Impact of FcγR variants on the response to alemtuzumab in multiple sclerosis

Abstract Allelic variants of genes encoding for the Fc gamma receptors IIIA and IIA have been associated with the clinical response to cell‐depleting antibodies in lymphoma patients. Here, we tested the hypothesis that FCGR3A and FCGR2A high‐affinity polymorphisms predict clinical outcomes to alemtuzumab therapy in 85 patients with relapsing‐remitting multiple sclerosis. No differences in clinical and MRI‐based efficacy parameters, the development of severe infusion‐associated reactions and secondary autoimmune diseases during a 2 year follow‐up was observed based on FCGR3A or FCGR2A polymorphisms. This study does not support the use of FCGR genetic variants to predict clinical outcomes to alemtuzumab.


Introduction
Alemtuzumab (Sanofi Genzyme, Cambridge, MA) is a lymphocyte-depleting humanized monoclonal IgG1 kappa antibody that selectively targets CD52, an antigen highly expressed on T and B cells. It is currently approved for highly active relapsing-remitting multiple sclerosis (RR-MS) in over 50 countries, and data from initial treatment cohorts as well as extension studies of phase III clinical trials provided evidence that alemtuzumab can induce prolonged disease remission with no evidence for disease activity in more than 50% of treated patients. 1,2 While the most common adverse events of alemtuzumab therapy are mild and moderate infusion-associated reactions (IARs), a major concern with its use is the high risk of developing secondary autoimmune diseases (SAD) which peak around year 3 after treatment initiation with thyroid autoimmune disease (30-40% of patients) and immune thrombocytopenia (1-3% of patients) being the most frequent conditions. 1,2 Identification of biomarkers that can predict the clinical efficacy of alemtuzumab or the occurrence of adverse events would be of great value for the management of patients with MS.
Lymphocyte-depleting antibodies such as alemtuzumab lyse target cells through IgG Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and by induction of apoptosis. ADCC and ADCP require engagement and cross-linking of activating FccR expressed by immune effector cells. In preclinical models, FccR-mediated cytotoxicity contributes substantially to the efficacy of depleting antibodies such as the anti-HER2 antibody trastuzumab, rituximab, and alemtuzumab, 3,4 and higher affinity allotypic forms of the human activating FccRIIIA as well as genotypic variants of the activating FccRIIA were shown to be associated with improved clinical outcomes in rituximab-treated lymphoma patients in some [5][6][7] but not all 8 studies.
The clinical efficacy of alemtuzumab to induce durable disease remission in MS is believed to involve the initial elimination of pathogenic lymphocytes. 9 While peripheral blood leukocytes are usually efficiently depleted by alemtuzumab, FccR binding affinities of alemtuzumab might additionally determine its efficacy to eliminate pathogenic T and B cells that are less accessible and reside at sites of inflammation such as lymphoid tissues or the central nervous system. 10 Expression of the high-affinity V allele with a valine at amino acid position 158 of FCGR3A results in tighter binding of the FccRIIIA to IgG1 and IgG3, whereas the low-affinity F allele with phenylalanine at this position is associated with decreased binding to IgG, resulting in less efficient ADCC. 11,12 Similarly, the high-affinity H allele with histidine at amino acid position 131 of FCGR2A results in greater affinity of FccRIIA for IgG2, whereas the low-affinity R allele correlates with decreased IgG binding. Here we investigated whether FccRIIIA and FccRIIA polymorphisms are associated with clinical outcomes to alemtuzumab treatment in patients with MS.

Patients and biomaterial
All patients were recruited in the Department of Neurology at the University Hospital M€ unster, Germany. 85 patients with RR-MS on alemtuzumab treatment (Table 1) were included in the current study. For some patients not all parameters investigated were available which resulted in analyses of n = 85x for some characteristics. These dropouts included: NEDA-3, one patient (F/F group); MRI stability, two patients (F/F and V/V groups) for FccRIIIA and NEDA-3, one patient (R/R group); MRI stability, two patients (H/R and R/R groups) for FccRIIA. PBMCs were isolated from ethylenediaminetetraacetic acid blood derived from these patients during blood draws between 2014 -2017 and cryopreserved as previously described. 13 Standard protocol approvals, registrations, and patient consents This study was performed according to the Declaration of Helsinki and approved by the local ethics committee (2014-398-f-S). All patients gave written informed consent.

