receptors, and targeting of tumor cells for destruction by immune effector cells, such as NK cells and macrophages, through ADCC and trogocytosis mechanisms, respectively.^2,4 In preclinical studies in NSCLC cell lines and xenograft mouse tumor models harboring EGFR Exon20ins mutations, amivantamab showed a dosedependent reduction in cell viability, inhibited EGFR and MET downstream signaling pathways, and decreased tumor volumes.³ Data from the preclinical studies that evaluated the mechanisms of action of amivantamab are summarized below.

Immune Cell Directing Activity

In Vitro Antibody-Dependent Cellular Cytotoxicity

Amivantamab has low levels of core fucose, which allows for tighter binding to human FcγRIIIa on NK cells and increases ADCC. In vitro ADCC assays using H292 and H1975 cells showed increased tumor cell lysis with amivantamab treatment compared with an EGFR-MET bispecific control with normal fucose levels. Testing with additional lung cancer cell lines, including those with WT or mutant EGFR and WT or amplified MET, showed similar results.²

In Vitro and In Vivo Anti-Tumor Activity by Trogocytosis

In vitro proliferation and apoptosis assays of amivantamab using H1975 cells and a MET amplified gastric carcinoma cell line (SNU5), in the presence of peripheral blood mononuclear cells (PBMCs), showed reduction in tumor cell viability (IC₅₀, 0.0054 mcg/mL and 0.0013 mcg/mL, respectively) and increase in apoptosis (IC₅₀, 0.0004 mcg/mL and 0.014 mcg/mL, respectively). In coculture of H1975 and PBMCs, amivantamab treatment showed increased macrophage inflammatory protein (MIP)1β in the presence of PBMCs, monocytes, or macrophages, and enhanced downregulation of EGFR, p-EGFR and MET receptors.⁴

In xenograft mouse models harboring H1975 and SNU5 tumor cells, amivantamab treatment reduced tumor growth by 75% (P<0.0001) and 96% (P<0.0001), respectively, compared with control. Fc-mediated monocyte and M1 and M2 macrophage interactions, and associated trogocytosis were also identified as key mediators of receptor downmodulation and antibody-mediated cancer cell apoptosis.⁴

Receptor Degradation

In Vitro Inhibition of EGFR and MET Phosphorylation and Downstream Signaling

Inhibition of EGFR and MET phosphorylation was evaluated in 7 NSCLC human tumor cell lines (including H1975) with EGFR primary activating mutations, EGFR T790M mutation (with or without MET gene amplification), WT EGFR and MET. Amivantamab showed dosedependent inhibition of EGFR and MET phosphorylation, with IC₅₀ values <100 nmol/L. Amivantamab inhibited both phospho-ERK (p-ERK) and phospho-AKT (p-AKT) in most of these cell lines, with low to sub nanomolar IC₅₀ values. However, there was weak or no inhibition of p-ERK or p-AKT in cell lines with MET gene amplification (H820 and H1993).²

In Vivo Reduction of Tumor Growth and Induction of Receptor Downmodulation

In the H1975-HGF xenograft mouse model bearing the EGFR L858R and T790M mutations, treatment with amivantamab significantly reduced tumor growth by 80% compared with treatment with vehicle (P<0.0001).²

Tumor lysates of the H1975-HGF xenograft mice treated with amivantamab showed significant reductions of 76% and 61% in levels of EGFR and MET, respectively (P<0.0001 and P=0.0007, respectively), and in the levels of phospho-EGFR (p-EGFR) and phosphoMET (pMET) compared with vehicle control demonstrating downmodulation of both receptor targets and downstream signaling.²

Inhibition of Ligand Binding

In Vitro Inhibition of Ligand Binding

In vitro assays showed simultaneous binding of amivantamab to the extracellular domains of EGFR (K_D: 1.4 nmol/L) and MET receptor (K_D: 40 nmol/L) with high affinity and similar potency compared with the parental monospecific bivalent antibodies.^2,17 Amivantamab was also shown to inhibit ligand binding of each receptor, with 50% inhibitory concentration (IC₅₀) values of 10 nmol/L for EGF binding to EGFR and 30 nmol/L for HGF binding to MET receptor.²

