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1.
J Biol Chem ; 296: 100641, 2021.
Article in English | MEDLINE | ID: mdl-33839159

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 ß-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.


Subject(s)
Antibodies, Bispecific/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Discovery , Lung Neoplasms/drug therapy , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Animals , Apoptosis , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/immunology , Female , Humans , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Proto-Oncogene Proteins c-met/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
Science ; 317(5844): 1554-7, 2007 Sep 14.
Article in English | MEDLINE | ID: mdl-17872445

ABSTRACT

Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.


Subject(s)
Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Allergens/immunology , Animals , Antibodies, Bispecific/immunology , Antibodies, Monoclonal/immunology , Antigens, CD20/immunology , Antigens, Plant , Autoantibodies/immunology , ErbB Receptors/immunology , Glycoproteins/immunology , Humans , Immunoglobulin Constant Regions/chemistry , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/metabolism , Immunoglobulin Heavy Chains , Macaca mulatta , Mice , Mutation , Myasthenia Gravis, Autoimmune, Experimental/immunology , Myasthenia Gravis, Autoimmune, Experimental/prevention & control , Protein Processing, Post-Translational , Receptors, Cholinergic/immunology
3.
J Immunol ; 177(1): 362-71, 2006 Jul 01.
Article in English | MEDLINE | ID: mdl-16785532

ABSTRACT

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.


Subject(s)
Antibodies, Monoclonal/metabolism , Antigens, CD20/immunology , Antigens, CD20/metabolism , Binding Sites, Antibody , Epitopes/immunology , Epitopes/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Antigens, CD20/genetics , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Antineoplastic Agents/metabolism , Binding Sites, Antibody/genetics , Cell Line , Cell Line, Tumor , Complement Pathway, Classical/genetics , Complement Pathway, Classical/immunology , Cytotoxicity, Immunologic/genetics , Epitope Mapping , Epitopes/genetics , Humans , Immunoglobulin G/metabolism , Lymphoma/immunology , Lymphoma/pathology , Lymphoma/therapy , Mice , Mice, Transgenic , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Peptide Fragments/metabolism
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