ABSTRACT
Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS)1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.
Subject(s)
Antibodies, Neutralizing/administration & dosage , Epitopes, T-Lymphocyte/immunology , Multiple Sclerosis/drug therapy , Natalizumab/administration & dosage , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Neutralizing/chemistry , Antibody Formation/drug effects , Antibody Formation/immunology , B-Lymphocytes/drug effects , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Integrin alpha4/antagonists & inhibitors , Integrin alpha4/immunology , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Protein Conformation/drug effects , T-Lymphocytes/chemistry , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Various important biological pathways are modulated by TGFß isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFß neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFß3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFß1 and TGFß2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFß1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFß2 to 2.83 Å. The structures provide further insight into the details of TGFß recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoform-specific antibodies using a fresolimumab scaffold.