Anti-Tumor Activity Against EGFR Exon20ins Mutations

In Vitro Reduction of Cell Viability, Receptor Levels, and Downstream Signaling

Ba/F3 Cells with EGFR Exon20ins Mutations

An in vitro assay evaluated the effect on cell viability in Ba/F3 cells harboring diverse Exon20ins EGFR mutations. Amivantamab, at concentrations ranging from 0.05 to 1 mg/mL, led to significant (P<0.0001), dosedependent reduction of cell viability of all 5 EGFR Exon20ins mutant cells (V769_D770insASV, D770delinsGY, H773_V774insH, Y764_V765insHH, and D770_N771insSVD), with IC₃₀ values in the low to sub nanomolar range (0.11.5 mg/mL for Exon20ins EGFRs and 0.9 for WT EGFR).^3,8

In Ba/F3 cells overexpressing the EGFR D770delinsGY and H773_V774insH Exon20ins mutations, amivantamab treatment showed a reduction in total EGFR levels compared with untreated cells, with significant reductions seen in p-EGFR, p-AKT, p-ERK, and phospho-S6 (p-S6). Furthermore, amivantamab treatment led to a significant accumulation of cells in G1 phase compared with vehicle-treated cells (P<0.0001) and induced apoptosis.³

Evaluation of the binding of amivantamab (0.1 mg/mL) in these cells using fluorescence-activated cell-sorting (FACS) analysis showed a decrease in plasma membrane-bound EGFR after amivantamab treatment compared with IgG1 control-treated cells (40% EGFR expression relative to control at 72 hours of exposure).³

Patient-Derived Cells with EGFR Exon20ins Mutations

In vitro testing of the antitumor activity of amivantamab was conducted in NSCLC patientderived cells (PDCs; DFCI-127, DFCI-58, YU-1163) harboring Exon20ins mutations. Results were generally consistent with those observed with in vitro testing in Ba/F3 cells bearing Exon20ins mutations. In DFCI-127 and DFCI-58 cells, amivantamab treatment led to decreases in total EGFR and MET levels, and inhibition of p-EGFR, p-MET, p-AKT, p-ERK, and p-S6. Also, compared with IgG1 controls, amivantamab inhibition cell growth and cell proliferation in a dose-dependent manner.³

Immunofluorescence binding assays in PDCs (DFCI-127 and DFCI-58) with EGFR Exon20ins mutations showed decreases in both plasma membrane-bound EGFR and the MET receptor following amivantamab treatment compared with IgG1-treated controls; additionally, amivantamab treatment led to internalization of both receptors.³

In Vitro Induction of Antibody-Dependent Cellular Cytotoxicity

In vitro ADCC assays in PDCs (DFCI-127 and YU-1163, bearing EGFR Exon20ins mutations) showed dose-dependent cytotoxicity in both PDC lines with amivantamab treatment. However, in both PDCs, this cytotoxicity was significantly diminished in the presence of an Fc receptor (FcR) blocker in both PDCs (P<0.0001), which indicates that interaction with FcRs on PBMC may be required for the antibodydependent cytotoxic effect of amivantamab.³

In Vivo Reduction of Tumor Growth, Receptor Levels, and Downstream Signaling

In xenograft mouse models created using Ba/F3 cells overexpressing the EGFR D770delinsGY and H773_V774insH Exon20ins mutations and PDCs DCFI-127 and YU-1163 bearing the EGFR P772insPNP and S768_D770dup Exon20ins mutations, respectively, treatment with amivantamab significantly decreased tumor volumes compared with treatment with IgG1 control (P<0.0001; vs Ba/F3 cells) or vehicle (P<0.0001; vs PDCs).³

Tumor lysates of xenograft mice treated with amivantamab showed significant reductions in levels of EGFR, MET receptor, p-EGFR, and p-MET compared with vehicle.³

A YHIM-1029 patient-derived xenograft model bearing the D770_N771insG Exon20ins mutation also showed a significant reduction in tumor volume with amivantamab treatment compared with vehicle (P<0.0001), along with downmodulation of EGFR and the MET receptor, inhibition of downstream signaling pathways, and an increase in apoptosis markers.³

LITERATURE SEARCH

A literature search of MEDLINE^®, Embase^®, BIOSIS Previews^®, and Derwent Drug File (and/or other resources, including internal/external databases) was conducted on
14 August 2024.