Evaluating Isatuximab Interference with Monoclonal Protein Detection By Immuno-Capture and Liquid Chromatography Coupled to High Resolution Mass Spectrometry in the Pivotal Phase 3 Multiple Myeloma Trial, Icaria-MM
Authors: Finn Greg , Macé Sandrine , Campana Frank , Le-Guennec Solenn , Muccio Stephané , Tavernier Alexandra , Rouchon Marie-Claude , Roccon Alain , Dai Shujia , Boutet Valérie , Mouret Dominique , Pradeilles Béatrice , Hugla Sébastien , Engelvin Graziella , DiNoto Deborah , van de Velde Helgi , Fedeli Olivier 
Published: November 2019

Recent advances in multiple myeloma (MM), have seen the advent of several therapeutic monoclonal antibodies (mAb) targeting antigens on malignant plasma cells such as CD38 and SLAMF7. Evaluation of depth of response through quantification of M-Protein, by Serum Protein Electrophoresis (SPEP) and Immuno-Fixation Electrophoresis (IFE) presents a particular challenge for clinical laboratories when therapeutic mAbs are used. Specifically, therapeutic mAbs can confound SPEP and IFE measurements when they overlap with patient serum M-protein. Such interference could lead to an inaccurate determination of depth of response according to International Myeloma Working Group (IMWG) criteria
Isatuximab (Isa), an IgG-kappa, anti-CD38 mAb, binds a specific epitope on CD38, which is widely expressed on MM cells. ICARIA-MM was a phase 3, prospective, randomized, open-label, active-controlled, multicenter study comparing isatuximab in combination with pomalidomide plus dexamethasone (Isa-Pd) to pomalidomide plus dexamethasone (Pd) in patients with relapsed/refractory multiple myeloma (RRMM). ICARIA-MM enrolled patients who had received ≥2 prior lines of therapy, including lenalidomide (len) and a proteasome inhibitor (PI). ICARIA-MM demonstrated that Isa-Pd significantly improved PFS, overall response and depth of response with a trend in overall survival benefit and a manageable toxicity profile in these patients.
To overcome the potential interference of isatuximab with M-Protein in patients enrolled in ICARIA-MM, we developed and validated a hybrid assay using Immuno-Capture and Liquid Chromatography coupled to High Resolution Mass Spectrometry (IC-LC-HRMS). This method enriches for serum immunoglobulins by immuno-capture. Heavy chains (HC) and light chains (LC) are then dissociated and sorted using Liquid Chromatography. M-Protein and isatuximab LC are analyzed using HRMS which allows to differentiate their monoisotopic intact mass.
A small number of patients (24/154; 15.6% in the Isa-Pd arm and 5/153; 3.3% in the Pd arm) fulfilled all criteria for CR (100% M-protein reduction on SPEP, bone marrow <5% plasma cells), but remained IFE positive as per IRC (near-CR category). Serial serum samples from 22 of 24 patients in the Isa-Pd arm were available for MS analysis. Separation of the M-protein and isatuximab signals was performed by IC-LC-HRMS. Using a semi-quantitative approach and applying a sensitivity threshold of 0.25g/dL for IFE-positivity (sensitivity threshold of IFE test per Covance central lab), we identified 11 of 22 patients as having M-protein levels below the threshold for IFE positivity, indicating that the true CR-rate was underestimated in the ICARIA-MM study due to interference.
Of the 11 patients with MS M-protein values below the IFE sensitivity threshold, 10 were IgG (8 IgGκ, 2 IgGλ) and one was lambda LC disease with undetectable baseline HC. Baseline myeloma isotype distribution was different in patients with MS M-protein values above IFE sensitivity threshold (4 IgA, 6 IgG, 1 kappa light chain). This confirms that M-protein interference mediated by isatuximab, an IgG kappa mAb, is predominantly seen in patients with IgG isotype (mainly IgGκ), and in LC patients in whom a detectable IgG heavy chain can now be identified. At primary analysis, 4 of 11 patients with MS M-protein values above IFE sensitivity level, versus 2 of 11 patients with MS M-protein values below IFE sensitivity level had progressed. Median PFS was 13.9mo in patients with MS M-protein values above IFE sensitivity level, versus 15.21mo in patients with MS M-protein values below IFE sensitivity level.
In conclusion, MS can be successfully used to separate endogenous M-protein from therapeutic isatuximab serum levels. In this subpopulation with excellent response to Isa-Pd, while most patients are still progression-free, a trend can be observed towards longer PFS in patients who would be considered IF negative after MS versus patients remaining IF positive.

Disclosures
Finn: Sanofi Oncology: Employment. Macé:Sanofi Oncology: Employment. Campana:Sanofi: Employment. Le-Guennec:Sanofi: Employment. Muccio:Sanofi Oncology: Employment. Tavernier:Sanofi Oncology: Employment. Rouchon:Sanofi Oncology: Employment. Roccon:Sanofi Oncology: Employment. Dai:Sanofi Oncology: Employment. Boutet:Sanofi Oncology: Employment. Mouret:Sanofi Oncology: Employment. Pradeilles:Sanofi Oncology: Employment. Hugla:Sanofi Oncology: Employment. Engelvin:Sanofi Oncology: Employment. DiNoto:Sanofi Oncology: Employment. van de Velde:Sanofi: Employment. Fedeli:Sanofi Oncology: Employment.

OffLabel Disclosure:
Isatuximab is an investigational agent and has not yet been approved by the US Food and Drug administration or any other regulatory agency.