Development of a Hydrashift 2/4 Isatuximab Assay to Mitigate Interference with Monoclonal Protein Detection on Immunofixation Electrophoresis in Vitro Diagnostic Tests in Multiple Myeloma

Introduction
Evaluation of multiple myeloma (MM) response through quantification of M-protein, by serum protein electrophoresis (SPEP) and immuno-fixation electrophoresis (IFE) is challenging for clinical laboratories because therapeutic monoclonal antibodies (mAbs) can confound IFE when they converge with serum M-protein. This can be misleading when interpreting patients’ response to therapy. Isatuximab (Isa), an IgG-kappa anti-CD38 mAb is approved based on the pivotal ICARIA-MM study in combination with pomalidomide (P) and dexamethasone (d), in the United States, the European Union, Canada, Australia, Switzerland and Japan for the treatment of adult patients with relapsed/refractory MM who have received at least two prior therapies including lenalidomide and a proteasome inhibitor. Recently, the Phase 3 IKEMA study evaluating Isa plus carfilzomib (K) and d met its primary endpoint at the first planned interim analysis, demonstrating significantly prolonged progression free survival (PFS) compared with Kd alone in patients with relapsed MM.

Methods
To overcome the interference mediated by Isa on the IFE test, we have developed an Isa-specific assay “HYDRASHIFT 2/4 Isa” using a highly specific rabbit, anti-Isa mAb. The HYDRASHIFT 2/4 Isa test is used in conjunction with the regular Hydragel IF procedure and the semi-automated Hydrasys 2 electrophoresis apparatus. The HYDRASHIFT 2/4 Isa test displaces Isa (IgG kappa) interference during the electrophoresis process by forming Isa/anti-Isa complex which is moved out of the gamma zone toward the alpha globulin fractions.

Results:
Sensitivity of the HYDRASHIFT 2/4 Isa assay was demonstrated on serum samples, including normal serum samples. Isa control and serum samples with different monoclonal components spiked with Isa (final concentrations in the range of 0.1 and 3.0 g/L) were analyzed with the HYDRASHIFT 2/4 Isa procedure used in conjunction with Hydragel 4 IF Acid Violet. The detection limit of Isa and/or the Isa / anti-Isa antibody complex visualized was 0.2 g/L. Importantly, efficient removal of Isa from the gamma globulin zone, with no trace signal evident, was demonstrated for all Isa concentrations tested, even for 3 g/L. Reproducibility of the HYDRASHIFT 2/4 Isa assay was also demonstrated between gels, between product lots, between instruments and on different test days. Ten different serum samples, including 1 normal serum sample and 9 serum samples with monoclonal components spiked with Isa at 1 g/L, were run using the HYDRASHIFT 2/4 Isa procedure in conjunction with Hydragel 4 IF Acid Violet. All samples gave 100% concordant results between gels on the different instruments and with different HYDRASHIFT 2/4 Isa lots. To evaluate specificity, 50 serum samples from MM patients were spiked with 1 g/L Isa and the anti-Isa mAb shifted Isa specifically with no impact on the patients’ M-spike, demonstrating 100% specificity. Finally, the HYDRASHIFT 2/4 Isa test was evaluated on 15 samples from treated patients enrolled in Isa clinical trials at different therapy cycles. The evaluated samples demonstrated efficient removal of Isa from G and Kappa tracks on the gamma globulins zone and visualization of the Isa/anti-Isa complex on alpha zone and/or Isa on ELP track. For the pre-treatment samples, there was as expected no Isa detection, and no impact on M-protein was observed, further confirming the specificity of the assay toward Isa.

Conclusions
Therapeutic mAb inclusion in MM treatment regimens offer patients significant improvements in clinical outcomes. With rapid evolution of therapeutic options in MM, there is a clear need for a standardized and reliable method to ensure authentic IFE-based clinical assessment. Development of the Isa-specific HYDRASHIFT 2/4 Isa assay offers the advantage of high clinical utility due to the simplicity of the method as an add-on to the conventional IFE In vitro diagnostic test. Consequently, this assay will facilitate the correct assessment of clinical outcomes for patients receiving Isa as part of their MM treatment. Submission for global regulatory clearance for Isa-specific HYDRASHIFT 2/4 assay is planned in 2020.