Comprehensive genetic and functional analysis of FcγRs in rituximab therapy for autoimmunity reveals a key role for FcγRIIIa on NK cells

B cell depletion using rituximab is widely used to treat autoimmune diseases, but patient response varies. The efficacy of rituximab is limited by the efficiency of depletion. Strategies to improve response include altering rituximab dosing, switching anti-CD20-mAb, alternative B cell targets, or non-B cell targeted therapies. Implementing an appropriate strategy requires understanding of the mechanism(s) of resistance to depletion and, if this varies between individuals, a means to test for it. Rituximab kills B cells via a variety of Fcγ receptor (FcγR)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), as well as non-FcγR mechanisms. We conducted a longitudinal cohort study in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) using two national registries. Qualitative and quantitative FCGR functional variants were measured using multiplexed ligation-dependent probe amplification, supplemented by novel FCGR2C assays. We provide consistent evidence that FCGR3A, specifically increased number of copies of the FCGR3A- 158V allele, was the major FcγR gene associated with rituximab response, including clinical response in RA and SLE and depth of B cell depletion in the combined cohort. In SLE, we provide preliminary data suggesting increased FCGR2C ORF copies were also associated with improved clinical response. Furthermore, we demonstrated the impact of disease status and concomitant therapies on both natural killer cell FcγRIIIa expression and rituximab-induced ADCC; demonstrating increased FcγRIIIa expression and FCGR3A genotype were independently associated with clinical response and B cell depletion. Our findings highlight the importance of enhancing FcγR-effector functions, may help stratify patients, and support ongoing development of next-generation CD20 depleting therapeutics. One Sentence Summary The high affinity FcγRIIIa allotype on NK cells explains depth of B cell depletion and clinical response in rituximab therapy for autoimmune disease ### Competing Interest Statement Dr Vital and Prof Emery have received honoraria and consulting fees from Roche within the last 3 years. All other authors declare no competing interest related to the work described in this manuscript. ### Funding Statement We thank the Medical Research Council (MRC) and Versus Arthritis for their joint funding of MATURA (grant codes 36661 and MR/K015346/1). MASTERPLANS was funded by the MRC (grant code MR/M01665X/1). The Leeds Biologics Cohort was part funded by programme grants from Versus Arthritis (grant codes 18475 and 18387), the National Institute for Health Research (NIHR) Leeds Biomedical Research Centre (BRC) and Diagnostic Evaluation Co-operative and the Ann Wilks Charitable Foundation. The BILAG-BR has received funding support from Lupus UK, and unrestricted grants from Roche and GSK. The functional studies were in part supported through a NIHR/HEFCE Clinical Senior Lectureship to AWM, a Versus Arthritis Foundation Fellowship (grant code 19764), the Wellcome Trust Institutional Strategic Support Fund to JIR and MYMY (204825/Z/16/Z) and NIHR Doctoral Research Fellowship to MYMY (DRF-2014-07-155). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All component studies were approved by a Research Ethics Committee (REC); North West Multicentre (00/8/053), North West Greater Manchester (09/H1014/64), COREC (04/Q1403/37), Leeds West (01/023, 05/MRE03/85 and 09/H1307/98) and Leeds East (04/Q1206/107, 10/H1306/88). All participants provided informed written consent and research was carried out in compliance with the Declaration of Helsinki. All experiments were performed in accordance with relevant protocols and regulations. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data associated with this study are available in the main text or the supplementary materials. Anonymised data and statistical codes can be shared and all requests should be made to AWM to ensure all users of the data adhere to the legal requirements and data sharing of personal data. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided that the original work is properly cited. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.