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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All requests for raw and analyzed data and materials will be promptly reviewed by the Institute for Research in Biomedicine to verify if the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper may be subject to patient confidentiality. Any data and materials that can be shared will be released via a Material Transfer Agreement. Source data of Fig. 1 are provided. Sequence data of the monoclonal antibodies isolated in this study have been deposited in GenBank (MN044260–MN044339). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE34 partner repository with the dataset identifier PXD013599. The X-ray structure factors and coordinates have been deposited in the Protein Data Bank (access numbers are 6FG1 and 6FG2).
Hao, L., Fang-Hong, S., Shi-Ying, H., Shun-Guo, Z. & Min-Ling, C. A review on clinical pharmacokinetics, pharmacodynamics, and pharmacogenomics of natalizumab: a humanized anti-alpha4 integrin monoclonal antibody. Curr. Drug Metab. 19, 1213–1223 (2018).
Chataway, J. & Miller, D. H. Natalizumab therapy for multiple sclerosis. Neurotherapeutics 10, 19–28 (2013).
Calabresi, P. A. et al. The incidence and significance of anti-natalizumab antibodies: results from AFFIRM and SENTINEL. Neurology 69, 1391–1403 (2007).
Bachelet, D. et al. Occurrence of anti-drug antibodies against interferon-beta and natalizumab in multiple sclerosis: A collaborative cohort analysis. PLoS One 11, e0162752 (2016).
Rup, B. et al. Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals: recommendations of the Innovative Medicines Initiative ABIRISK consortium. Clin. Exp. Immunol. 181, 385–400 (2015).
Link, J. et al. Clinical practice of analysis of anti-drug antibodies against interferon beta and natalizumab in multiple sclerosis patients in Europe: A descriptive study of test results. PLoS One 12, e0170395 (2017).
Dunn, N. et al. Rituximab in multiple sclerosis: Frequency and clinical relevance of anti-drug antibodies. Mult. Scler. 24, 1224–1233 (2018).
Quistrebert, J. et al. Incidence and risk factors for adalimumab and infliximab anti-drug antibodies in rheumatoid arthritis: a European retrospective multicohort analysis. Semin. Arthritis Rheum. 48, 967–975 (2018).
Jensen, P. E. H. et al. Detection and kinetics of persistent neutralizing anti-interferon-beta antibodies in patients with multiple sclerosis. Results from the ABIRISK prospective cohort study. J. Neuroimmunol. 326, 19–27 (2019).
Zare, N., Zarkesh-Esfahani, S. H., Gharagozloo, M. & Shaygannejad, V. Antibodies to interferon beta in patients with multiple sclerosis receiving CinnoVex, rebif, and betaferon. J. Korean Med. Sci. 28, 1801–1806 (2013).
Murdaca, G. et al. Immunogenicity of infliximab and adalimumab: what is its role in hypersensitivity and modulation of therapeutic efficacy and safety? Expert Opin. Drug Saf. 15, 43–52 (2016).
Traggiai, E. et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 10, 871–875 (2004).
Yu, Y., Schurpf, T. & Springer, T. A. How natalizumab binds and antagonizes alpha4 integrins. J. Biol. Chem. 288, 32314–32325 (2013).
Jensen, K. K. et al. Improved methods for predicting peptide binding affinity to MHC class II molecules. Immunology 154, 394–406 (2018).
Paul, S. et al. Development and validation of a broad scheme for prediction of HLA class II restricted T cell epitopes. J. Immunol. Methods 422, 28–34 (2015).
de la Hera, B. et al. Natalizumab-related anaphylactoid reactions in MS patients are associated with HLA class II alleles. Neurol. Neuroimmunol. Neuroinflamm 1, e47 (2014).
Pan, Y., Yuhasz, S. C. & Amzel, L. M. Anti-idiotypic antibodies: biological function and structural studies. FASEB J. 9, 43–49 (1995).
van Schie, K. A. et al. Neutralizing capacity of monoclonal and polyclonal anti-natalizumab antibodies: The immune response to antibody therapeutics preferentially targets the antigen-binding site. J. Allergy Clin. Immunol. 139, 1035–1037.e6 (2017).
Foote, J. & Milstein, C. Kinetic maturation of an immune response. Nature 352, 530–532 (1991).
Hamze, M. et al. Characterization of CD4 T cell epitopes of infliximab and rituximab identified from healthy donors. Front Immunol. 8, 500 (2017).
Harding, F. A., Stickler, M. M., Razo, J. & DuBridge, R. B. The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs 2, 256–265 (2010).
Tiller, T. et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. Methods 329, 112–124 (2008).
Lefranc, M. P. et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res. 37, D1006–D1012 (2009).
Yaari, G., Uduman, M. & Kleinstein, S. H. Quantifying selection in high-throughput Immunoglobulin sequencing data sets. Nucleic Acids Res. 40, e134 (2012).
Gupta, N. T. et al. Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data. Bioinformatics 31, 3356–3358 (2015).
Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 (1997).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
Bricogne, G. et al. BUSTER version 2.11.7. (Global Phasing Ltd, Cambridge, United Kingdom, 2017).