DNA isolation
DNA was isolated using Qiagen's DNeasy Blood & Tissue Kit (Cat No.: 69506) according to the manufacturer's recommendation.

PCR testing and optimization
PCR primers were designed to amplify the region of the two SNPs of interest in FCGR2A (NP_067674.2 position 166) and FCGR3A (NP_000560.5, position 212). Due to high homology of FCGR3A, primers were designed in a region allowing the specific amplification of the FCGR3A gene. The PCRs were tested on a gradient and the optimal annealing temperature was chosen.
The analytical procedure was tested using six blood samples derived from donors with known FCGR2A and FCGR3A genotypes recruited at the University Hospital Erlangen, Germany. The results of the test runs are shown in Table 2.

PCR amplification
PCR products were amplified in a 25 µL reaction volume containing 0.3 µmol/L of each primer, 1.5 µmol/L MgCl 2 , 200 µmol/L of each dNTP, 0.02 U µl -1 DNA polymerase (kappa robust; kappa Biosystems, KK5024), and 25 ng DNA of the sample. PCR cycle conditions are given in Table 3 and 4.

Sanger sequencing
The PCR products were purified, and bi-directionally Sanger sequenced at Microsynth (Balgach, Switzerland) using the two PCR primers for FCGR2A. In case of the FCGR3A gene an internal forward primer (FCGR3A_-For02: GGGGTGTCTGTGTCTTTCAG) as well as the reverse PCR primer were used for the Sanger sequencing. The sequences were aligned against the reference sequences derived from GenBank (and any SNPs were recorded with Soft Genetics Mutation Surveyor v4.0.9 software).

Statistical analysis
The distributions of positive versus negative clinical and radiological outcomes for patients in different genotypic groups were compared using the two-sided Fisher's exact test via Prism version 7.0a for Mac OSX (Graphpad Software, Inc) and corrected p-values (Bonferroni correction) were calculated with the p.adjust function of the "stats" package (version 3.6.0) with R (http://www.R-project.org). A p-value < .05 was considered statistically significant.

Clinical outcomes
The total number of patients who experienced at least one relapse during the first two years of the follow-up period was 34 (40%). Twenty patients (24%) met the definition for confirmed disease progression (CDP   genotyped as FCGR2A 131 H/H, 36 (42%) were heterozygous FCGR2A 131 R/H carriers, and 28 (33%) were homozygous for FCGR2A 131 R/R (Fig. 1). Frequency distribution of allelic variants in our study were similar to those reported for Caucasians in previous studies. 5,6 Association of FCGR genotypes with clinical outcomes Twenty-seven percent of patients with the FCGR3A 158 V/V genotype did not experience any relapses during the 2 year follow-up period as opposed to 67% for FCGR3A 158 F/F and 63% for FCGR3A F/V carriers. Absence of CDP was documented for 63% of patients with V/V, 87 % of patients with F/F and 71% of patients with F/V genotypes. Sixty percent (V/V), 69% (F/F) and 66% (F/V) respectively showed MRI stability as defined by no new or enlarging T2 or gadolinium-enhancing lesion. NEDA-3 criteria were met by 18% of the V/V group versus 59% (F/F) and 49% (F/V). Severe infusionassociated reactions were experienced by 36% of patients with V/V, 21% with F/F and 46% with F/V genotypes. In 55% of V/V secondary autoimmune diseases occurred as opposed to 30% of F/F and 20% of F/V ( Fig. 2A). Forty seven percent of the FCGR2A 131 H/H carriers were relapse-free during the first 2 years after alemtuzumab treatment initiation compared to 57% of R/R and 69% of H/R carriers. Absence of CDP was noted in 71% (H/H), 68% (R/R) and 86% (H/R) of carriers, respectively. 52% (H/H), 56% (R/R) 83% (H/R) depicted stable MRI activity and NEDA-3 status was achieved in 38% (H/H), 41% (H/R) and 61% (R/R) of study participants. Severe IAR occurrence was distributed as follows: 33% (H/H), 29% (R/R) and 42% (H/R). 24% (H/H), 36% (R/R), and 25% (H/R) developed SADs (Fig. 2B). Moreover, no statistically significant differences in clinical efficacy outcomes, the development of severe IARs and SADs were observed in patients homozygous for both high-affinity variants (FCGR3A 158 V/V; FCGR2A 131 H/ H, designated V/V 3A -H/H 2A ) as compared to patients homozygous for both low-affinity FccRs (FCGR3A 158 F/ F; FCGR2A 131 R/R, designated F/F 3A -R/R 2A ) (Fig. 3).
Altogether, FCGR3A and FCGR2A genotypes were not significantly associated with any of the aforementioned clinical or radiological outcomes following alemtuzumab therapy.

