Overcoming the Interference of Daratumumab with Immunofixation Electrophoresis (IFE) Using an Industry-Developed Dira Test : Hydrashift 2/4 Daratumumab
Authors: Caillon Helene , Irimia Alix , Simon Jason S. , Axel Amy , Sasser Kate , Scullion Matthew James , Ligneel Thierry , Nouadje Georges , Moreau Philippe , Dejoie Thomas 
Published: October 2019

Abstract
Background : Detection and quantification of monoclonal component (M-spike) by serum protein electrophoresis (SPE) and immunofixation (IFE) are essential for response evaluation in multiple myeloma (MM) according to the International Myeloma Working Group (IMWG) criteria. Recent clinical trials on daratumumab, an IgG Kappa anti-CD38 monoclonal antibody, have shown impressive results with deep responses. However daratumumab may be detected on serum protein electrophoresis (SPE) and immunofixation (IFE) assays used for monitoring disease monoclonal immunoglobulins (M protein). This can lead to false positive SPE and IFE assay results for patients with IgG kappa myeloma protein impacting initial assessment of complete responses (CR) by International Myeloma Working Group (IMWG) criteria. Differentiating therapeutic monoclonal antibodies, such as daratumumab, from endogenous monoclonal protein can be challenging when both molecules co-migrate or migrate closely on electrophoresis. The availability of a specific, anti-daratumumab antibody has provided the opportunity to overcome this interference and to correctly assess biochemical response. Indeed, Mc Cudden and al. in collaboration with Janssen developed a technique, the Daratumumab Interference Reflex Assay (DIRA) test, which has been utilised in daratumumab clinical trials. Given the need for a commercially available automated and standardized test, we evaluated a new commercial DIRA kit test being developed by Sebia (Lisses, France): the Hydrashift 2/4 daratumumab.
Objective: The aim of this study is to evaluate the Hydrashift 2/4 daratumumab in comparison with our laboratory developed DIRA test for the displacement of daratumumab on IFE.
Design and methods: The Hydrashift 2/4 daratumumab assay was prepared by Sebia using the anti-daratumumab antibody produced by Janssen and modified to allow a migration of daratumumab/anti-daratumumab complexes toward the α-globulin fraction on IFE. IFE technical procedures, migration, and staining programs were performed according to the manufacturer instructions, and run on the standard Sebia, Hydrasys plateform, with the HYDRAGEL 4 IF kit. In addition to the regular procedure, an additional applicator to apply the anti-daratumumab antibody was used. Analytical performances including sensitivity, specificity and comparisons with the original DIRA test were assessed on 31 samples from ongoing daratumumab clinical trials.
Results: Serum samples from 309/324 (95.4%) patients assessed demonstrated a positive IFE at diagnosis. In 119/309 (38,5%) of cases, the M-spike partially or totally co-migrated with daratumumab detected in serum. Of these, MM cases displayed an isotype other than IgG Kappa or Kappa light chains did not require a DIRA test during follow-up for response assessment as a standard IFE could clearly show if initial monoclonal component was still detectable or not. From our experience, an anti-daratumumab displacement assay was only required for IgG Kappa MM or Kappa Light Chain MM (LCMM) when standard IFE could not distinguish daratumumab from endogenous M-spike. This situation represented 66 cases (21,4 %, i.e. 66 on 309).
The Hydrashift 2/4 daratumumab assay showed excellent concordance (100%) with the laboratory developed test on the 31 samples tested (i.e. 17 negative DIRA, 10 positive DIRA and 4 doubtful DIRA). Daratumumab/anti-daratumumab complexes were detected in the α-globulin fraction with a sensibility of 200 mg/L. Daratumumab/anti-daratumumab complex was difficult to visualize when daratumumab concentrations were less than 200 mg/L but daratumumab was shown to be completely removed from the gamma globulin fraction with no trace left for all tested patients. For 48 samples tested on diagnosis, the anti-daratumumab shifted specifically the daratumumab with no effect on the patients' M-spike (100% specificity).
Conclusion: With the growing application of monoclonal antibodies such as daratumumab in the treatment of multiple myeloma, the development of validated, widely available assays to overcome antibody interference will become increasingly important. The Hydrashift 2/4 daratumumab test provides the opportunity to automate and standardize the displacement of daratumumab interference and help with the correct interpretation of IFE results for clinical outcome measures.

Disclosures
Simon: Janssen: Employment. Axel:Janssen Pharmaceuticals Research and Development: Employment. Sasser:Johnson & Johnson: Equity Ownership; Janssen Pharmaceuticals R&D: Employment. Scullion:Janssen: Employment. Ligneel:Sebia: Employment. Nouadje:Sebia: Employment. Moreau:Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Takeda: Honoraria; Celgene: Honoraria.