Spits, H. et al. Characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned CTL lines. Hybridoma 2, 423–437 (1983).
Watson, A. J., DeMars, R., Trowbridge, I. S. & Bach, F. H. Detection of a novel human class II HLA antigen. Nature 304, 358–361 (1983).
Latorre, D. et al. T cells in patients with narcolepsy target self-antigens of hypocretin neurons. Nature 562, 63–68 (2018).
Bassani-Sternberg, M., Pletscher-Frankild, S., Jensen, L. J. & Mann, M. Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation. Mol. Cell Proteomics 14, 658–673 (2015).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).
The authors would like to thank the patients for their participation in the study. We would like to thank M. Nussenzweig (Rockefeller University) for providing reagents for antibody cloning and expression, and Servizio Tipizzazione of the IRCCS San Matteo Hospital Foundation, Pavia, Italy, for HLA typing. This work was supported by the Swiss National Science Foundation (grant no. 176165 to A.L.) and by the Innovative Medicines Initiative Joint Undertaking ABIRISK (Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk) project under grant agreement no. 115303, resources of which are composed of financial contribution from the European Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA Companies. A.L. and F.S. are supported by the Helmut Horten Foundation.
V.M., T.B., S.P., J.L.P., P.F. and J.D. are all employees of Sanofi. F.D. has participated in meetings sponsored by or received honoraria for acting as an advisor/speaker for Biogen Idec, Celgene, Genzyme-Sanofi, Merck, Novartis Pharma, and Roche. His institution has received research grants from Biogen and Genzyme Sanofi. He is section editor of the MSARD Journal (Multiple Sclerosis and Related Disorders). A.L. is a Senior Vice President and Senior Research Fellow at Vir Biotechnology, Inc.
Peer review information: Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Alignment of NZM heavy and light chain variable regions (NZM VH and NZM VL) to the human scaffold antibody counterparts (21/28’CL and REI) used for NZM humanization. Mutated residues are shown in red. Dots indicate the same residue. b, Scheme of the 8 heavy and 8 light chains variants of NZM that were combined in an 8x8 matrix to express 64 different NZM CDR swap variants. c, Cluster analysis of binding of 30 antibodies isolated from patient A to the 64 NZM swap variants by ELISA. BAbs and NAbs are indicated on the x-axis in red and black, respectively. The NZM swap variants are shown on the right y-axis (H, heavy chain; L, light chain; 1, CDR1; 2, CDR2; 3, CDR3). Optical density (OD) values are shown with a two-color gradation scale from minimum (white) to maximum (blue).
Extended Data Fig. 2 Structural details of the interaction of NZM with a NAb, a BAb and α4-integrin.
a, Closer view of the interaction interface between NZM and NAA32 (left) and NAA84 (right). Epitope and paratope residues are shown in solid sticks. Proteins are displayed in ribbon diagram. The empty space in the interface between the NZM and NAA32 or NAA84 is represented as orange or purple surface, respectively, in two different orientations b, Superimposition of the antigen-binding fragment of NZM in complex with NAA84 (NAb, green), NAA32 (BAb, cyan) and α4-integrin (orange). NZM heavy and light chains are shown in salmon and slate blue, respectively. Proteins are displayed in ribbon diagram.
Flow cytometry analysis of memory CD4+ T cells at day 12 after ex-vivo stimulation with irradiated autologous monocytes untreated (upper panels) or pre-pulsed with NZM peptide pool (lower panels). CFSElowCD25+ICOS+ T cells reactive to NZM peptide pool were FACS-sorted and cloned by limiting dilution (representative of n = 2 biologically independent samples).
Extended Data Fig. 4 MHC restriction of NZM-reactive CD4+ T cell clones and peptide-MHC-II binding affinity predictions of NZM and deimmunized variants.
a, MHC restriction of NZM-reactive T cell clones. NZM-specific CD4+ T cell clones isolated from patient A (upper panel) and patient B (lower panel) were stimulated with antigen-pulsed autologous APCs in the absence or presence of blocking anti-MHC-II antibody (anti-HLA-DR, clone L243; anti-HLA-DQ, clone SPVL3; anti-HLA-DP, clone B7/21). Proliferation was measured on day 3 after a 16-h pulse with [3H]-thymidine, and is expressed as counts per minute (cpm). Inhibition of T cell proliferation was > 80% only in the presence of the anti-HLA-DR antibody. b and c, Predicted binding affinities of all theoretical 15mer peptides derived from NZM heavy chain (HC) and light chain (LC) to HLA-DRB1 alleles carried by the two patients (b), or to a reference set of nine HLA-DRB1 and HLA-DRB3/4/5 alleles (c). The affinities are shown as reciprocal IC50 (nM) values. The dotted lines define the thresholds of high-affinity binding set at 100 nM and low-affinity binding set at 300 nM. d, Predicted binding affinities of 15mer peptides spanning the light chain CDR2 region of NZM variants to HLA-DRB1 alleles carried by patient A (DRB1*14:01 and DRB1*16:01) and patient B (DRB1*0701). The affinities are shown as reciprocal median IC50 (nM) values. The dotted lines define the thresholds of high-affinity binding set at 100 nM and low-affinity binding set at 300 nM.