Discussion
Our report is the first investigation into the impact of FccR polymorphisms on clinical outcomes to lymphocyte-depleting antibody therapy in patients with MS. No differences in clinical efficacy parameters, the development of severe IARs and SADs in response to alemtuzumab therapy were observed in patients with RR-MS based on FCGR3A or FCGR2A polymorphisms. These data indicate that FCGR3A and FCGR2A polymorphisms are not associated with clinical outcomes to alemtuzumab in MS.
Our study was motivated by epidemiological investigations that reported an association between the low affinity FCGR3A 158 F/F genotype in patients with non-Hodgkin B-cell lymphoma and a poor clinical response to rituximab, presumably due to incomplete ADCC-mediated depletion of tumor cells. [5][6][7]14 In vitro studies demonstrated that alemtuzumab depletes human lymphocytes through both Fc-mediated ADCC and CDC. 4 In vivo, cell depletion by alemtuzumab is reported to be predominantly mediated by ADCC and largely independent of complement as reported in transgenic mice expressing human CD52 15 and in an experimental murine model of T cell leukemia. 16 While the mechanisms by which alemtuzumab depletes lymphocytes in humans and exerts its beneficial effects in MS are incompletely understood and might require complement-dependent pathways, our data should not be interpreted to minimize the impact of ADCC as an important mechanism of action. Rather, FccR polymorphisms may not strongly influence the clinical efficacy of alemtuzumab therapy. Our findings are based on a relatively small number of subjects (n = 85) and clearly require validation in larger cohorts. Moreover, we cannot exclude potentially confounding effects of neutralizing antibodies to alemtuzumab which may occur in individual patients within the first 2 years after treatment initiation. 17 Given the clinical efficacy and increasing use of lymphocyte-depleting antibodies in the therapeutic landscape of MS and other neurological diseases, our initial assessment provides incentive to conduct larger prospective studies in order to evaluate the impact of FccR polymorphisms on clinical outcomes and in vivo mechanisms of therapeutic antibodies in MS.

Author contributions
Christian W. Keller: Study concept and design, acquisition of data, analysis and interpretation, writing of the manuscript. Tobias Ruck: Study concept and design, acquisition of data, analysis and interpretation, critical revision of the manuscript for important intellectual content. Donal McHugh: Study concept and design, acquisition of data, analysis and interpretation, critical revision of the manuscript for important intellectual content. Steffen Pfeuffer: Acquisition of data, critical revision of the manuscript for important intellectual content. Catharina C. Gross: Acquisition of data, critical revision of the manuscript for important intellectual content. Catharina Korsukewitz: Acquisition of data, critical revision of the manuscript for important intellectual content. Nico Melzer: Acquisition of data, critical revision of the manuscript for important intellectual content. Luisa Klotz: Acquisition of data, critical revision of the manuscript for important intellectual content. Sven G. Meuth: Critical revision of the manuscript for important intellectual content. Christian M€ unz: Critical revision of the manuscript for important intellectual content. Falk Nimmerjahn: Critical revision of the manuscript for important intellectual content. Heinz Wiendl: Critical revision of the manuscript for important intellectual content. Jan D. L€ unemann: Study concept and design, analysis and interpretation, writing of the manuscript, study supervision. For some characteristics not all patient data was available. For the following analyses the n was: 20, NEDA-3 (1 patient missing, F/F 3A -R/R 2A group); 20, MRI stability (1 patient missing, F/F 3A -R/R 2A group). The remaining analyses were carried out on all 21 patients. Shown are percentages of the respective genotypic groups as bargraphs and absolute numbers of patients in white. Statistical analysis: Two-sided Fischer's exact test was applied on absolute patient numbers. A p-value < .05 was considered statistically significant. Abbreviations: CDP, confirmed disability progression; MRI, magnetic resonance imaging; NEDA, no evidence of disease activity; IAR, infusion-associated reactions; SAD, secondary autoimmune disease.